CN101238224A - Expression profiles of peripheral blood mononuclear cells for inflammatory bowel diseases - Google Patents
Expression profiles of peripheral blood mononuclear cells for inflammatory bowel diseases Download PDFInfo
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Abstract
The present invention is directed to the identification of PBMC- and IBD- associated biomarkers that may be used to diagnose inflammatory bowel disease, and optionally, distinguish between PBMCs isolated from a patient with Crohn's disease and PBMCs isolated from a patient with ulcerative colitis. The present invention is further directed to methods of screening, including high throughput methods of screening, for regulatory agents capable of regulating the activity of PBMC- and IBD-associated biomarkers. Provided are compositions of PBMC- and IBD-associated biomarkers, including regulatory agents of at least one PBMC- and IBD-associated biomarker for methods of diagnosis, prognosis, therapeutic intervention and prevention of an inflammatory bowel disease, e.g., Crohn's disease and ulcerative colitis. Moreover, the present invention is also directed to methods that can be used to assess the efficacy of test compounds and therapies in the treatment inflammatory bowel disease, i.e., Crohn's disease or ulcerative colitis.
Description
[0001] the application requires No. the 60/687th, 331, the U.S. Provisional Patent Application of on June 6th, 2005 application and the benefit of priority of No. the 60/692nd, 295, the U.S. Provisional Patent Application of applying on June 20th, 2005; The content whole of these two parts of provisional application is hereby incorporated by.
Background of invention
Invention field
[0002] the present invention relates to from the isolating peripheral blood lymphocytes of patients with inflammatory bowel (peripheral blood mononuclear cell, expression pattern analysis PBMC) and the discriminating that can distinguish the patient's who suffers from one of two class inflammatory bowels (being Crohn's disease and ulcerative colitis) PBMC open gene label.
Related background art
[0003] ulcerative colitis (ulcerative colitis, UC) and Crohn's disease (Crohn ' sdisease, CD) be that (inflammatory boweldisease IBD), has several population counies and Clinical symptoms to two kinds of common chronic and recurrent inflammatory bowels.But there are significant differences in UC and CD aspect tissue injury, point out these two kinds of diseases that distinct pathogenic process is arranged.What usually a kind of IBD cause of disease that proposes was a mucomembranous immune system to normal intestinal flora is not suitable for activation (Podolsky (2002) N.Engl.J.Med.347:417-29).Reply relevant saturating wall with the Thl-type, the granulomatous inflammation process is the feature of CD, and the inflammation among the UC often is confined to mucous membrane, and contains the plasmocyte (Podolsky, ibid) that seems to reply with Th2 relevant immunoglobulin,exocrine in a large number.The combination that these two kinds of diseases all are wherein E﹠H factors can determine the complex disease (Bouma and Strober (2003) Nat.Rev.Immunol.3:521-33) of individuality to the susceptibility of disease.
That [0004] uses that oligonucleotide microarray existed in the ability of the quantitative whole express spectra of rna level has been applied to study the gastrointestinal tissue of the excision that derives from CD and UC patient recently transcribes label (Lawrance etc., (2001) Hum.Mol.Genet.10:445-56; In addition referring to Warner and Dieckgraefe (2002) Inflamm.Bowel.Dis.8:140-57).These researchs identify the gene that relates to the inflammatory reaction that generally changes in IBD.In addition, these studies show that, it is fully different that the gastrointestinal tissue that is obtained by CD and UC patient transcribes group, and the genome that identifies seems to distinguish the UC tissue and CD organizes.
[0005] opposite with the gastrointestinal tissue of excision or biological biopsy, peripheral blood is the cell tissue source that obtains very easily, can be used for distinguishing UC and CD.Circulation peripheral blood lymphocytes (PBMC) is responsible for monitoring the infection and the disease sign of body comprehensively.Therefore, PBMC can be used as evaluation as the alternative tissue of the disease inducible gene expression of the patient's condition or seriousness mark (about the generality summary referring to Rockett etc., (2004) Toxicol.Appl.Pharmacol.194:189-99).Maas and colleagues identify autoimmune disease (as rheumatoid arthritis, systemic lupus erythematous, type i diabetes and multiple sclerosis) patient's common PBMC spectrum (Maas etc., (2002) J.Immunol.169:5-9).Twine and colleagues show, under non-autoimmune disease background, derive from renal cell carcinoma (renal cell carcinoma, RCC) patient's PBMC shows with healthy volunteer's the distinct disease-related of group of transcribing and transcribes group (Twine etc., (2003) Cancer Res.63:6069-75).Mannick and colleagues have studied 7 CD patients and 5 UC patients' PBMC express spectra recently with 2400 gene cDNA microarraies, several genes (Mannick etc., (2004) Clin.Immunol.112:247-57) that present differential expression between these diseases have been described; Before, reported that other gene is conditioned (Gijsbers etc., (2004) Eur.J.Immunol.34:1992-2000 in the peripheral blood lymphocytes of cd patient on the mRNA level; Hori etc., (2002) J.Gastroenterol.Hepatol.17:1070-77; Gonsky etc., (1998) J.Immunol.160:4914-22).
[0006] although proposed the IBD that the periphery inflammatory component relates to these two kinds of forms, but also the health volunteer is not composed with the open gene of the cycle P BMC of the patient with certified IBD diagnostic result of histology (perhaps be the CD form, perhaps be the UC form) so far and be successfully used to develop the gene sorter (gene classifier) that allows the differentiation disease.Up to now, the ability of PBMC associated retroviral group diagnosis IBD and/or differentiation CD and UC is still the unknown in this area.
[0007] whether different must being enough to can be distinguished them based on the gene expression profile among the independent PBMC by the gene expression profile among the PBMC that determines CD and UC patient, and can be used for distinguishing IBD patient and health volunteer and optional PBMC-and the IBD-associated retroviral gene expression profile that is used to distinguish CD patient and UC patient by providing, the invention solves this problem.Therefore, the relative noninvasive method that the peripheral blood of patients monocyte is transcribed spectrum analysis that relates to of the present invention has diagnosis, prognosis and/or the monitoring that helps inflammatory bowel and/or multi-form IBD (being CD and UC).
Summary of the invention
[0008] in one aspect, the discriminating and the classification that the present invention is based on numerous PBMC-and IBD-relevant biomarkers (are for example shown listed PBMC-of 1-4 and IBD-relevant biomarkers, they there are differences expression respectively under following four kinds of situations: 1) PBMC with the experimenter who does not have IBD substantially (for example health volunteer) compares, differential expression in the PBMC of patients with inflammatory bowel, 2) PBMC with the experimenter who does not have IBD substantially (for example health volunteer) compares, differential expression in the PBMC of cd patient, 3) PBMC with the experimenter who does not have IBD substantially (for example health volunteer) compares, differential expression in the PBMC of patients of ulcerative colitis, with 4) compare differential expression in the PBMC of cd patient with patients of ulcerative colitis).PBMC-and the IBD-relevant biomarkers that is listed among the table 1-4 provided by the invention also is classified as I group, II group, III group and IV group, whether basis of classification is respectively them can be used for diagnosis, prognosis or monitoring best: 1) suffer from patient's the development (I organizes biomarker, and this paper is also referred to as " common biomarker (commonbiomarkers) " group) of the IBD of Crohn's disease or ulcerative colitis form; 2) suffer from the patient of Crohn's disease development (II organizes biomarker, this paper be also referred to as " CD biomarker " group); Or 3) (III organizes biomarker to suffer from the patient's of ulcerative colitis development; This paper is also referred to as " UC biomarker " group); And/or whether they can be used to distinguish IBD patient best and suffer from Crohn's disease or ulcerative colitis (IV group; This paper is also referred to as " CDvUC biomarker " group).In addition, the PBMC-and the IBD-relevant biomarkers (this paper is also referred to as " classification biomarker (classifyingbiomarkers) " group) of listing in table 5 and being categorized as V group biomarker also can be used for distinguishing cd patient and patients of ulcerative colitis.These PBMC-and IBD-relevant biomarkers can be the integral part of IBD disease approach again, and therefore can be used as the new treatment target of treatment inflammatory bowel (being Crohn's disease or ulcerative colitis).
[0009] therefore, the present invention relates to as the polynucleotide of the PBMC-of inflammatory bowels such as Crohn's disease and/or ulcerative colitis and IBD-relevant biomarkers (it can be classified as I group, II group, III group, IV group and/or V group biomarker), by the polypeptide of described polynucleotide encoding and fragment, homologue and the isotype of described polynucleotide or polypeptide.The invention still further relates to anti-PBMC-of the present invention and IBD biomarker antibody, comprise the array of biomarker of the present invention and/or relate to the purposes of the mensuration (for example microarray assays, Q-PCR mensuration, nucleic acid reporter molecules mensuration etc.) of biomarker of the present invention.In addition, the express spectra that the present invention relates to use these PBMC-and IBD-relevant biomarkers illustrates the existence or the risk of inflammatory bowels such as Crohn's disease and/or ulcerative colitis.With regard to inflammatory bowels such as Crohn's disease and/or ulcerative colitis, these PBMC-and IBD-relevant biomarkers also can be used for expression level difference is associated with bad or favourable prognosis.PBMC-and IBD-relevant biomarkers also can be used for estimating the effect of IBD treatment or therapy.About IBD (for example Crohn's disease, ulcerative colitis etc.) treatment, PBMC-of the present invention and IBD-relevant biomarkers also can be used for screening the test compound that can improve IBD, and/or itself can be used as curative.
[0010] in one aspect, the invention provides PBMC-and IBD-relevant biomarkers, their expression level is represented their amount or activity, and is relevant with the existence of inflammatory bowel (for example Crohn's disease or ulcerative colitis).PBMC-of the present invention and IBD-relevant biomarkers can be polynucleotide (for example DNA, cDNA, mRNA), by the polypeptide of described polynucleotide encoding and fragment, homologue and the isotype of described polynucleotide or polypeptide.In certain embodiments, the inventive method is implemented by detecting the polynucleotide transcribed or the situation that exists of its part, and the polynucleotide of wherein having transcribed comprise PBMC-and IBD-relevant biomarkers.Perhaps, can detect by the proteic situation that exists that detects corresponding to PBMC-and IBD-relevant biomarkers gene or RNA material (promptly by its coding).These methods also can be carried out on protein level; That is to say, can estimate PBMC-and the proteic protein expression level of IBD-relevant biomarkers, be used for diagnosis, prognosis and/or monitoring purpose, or be used for the shaker test compound, or as curative.
[0011] in certain embodiments, the inventive method is used the many groups biomarker comprise PBMC-and IBD-relevant biomarkers more than a kind.In one embodiment, the invention provides the one group of biomarker that comprises multiple PBMC-and IBD-relevant biomarkers.In one embodiment, one group of biomarker of the present invention comprises at least 2 kinds of PBMC-and IBD-relevant biomarkers.In one embodiment, one group of biomarker of the present invention comprises at least 3 kinds of PBMC-and IBD-relevant biomarkers.In one embodiment, one group of biomarker of the present invention comprises at least 4 kinds of PBMC-and IBD-relevant biomarkers.In one embodiment, one group of biomarker of the present invention comprises at least 5 kinds of PBMC-and IBD-relevant biomarkers.In one embodiment, one group of biomarker of the present invention comprises at least 6 kinds of PBMC-and IBD-relevant biomarkers.In one embodiment, one group of biomarker of the present invention comprises at least 7 kinds of PBMC-and IBD-relevant biomarkers.In one embodiment, one group of biomarker of the present invention comprises at least 8 kinds of PBMC-and IBD-relevant biomarkers.In one embodiment, one group of biomarker of the present invention comprises at least 9 kinds of PBMC-and IBD-relevant biomarkers.In one embodiment, one group of biomarker of the present invention comprises at least 10 kinds of PBMC-and IBD-relevant biomarkers.In one embodiment, one group of biomarker of the present invention comprises at least 11 kinds of PBMC-and IBD-relevant biomarkers.In one embodiment, one group of biomarker of the present invention comprises at least 12 kinds of PBMC-and IBD-relevant biomarkers.In other embodiments, the biomarker group comprises common biomarker, CD biomarker, UC biomarker, CDvUC biomarker and/or classification biomarker.The biomarker group also is provided, and it comprises and is selected from following biomarker: I group biomarker, II group biomarker, III group biomarker, IV group biomarker and/or V group biomarker.The technician will recognize that one group of biomarker of the present invention can comprise the PBMC-of the present invention and the IBD-relevant biomarkers of any amount and arbitrary combination, V group biomarker especially of the present invention.Therefore, in other non-limiting embodiments of the present invention, one group of biomarker of the present invention comprises at least 2 kinds, at least 3 kinds, at least 4 kinds, at least 5 kinds, at least 6 kinds, at least 7 kinds, at least 8 kinds, at least 9 kinds, at least 10 kinds, at least 11 kinds or at least 12 kinds of classification biomarkers, for example the classification biomarker of V group.For example, one group of non-limiting biomarker of the present invention can comprise immunoglobulin heavy chain constant region γ 1 and immunoglobulin (Ig) κ constant region biomarker.Of the present invention another organized non-limiting biomarker can comprise people 28S ribosome-RNA(rRNA) 5 ' district, C type Protein-tyrosine-phosphatase receptor associated protein(RAP), H3 histone family member K, integrin β 3 (platelet glycoprotein IIIa, antigens c D61) and H2B histone family member Q biomarker.Of the present invention another organizes that non-limiting biomarker can comprise immunoglobulin heavy chain constant region γ 1, granzyme K (granzyme K), mutL homologue 3, NGAL (lipocalin2), CXCL5, serum is deprived and replied phosphatidylserine conjugated protein (serum deprivationresponse phosphatidylserine binding protein) and H3 histone family member K biomarker.In one embodiment, one group of biomarker of the present invention provides at least 70% tolerance range (more preferably at least 80% tolerance range in the following areas, at least 90% tolerance range most preferably): (a) determining whether the patient suffers from the IBD of (1) Crohn's disease or ulcerative colitis form, (2) Crohn's disease, and/or (3) ulcerative colitis aspect, and/or (b) suffer from Crohn's disease or suffer from aspect the ulcerative colitis distinguishing IBD patient.In another aspect of this invention, obtain for information about in concrete experimenter's sample of (process of for example diagnosis, prognosis, monitor therapy and/or disease etc.), measure the PBMC-of the present invention more than a kind and the expression level of IBD-relevant biomarkers in expectation.
[0012] in certain embodiments, the comparative descriptions of relative expression's level of at least a PBMC-and IBD-relevant biomarkers the seriousness of inflammatory bowel such as Crohn's disease and/or ulcerative colitis, this relatively allows to diagnose, prognosis and monitoring analysis.For example, the PBMC-of the various disease state of progress of IBD (and/or UC or CD) and the method that a kind of long-term prognosis relatively is provided of IBD-relevant biomarkers express spectra comprise any expectation time length of breaking out or showing effect in these diseases.In another example, can estimate concrete treatment plan, comprise whether concrete medicine works to improve concrete patient's long-term prognosis.
[0013] PBMC-and IBD-relevant biomarkers also can be used as treatment target or curative.Therefore, the non-mechanism that is limited to, certain methods of the present invention is based in part on following principle: when PBMC-of the present invention and IBD-biomarker when being similar to or being similar to substantially the horizontal expression from the isolating PBMC of experimenter's (being the health volunteer) that do not have IBD substantially, their expression is regulated can improve inflammatory bowel.The discovery of these differential expression patterns of respectively organizing biomarker of various PBMC-and IBD-relevant biomarkers or these biomarkers allows the screening target to be to regulate the test compound of the pattern of embodying; The compound that for example can change better or improve the express spectra of prognosis to the express spectra with prognosis mala into screens.
[0014] about these embodiments, some PBMC-and IBD-relevant biomarkers can comprise regulates biomarker active or that express when determining to have the response treatment plan.Perhaps, the adjusting active or that express of PBMC-and IBD-relevant biomarkers can be associated with the diagnosis or the prognosis of inflammatory bowels such as Crohn's disease and/or ulcerative colitis.In addition, conditioning agent of the present invention, the conditioning agent of for example at least a PBMC-and IBD-relevant biomarkers (for example PBMC-and IBD-related polynucleotides and/or polypeptide, relevant PBMC-and IBD-related polynucleotides and/or polypeptide (for example inhibitory polynucleotide, inhibitory polypeptide (for example antibiont traget antibody)), small molecules etc.) can be used as medicine and uses.In another embodiment of the invention, conditioning agent of the present invention can with one or more other therapeutic composition couplings of the present invention.As described below these compounds are formulated as pharmaceutical composition.Use the unconventionality expression that this therapeutic regulation agent is adjustable to less a kind of PBMC-and IBD-relevant biomarkers, and therefore can be used for improving or suppressing inflammatory bowels such as Crohn's disease and/or ulcerative colitis.In another embodiment of the invention, one or more conditioning agents of the present invention or other therapeutic composition can with one or more other known curative or composition coupling.
The accompanying drawing summary
[0015] Fig. 1: differentiate functional annotation and classification relevant, that UC is relevant and the transcript of variant expression between UC and CD into CD.A) shown CD to normal ANCOVA relatively in (grey band), UC to normal ANCOVA relatively in (black stripe) and UC to the CD ANCOVA typical approach (x axle) of overexpression in (white ribbon) relatively.In these figure, the negative logarithm (y axle) of mapping p value is so that highlight more significant dependency.The approach (x axle) of inquiry is as follows: the processing of (1) amyloid, (2) apoptotic signal transduction, (3) arginine and proline(Pro) metabolism, (4) B-cell receptor signal transduction (NIg), (5) heart beta-adrenergic signal transduction, (6) chemokine signal transduction, (7) death receptor signal transduction, (8) ERK/MAPK signal transduction, (9) fatty acid metabolism, (10) Cycle Regulation (G1/S), (11) Cycle Regulation (G2/M), (12) g protein coupled receptor signal transduction, (13) glutamic acid metabolism, (14) Histidine metabolism, (15) IGF-1 signal transduction, (16) IL-2 signal transduction, (17) IL-4 signal transduction, (18) inositol monophosphate metabolism, (19) insulin receptor signal transduction, (20) integrin signal transduction, (21) Interferon, rabbit signal transduction, (22) JAK/STAT signal transduction, (23) NF-κ B signal transduction, (24) nitrogen metabolism, (25) p38MAPK signal transduction, (26) PI3K/AKT signal transduction, (27) PPAR signal transduction, (28) Prostaglandins and Leukotrienes metabolism, (29) purine metabolism, (30) pyrimidine metabolic, (31) starch and Sucrose Metabolism, (32) TXi Baoshouti signal transduction, (33) tryptophan metabolism, (34) tyrosine metabolism and (35) VEGF signal transduction.B) use Ingenuity path analysis system (Ingenuity, Mountain View, CA) the CD specific transcriptional thing among the functional annotation PBMC; Transcript is showing with pie graph at the relative distribution in each selected functional category of CD genes involved.C) the UC specific transcriptional thing among manual function's note PBMC has provided the relative distribution of immunoglobulin (Ig) to transcript in the NIg classification.
[0016] Fig. 2: use the PBMC spectrum to carry out the supervision classification prediction of CD and UC.A) shown the relative overall accuracy (■ of many groups biomarker (x axle) of forming by 2-20 gene sorter; The y axle), the tolerance range of CD classification (
The y axle) and UC classification tolerance range (▲; The y axle).B) shown that sample test concentrates weighted voting classification distribution result.The degree of confidence of supporting CD supports the classification assigned confidence degree of UC to represent with negative value with on the occasion of expression.The overall accuracy that classification is distributed is 100% in test set, under situation only based on the expression pattern in the PBMC that microarray analysis obtains, there are 14 quilts correctly to be categorized as Crohn's disease in 14 cd patients, have 6 quilts correctly to be categorized as UC among 6 UC patients.Indicated actual source (the CD patient=preceding 14 white ribbons of PBMC express spectra; UC patient=back 6 black stripe).
[0017] PCR in real time of sorter transcript level check in Fig. 3: CD and the UC sample sets.A) shown the average rising multiple (y axle) of gene sorter transcript (x axle), in CD, transferred to detect, by Affymetrix microarray hybridization (Affymetrix; Bai Zhu) or quantitative real-time RT-PCR (TAQMAN_; Black post) detects.B) shown the average rising multiple (y axle) of gene sorter transcript (x axle), in UC, transferred to detect, by Affymetrix microarray hybridization (Affymetrix; Bai Zhu) or quantitative real-time RT-PCR (TAQMAN_; Black post) detects.
[0018] Fig. 4: that uses the Q-PCR analysis transcribes spectrum to Crohn's disease or the discriminating of patients of ulcerative colitis classification and the comparison of logic analysis.(A) 20 training sets (training set) or (B) logic analysis of 20 dependence test collection (test set) or the tolerance range (y axle) of discriminatory analysis have been shown.
Detailed Description Of The Invention
[0019] although the expression pattern analysis of gastrointestinal tissue's biopsy samples has identified be can be used for distinguishing the existence that these two kinds of inflammatory bowel diseases are the stomach and intestine associated retroviral group of Crohn disease (CD) or ulcerative colitis (UC), needed gastrointestinal tissue biopsy samples becomes these diagnostic methods and has no attraction. Cell in the peripheral blood, the PMBC (PBMC) that specifically circulates obtains easily more than gastrointestinal tissue's biopsy samples. Because PBMC is responsible for monitoring infection and the disease sign of body comprehensively, and IBD obviously relates to inflammatory process, so PBMC can be used as the substitute of gastrointestinal tissue, is used for estimating and can be used for measuring the state of IBD or tissue and the disease association of seriousness transcribed group.
[0020] the present invention relates to use at least a " open gene label (transcriptional gene signature) " (this paper is also referred to as " gene label (gene signature) ", " express label (expression signature) ", " transcribing group (transcriptome) ", " composing (proille) " or " gene profile (gene profile) ") of PBMC, it is the relevant open gene label of PBMC, be the PBMC express spectra, determine whether the patient suffers from inflammatory bowel disease. The invention still further relates to and use the PBMC correlation to transcribe group selection to determine that IBD patient suffers from Crohn disease or suffers from ulcerative colitis. The present invention is based on PBMC relevant relevant with IBD (for example Crohn disease relevant and/or ulcerative colitis is correlated with) transcribe the discovery of group. Specifically, the present invention is based on the discriminating of PBMC-and IBD-relevant biomarkers, these biomarkers can be the purposes aspect diagnosis, prognosis, monitoring and/or the treatment of Crohn disease and/or ulcerative colitis and be classified as five groups (I group, II group, III group, IV group and V groups) at IBD based on it.
[0021] term used herein " biomarker (biomarker) ", " gene classifier (gene classifier) " or " PBMC-and IBD-relevant biomarkers (PBMC-and IBD-associated biomarker) " etc. comprise and polynucleotides (being gene, transcript, EST etc.) or the peptide molecule of substantially comparing the significantly adjusting of amount quilt (namely raising or downward modulation) in the experimenter's who suffers from inflammatory bowel disease (being Crohn disease and/or ulcerative colitis) PMBC without the experimenter (such as the health volunteer) of IBD. In certain embodiments, PBMC-of the present invention and IBD-relevant biomarkers comprise polynucleotides, the corresponding gene outcome of described polynucleotides and fragment, homologue and the isotype of described polynucleotides or gene outcome of I group biomarker (being also referred to as " common biomarker "), II group biomarker (being also referred to as " Crohn disease relevant biomarkers ", " CD relevant biomarkers ", " CD specific biological mark " or " CD biomarker "), III group biomarker (being also referred to as " ulcerative colitis relevant biomarkers ", " UC relevant biomarkers ", " UC specific biological mark " or " UC biomarker "), IV group biomarker (being also referred to as " CDvUC biomarker ") and V group biomarker (being also referred to as " classification biomarker ").
[0022] PBMC-of the present invention and IBD-relevant biomarkers can be and substantially compare in CD patient's PBMC without the experimenter's of IBD PBMC and/or compare in UC patient's PBMC polynucleotides, the corresponding gene outcome of described polynucleotides and fragment, homologue and the isotype of described polynucleotides or gene outcome of significantly being regulated (namely raising or downward modulation) with the basic PBMC without the experimenter of IBD. In one embodiment, PBMC-and IBD-relevant biomarkers comprise and are categorized as PBMC-and the IBD-relevant biomarkers that I organizes common biomarker.
[0023] PBMC-of the present invention and IBD-relevant biomarkers also comprise the Crohn disease relevant biomarkers. Term used herein " Crohn disease relevant biomarkers " or " CD biomarker " comprise with the amount of substantially comparing in the PMBC of cd patient without the situation among the experimenter's of IBD the PBMC is significantly regulated polynucleotides, the corresponding gene outcome of described polynucleotides and fragment, homologue and the isotype of described polynucleotides or gene outcome of (namely raising or downward modulation). In addition, and substantially compare without the situation among the experimenter's of IBD the PBMC, the CD biomarker is not significantly regulated in the PMBC of patients of ulcerative colitis. In certain embodiments, Crohn disease relevant biomarkers of the present invention comprises PBMC-and the IBD-relevant biomarkers that is classified as II group biomarker, can find that wherein some subgroup is sorted in the tabulation of IV group and V group biomarker.
[0024] PBMC-of the present invention and IBD-relevant biomarkers also comprise the ulcerative colitis relevant biomarkers. Term used herein " ulcerative colitis relevant biomarkers " or " UC biomarker " comprise with the amount of substantially comparing in ulcerative colitis patient's PMBC without the experimenter's of IBD PBMC is significantly regulated polynucleotides, the corresponding gene outcome of described polynucleotides and fragment, homologue and the isotype of described polynucleotides or gene outcome of (namely raising or downward modulation). In addition, compare with the basic PBMC without the experimenter of IBD, the amount of UC biomarker in the PMBC of cd patient significantly do not regulated. In certain embodiments, ulcerative colitis relevant biomarkers of the present invention comprises PBMC-and the IBD-relevant biomarkers that is classified as III group biomarker, can find that wherein some subgroup is sorted in the tabulation of IV group and V group biomarker.
[0025] PBMC-of the present invention and IBD-relevant biomarkers comprise the CDvUC biomarker. Term used herein " CDvUC biomarker " comprises with the amount of substantially comparing in the PMBC of ulcerative colitis or cd patient without the experimenter's of IBD PBMC is significantly regulated polynucleotides, the corresponding gene outcome of described polynucleotides and fragment, homologue and the isotype of described polynucleotides or gene outcome of (namely raising or downward modulation), and they can distinguish the PMBC and the PMBC that separates from patients of ulcerative colitis that separates from cd patient. For example, compare with its expression in the experimenter who suffers from a kind of inflammatory bowel disease (for example Crohn disease), the CDvUC biomarker can significantly be regulated in the experimenter who suffers from another kind of inflammatory bowel disease (for example ulcerative colitis). Perhaps, compare with the experimenter who suffers from a kind of inflammatory bowel disease (for example Crohn disease), the CDvUC biomarker can be regulated in the experimenter who suffers from another kind of inflammatory bowel disease (for example ulcerative colitis) in the opposite direction. In certain embodiments, the property distinguished CDvUC biomarker comprises IV group biomarker, can find that wherein some subgroup is sorted in the tabulation of V group biomarker.
[0026] PBMC-of the present invention and IBD-relevant biomarkers can be categorized as less CDvUC biomarker group, belong to classification biomarker group." classification biomarker group " used herein comprises one group of fragment, homologue and isotype that can be used for distinguishing corresponding gene product and the described polynucleotide or the gene product of the polynucleotide of cd patient and patients of ulcerative colitis, described polynucleotide.In certain embodiments, a group categories biomarker is that a group categories is the biomarker of V group biomarker.
[0027] preferably, for purpose of the present invention, the PBMC-of the present invention of significantly adjusting of quilt (promptly raising or downward modulation) and the expression level of IBD-relevant biomarkers are increased respectively or reduce and reach intensity of anomaly, wherein expression level is unusual, for example exceeds the identical PBMC-among the health volunteer PBMC and the standard deviation of IBD-relevant biomarkers.More preferably, with respect to the health volunteer, PBMC-that is significantly regulated and IBD-relevant biomarkers are upward or downward and reach 1.5,2,3 or 4 times of unusual at least changes or higher.
[0028] PBMC-that includes in as I group, II group, III group, IV group and V group biomarker and UniGene accession number, title and their the adjusting direction (promptly raising or downward modulation) of IBD-relevant biomarkers are listed in following table 1, table 2, table 3, table 4 and table 5 respectively.
| The PBMC-and the IBD-relevant biomarkers of table 1.I group; Common biomarker | ||||||
| Accession number | Title | Direction in the two | CD is with respect to normal multiple difference | CD is with respect to normal ANCOVA p value | UC is with respect to normal multiple difference | UC is with respect to normal ANCOVA p value |
| Hs.75716 | Serine (or halfcystine) proteinase inhibitor, clade B (ovalbumin), the |
↑ | 6.93 | 6.84E-12 | 3.35 | 3.87E-05 |
| Hs.154654 | Cytochrome P450, subfamily I (two _ English is derivable), |
↑ | 3.31 | 6.49E-10 | 2.37 | 2.81E-05 |
| Hs.79516 | Brain signal protein I abundant, that symphysis connects | ↑ | 1.94 | 6.81E-09 | 2.13 | 2.61E-09 |
| Hs.177781 | Unknown | ↑ | 1.88 | 6.34E-08 | 1.92 | 3.99E-07 |
| Hs.104624 | Aquaporin 9 | ↑ | 1.88 | 9.92E-06 | 2.02 | 9.06E-06 |
| Hs.20084 | Retinoid X acceptor, α | ↑ | 1.80 | 1.33E-06 | 1.68 | 9.18E-05 |
| Hs.2161 | |
↑ | 1.74 | 2.65E-05 | 1.81 | 5.05E-05 |
| (C5a part) | ||||||
| Hs.865 | RAP1A, RAS oncogene family member | ↑ | 1.64 | 5.95E-10 | 1.53 | 1.01E-06 |
| Hs.177486 | Amyloid beta (A4) precursor protein (proteolytic enzyme nexin-II, alzheimer's disease (Alzheimer disease) | ↑ | 1.63 | 6.11E-10 | 1.60 | 4.81E-08 |
| Hs.198282 | Phosphatide synthetase 1 (scramblase 1) | ↑ | 1.60 | 3.90E-05 | 1.76 | 1.09E-05 |
| Hs.288555 | ELK3, ETS-domain protein (SRF accessory protein 2) | ↑ | 1.59 | 4.25E-10 | 1.51 | 2.75E-07 |
| Hs.101695 | NCK connects |
↑ | 1.57 | 2.72E-14 | 1.51 | 1.08E-10 |
| Hs.198282 | The |
↑ | 1.56 | 1.02E-05 | 1.55 | 7.12E-05 |
| Hs.285313 | The nuclear promoter element is conjugated protein | ↓ | 2.78 | 8.63E-05 | 5.65 | 9.23E-09 |
| Hs.151411 | KIAA0916 albumen | ↓ | 2.47 | 7.92E-05 | 3.21 | 6.01E-06 |
| Hs.20072 | Myosin modulability light chain interact protein | ↓ | 2.45 | 3.21E-07 | 2.47 | 2.73E-06 |
| Hs.81248 | CUG triplex tumor-necrosis factor glycoproteins, |
↓ | 2.44 | 8.74E-08 | 2.52 | 4.84E-07 |
| Hs.211610 | CUG triplex tumor-necrosis factor glycoproteins, |
↓ | 2.27 | 5.40E-06 | 2.62 | 1.77E-06 |
| Hs.86896 | The albumen 3 (bromodomain containing 3) that contains the Bu Luomo structural domain | ↓ | 2.24 | 1.22E-06 | 2.26 | 8.83E-06 |
| Hs.100555 | DEAD/H (Asp-Glu-Ala-Asp/His) frame polypeptide 18 (Myc-regulates) | ↓ | 2.24 | 3.89E-05 | 2.48 | 3.11E-05 |
| Hs.143601 | Putative protein hCLA-iso | ↓ | 2.20 | 1.20E-07 | 2.16 | 2.42E-06 |
| Hs.149436 | Kinesin family member 5B | ↓ | 2.15 | 2.66E-05 | 2.56 | 4.15E-06 |
| Hs-239483 | Unknown | ↓ | 2.10 | 2.47E-09 | 2.42 | 2.47E-10 |
| Hs.78909 | Zinc finger protein 36, |
↓ | 2.03 | 1.46E-07 | 2.06 | 1.18E-06 |
| Hs.85273 | Retinoblastoma binding protein white 6 | ↓ | 2.01 | 6.23E-05 | 2.56 | 1.64E-06 |
| Hs.219614 | F-frame and leucine enrichment |
↓ | 1.95 | 9.04E-07 | 2.41 | 1.11E-08 |
| Hs.153834 | Pumilio homologue 1 (fruit bat) | ↓ | 1.92 | 6.49E-08 | 1.92 | 8.29E-07 |
| Hs.18827 | Cylindroma (scarf (turban) neoplastic syndrome) | ↓ | 1.91 | 7.70E-06 | 2.13 | 2.93E-06 |
| Hs.127287 | KIAA0794 albumen | ↓ | 1.83 | 3.31E-05 | 1.95 | 4.07E-05 |
| Hs.73090 | The nf (p49/p100) of κ light chain polypeptide genetic enhancer in the |
↓ | 1.81 | 3.63E-06 | 1.77 | 4.93E-05 |
| Hs.373557 | The part transcript that comprises the THC211630 gene | ↓ | 1.81 | 3.43E-05 | 2.18 | 1.32E-06 |
| Hs.75243 | The |
↓ | 1.76 | 1.11E-06 | 1.86 | 1.65E-06 |
| Hs.118174 | 34 peptide |
↓ | 1.76 | 4.63E-05 | 2.35 | 6.54E-08 |
| Hs.37096 | |
↓ | 1.74 | 8.38E-07 | 1.91 | 2.96E-07 |
| Hs.3530 | FUS interact protein (Serine-arginine enrichment) 1 | ↓ | 1.72 | 2.20E-05 | 1.89 | 7.47E-06 |
| Hs.83484 | Meisl | ↓ | 1.70 | 2.26E-06 | 1.78 | 3.70E-06 |
| Hs.294014 | Unknown | ↓ | 1.68 | 5.43E-06 | 1.81 | 2.84E-06 |
| Hs.183418/ 214291/355 896 | Cell |
↓ | 1.64 | 1.42E-05 | 1.88 | 9.80E-07 |
| Hs.77256 | The enhanser of zeste homologue 2 (fruit bat) | ↓ | 1.63 | 7.72E-06 | 1.71 | 8.73E-06 |
| Hs.278426 | The PDGFA associated |
↓ | 1.60 | 6.59E-07 | 1.57 | 1.61E-05 |
| Hs.10351 | KIAA0308 albumen | ↓ | 1.60 | 8.99E-06 | 1.78 | 1.10E-06 |
| Hs.18368 | The SR rich protein | ↓ | 1.57 | 1.30E-05 | 1.83 | 2.43E-07 |
| Hs.2173 | Fucosyltransferase 4 (α (1,3) fucosyltransferase, marrow specificity) | ↓ | 1.54 | 4.08E-05 | 1.70 | 6.69E-06 |
| Hs.152601 | UDP-glucose, UGCG | ↓ | 1.54 | 3.35E-05 | 1.73 | 2.72E-06 |
| Hs.243901 | Unknown | ↓ | 1.51 | 8.84E-05 | 1.68 | 1.29E-05 |
1The abnormal expression of these PBMC-and IBD-biomarker is for example compared with healthy person in CD patient and UC patient's PBMC and is all significantly raised (↑) or downward modulation (↓).
| The PBMC-and the IBD-relevant biomarkers of table 2:II group; The CD biomarker | |||||
| Accession number | Title | Direction in CD | CD is with respect to normal multiple difference | CD is with respect to normal p value | Has significance (p≤0.0001) |
| Hs.72933 | |
↑ | 2.48 | 1.91E-05 | |
| Hs.83381 | Guanine-nucleotide-binding protein (G albumen), |
↑ | 2.30 | 4.02E-09 | |
| Hs.119257 | Emsl sequence ((the p80/85src substrate) that mammal tumor is relevant with squamous cell cancer | ↑ | 2.28 | 1.53E-08 | |
| Hs.87149 | Integrin, β 3 (platelet glycoprotein IIIa, antigens c D61) | ↑ | 2.22 | 6.10E-06 | |
| Hs.90061 | |
↑ | 2.18 | 1.61E-07 | |
| Hs.155097 | Carbonic anhydrase II | ↑ | 2.17 | 2.37E-06 | |
| Hs.81564 | Platelet factor 4 (chemokine (C-X-C motif) part 4) | ↑ | 2.14 | 4.03E-09 | |
| Hs.90786 | ATP-is in conjunction with expression cassette, subfamily C (CFTR/MRP), and the |
↑ | 2.12 | 4.00E-05 | |
| Hs.279843 | MutL homologue 3 (intestinal bacteria) | ↑ | 2.12 | 1.26E-08 | |
| Hs.88474 | Prostaglandin(PG)-endoperoxide synthase 1 (PGG/H synthase and cyclo-oxygenase) | ↑ | 2.06 | 4.95E-06 | |
| Hs.75106 | Bunch albumen (clusterin) (complement cracking inhibitor, SP-40,40, sulfated |
↑ | 2.00 | 7.02E-06 | |
| Hs.249216 | The H2B histone family, member J | ↑ | 1.98 | 9.64E-08 | |
| Hs.41267 | Karyomit(e) 21 open reading-frame (ORF)s 7 | ↑ | 1.97 | 1.39E-06 | |
| Hs.326035 | Early growth |
↑ | 1.95 | 6.86E-05 | |
| Hs.271473 | Hide collagen in cancer, being raised, embrane-associated |
↑ | 1.87 | 2.30E-06 | |
| Hs.84171 | The myeloproliferative leukemia virus oncogene | ↑ | 1.84 | 1.14E-06 | |
| Hs.77890 | Guanylate |
↑ | I.82 | 1.17E-06 | |
| Hs.1395 | Early growth reactive protein 2 (Krox-20 homologue, fruit bat) | ↑ | 1.79 | 5.01E-05 | |
| Hs.204238 | NGAL 2 (oncogene 24p3) | ↑ | 1.79 | 5.19E-05 | |
| Hs.88474 | Prostaglandin(PG)-endoperoxide synthase 1 (PGG/H synthase and cyclo-oxygenase) | ↑ | 1.76 | 3.21E-07 | |
| Hs.26530 | Serum is deprived response protein (phosphatidylserine is conjugated protein) | ↑ | 1.69 | 8.41E-07 | |
| Hs.193700 | Consensus sequence comprises gb:AL110164.1 | ↑ | 1.67 | 1.12E-06 | |
| Hs.2164 | Thromboblast basic protein (chemokine (C-X-C motif) part 7) | ↑ | 1.66 | 1.18E-06 | |
| Hs.8302 | 4 half LIM |
↑ | 1.66 | 3.70E-05 | |
| Hs.64016 | Protein S (α) | ↑ | 1.62 | 7.63E-06 | |
| Hs.87149 | Integrin, β 3 (platelet glycoprotein IIIa, antigens c D61) | ↑ | 1.61 | 3.55E-05 | |
| Hs.114360 | Transforming growth factor-beta stimulatory protein(SP) TSC-22 | ↑ | 1.59 | 1.10E-06 | |
| Hs 149846 | Integrin, |
↑ | 1.58 | 1.60E-05 | |
| Hs.6721 | Monoglyceride lipase | ↑ | 1.56 | 1.57E-05 |
| Hs.77899 | Tropomyosin 1 (α) | ↑ | 1.56 | 4.16E-05 | |
| Hs.114231 | C-type agglutinin receptor-2 | ↑ | 1.56 | 3.31E-06 | |
| Hs.7917 | The possible lineal homologue of mouse hypoxia inducible gene 1 | ↑ | 1.55 | 1.79E-06 | |
| Hs 261023 | Putative protein FLJ20958 | ↑ | 1.53 | 1.25E-06 | |
| Hs.22116 | CDC14 cell division cycle protein 14 homologue B (yeast saccharomyces cerevisiae) | ↑ | 1.51 | 4.87E-05 | |
| Hs.433622 | Follistatin sample albumen 1 | ↑ | 1.51 | 7.19E-05 | |
| Hs.183125 | Killer cell agglutinin receptor subfamily F, the member 1 | ↓ | 2.57 | 4.03E-07 | |
| Hs.334837 | Williams Beuren syndrome chromosomal region 20C, Williams-Beuren syndrome critical area protein 20 copy B | ↓ | 2.21 | 7.94E-06 | |
| Hs.8272 | Prostaglandin d 2 synthase 21kDa (brain) | ↓ | 2.17 | 6.12E-08 | |
| Hs.64746 | Chlorion born of the same parents interior passageway 3 | ↓ | 2.07 | 2.17E-05 | |
| Hs.8272 | Prostaglandin d 2 synthase 21kDa (brain) | ↓ | 2.06 | 4.36E-07 | |
| Hs.334837 | Williams Beuren syndrome chromosomal region 20A, B and C | ↓ | 1.94 | 4.80E-05 | |
| Hs.406306 | Williams Beuren syndrome chromosomal region 20A, B and C | ↓ | 1.88 | 4.85E-05 | |
| Hs.3066 | Granzyme K (serine protease, granzyme 3; Casease II) | ↓ | 1.87 | 6.01E-05 | |
| Hs.381613 | The albumen composition 1 that connector is relevant, γ 2 subunits | ↓ | 1.85 | 1.06E-05 | |
| Hs.355888 | Phospholipase C, β 2 | ↓ | 1.83 | 7.22E-06 | |
| Hs.88411 | Natural cytotoxicity triggers acceptor 3 | Little | 1.82 | 1.22E-05 | |
| Hs.406306 | Williams Beuren syndrome chromosomal region 20A, B and C | ↓ | 1.80 | 3.32E-05 | |
| Hs.194669 | The enhanser of zeste homologue 1 (really | ↓ | 1.80 | 5.58E-05 |
| Fly) | |||||
| Hs.99491 | The RAS guanyl-discharges albumen 2 (calcium and DAG-regulate) | ↓ | 1.77 | 5.81E-07 | |
| Hs.349256 | Paired immunoglobulin-like receptor β | ↓ | 1.73 | 9.12E-05 | |
| Hs.13377 | The albumen 3 that contains the abhydrolase structural domain | ↓ | 1.70 | 5.09E-05 | Neutrophilic granulocyte |
| Hs.274 | The casein kinase that megalokaryocyte is relevant | ↓ | 1.68 | 3.10E-06 | Monocyte/lymphocyte |
| Hs.323817 | DKFZP547E1010 albumen | ↓ | 1.66 | 2.68E-06 | |
| Hs.8272 | Prostaglandin d 2 synthase 21kDa (brain) | ↓ | 1.64 | 7.56E-06 | |
| Hs.288126 | Spondin 2, extracellular matrix protein | ↓ | 1.64 | 6.34E-06 | |
| Hs.8182 | Putative protein MGC17528, the gene 1 of cynapse nuclear expression | ↓ | 1.63 | 1.44E-07 | |
| Hs.31834 | Consensus sequence comprises gb:AI052536 | ↓ | 1.63 | 2.88E-05 | |
| Hs.234569 | ζ-chain (TCR) related protein kinase 70 kDa | ↓ | I.61 | 3.24E-05 | Monocyte/lymphocyte |
| Hs.167988 | Nerve cell adhesion molecule 1 | ↓ | 1.61 | 2.19E-05 | |
| Hs.75196 | HLA-B associated retroviral thing 8 | ↓ | 1.61 | 6.42E-05 | |
| Hs.180948 | The KIAA0570 gene product | ↓ | 1.60 | 4.03E-05 | |
| Hs.356684 | Putative protein DKFZp762C186 | ↓ | 1.58 | 3.27E-06 | Eosinophilic granulocyte/neutrophilic granulocyte |
| Hs.29288 | In-β-N-acetylglucosaminidase | ↓ | 1.55 | 1.69E-05 | |
| Hs.313844 | Consensus sequence comprises gb:AW069290 | ↓ | 1.55 | 8.69E-05 | |
| Hs.100293 | O-connection N-acetyl-glucosamine (GlcNAc) transferring enzyme (UDP-N-acetylglucosamine: polypeptide-N-acetyl-glucosamine transferring enzyme) | ↓ | 1.53 | 1.41E-05 | |
| Unknown | Consensus sequence comprises gb:NM_024957.1 | ↓ | 1.52 | 6.06E-07 | |
| Hs.170160 | RAB2, member RAS oncogene man | ↓ | 1.52 | 2.47E-06 | Neutrophil granule |
| Family's sample | Cell |
2Compare with healthy person, these PBMC-and IBD-biomarker be the abnormal expression of (promptly not in UC patient's PBMC) in CD patient's PBMC only, is for example significantly raised (↑) or downward modulation (↓).
| The PBMC-and the IBD-relevant biomarkers of table 3:III group; The UC biomarker | |||||
| Accession number | Title | Direction in UC | UC is with respect to normal multiple difference | UC is with respect to normal p value | Has significance (p≤0.0001) |
| Hs.300697 | Immunoglobulin heavy chain constant region γ 3 (G3m mark) | ↑ | 4.65 | 2.70E-12 | |
| N/A | Consensus sequence comprises gb:X51887 (immunoglobulin (Ig) κ orphon) | ↑ | 2.75 | 7.83E-05 | |
| Hs.406565 | Immunoglobulin (Ig) κ constant region | ↑ | 2.63 | 2.05E-06 | |
| Hs.76325 | Immunoglobulin (Ig) J polypeptide, the albumen that is connected of immunoglobulin (Ig) α and μ polypeptide | ↑ | 2.48 | 4.70E-06 | |
| s-406565 | Immunoglobulin (Ig) κ constant region | ↑ | 2.46 | 4.66E-08 | |
| Hs.102950 | Coatmer albumen mixture, subunit γ, immunoglobulin (Ig) λ J3 | ↑ | 2.32 | 3.12E-06 | |
| Hs.406565 | Immunoglobulin (Ig) κ constant region | ↑ | 2.21 | 5.90E-10 | |
| Hs.102950 | Coatmer albumen mixture, subunit γ, immunoglobulin (Ig) λ J3 | ↑ | 2.19 | 3.33E-07 | |
| N/a | Consensus sequence comprises gb:AJ408433 (the part IGKV gene of immunoglobulin kappa chain variable region) | ↑ | 2.16 | 1.92E-05 | |
| Hs.406565 | Immunoglobulin (Ig) κ constant region | ↑ | 2.16 | 7.59E-07 | |
| Hs.102950 | Coatmer albumen mixture, subunit γ, immunoglobulin (Ig) λ J3 | ↑ | 2.10 | 4.74E-06 | |
| Hs.406565 | Immunoglobulin (Ig) κ constant region | ↑ | 2.05 | 1.99E-06 | |
| Hs.381417 | Consensus sequence comprises | ↑ | 1.97 | 3.63E-06 | |
| Gb:AF103529.1 (immunoglobulin kappa light chain variable region) | |||||
| Hs.348935 | Immunoglobulin (Ig) |
↑ | 1.89 | 1.65E-06 | |
| Hs.405944 | Immunoglobulin (Ig) λ locus | ↑ | 1.83 | 2.53E-05 | |
| Hs.102950 | Coatmer albumen mixture, subunit γ, immunoglobulin (Ig) |
↑ | 1.82 | 1.55E-05 | |
| Hs.405944 | Immunoglobulin (Ig) λ locus | ↑ | 1.75 | 7.51E-06 | |
| Hs.406565 | Immunoglobulin (Ig) κ constant region | ↑ | 1.71 | 1.12E-07 | |
| Hs.381418 | Consensus sequence comprises gb:AF103530.1 (immunoglobulin kappa light chain variable region) | ↑ | 1.70 | 3.11E-07 | |
| Hs.406565 | Immunoglobulin (Ig) κ constant region | ↑ | 1.59 | 4.25E-06 | |
| Hs.102950 | Coatmer albumen mixture, subunit γ, immunoglobulin (Ig) λ J3 | ↑ | 1.50 | 2.91E-05 | |
| Hs.107213 | Formin |
↓ | 1.75 | 4.27E-05 |
3Compare with healthy person, these PBMC-and IBD-biomarker be the abnormal expression of (promptly not in CD patient's PBMC) in UC patient's PBMC only, is for example significantly raised (↑) or downward modulation (↓).
| The PBMC-and the IBD-relevant biomarkers of table 4.IV group; The CDvUC biomarker | ||||
| Accession number | Title | Specificity | Multiple difference | ANCOVA p value |
| Hs.90061 | |
Crohn's disease | 2.08 | 2.55E-07 |
| Hs.279843 | MutL homologue 3 (intestinal bacteria) | Crohn's disease | 2.00 | 2.75E-08 |
| Hs.88474 | Prostaglandin(PG)-endoperoxide synthase 1 (PGG/H synthase and cyclo-oxygenase) | Crohn's disease | 1.93 | 1.18E-05 |
| Hs.73769 | Folacin receptor 1 (adult) | Crohn's disease | 1.93 | 6.82E-11 |
| Hs.89714 | Chemokine (C-X-C motif) |
Crohn's disease | 1.85 | 3.71E-05 |
| Hs.83381 | Guanine-nucleotide-binding protein (G albumen), γ 11 | Crohn's disease | 1.79 | 8.04E-06 |
| Hs.2359 | Dual |
Crohn's disease | 1.76 | 5.30E-05 |
| Hs.204238 | NGAL 2 (oncogene 24p3) | Crohn's disease | 1.75 | 4.35E-05 |
| Hs.81564 | Platelet factor 4 (chemokine (C-X-C | Crohn's disease | 1.74 | 4.38E-06 |
| Motif) part 4) | ||||
| Hs.119257 | Emsl sequence ((the p80/85src substrate) that mammal tumor is relevant with squamous cell carcinoma | Crohn's disease | 1.70 | 8.21E-05 |
| Hs.26530 | Serum is deprived response protein (phosphatidylserine is conjugated protein) | Crohn's disease | 1.66 | 5.34E-07 |
| Hs.303023 | Tubulin, β 1 | Crohn's disease | 1.65 | 8.39E-05 |
| Hs.23581 | The leptin receptor gene associated protein | Crohn's disease | 1.61 | 7.52E-05 |
| Hs.249216 | The H2B histone family, member J | Crohn's disease | 1.61 | 6.91E-05 |
| Hs.77439 | Protein kinase, cAMP-is dependent, modulability, II type, β | Crohn's disease | 1.59 | 4.09E-05 |
| Hs.2178 | The H2B histone family, member Q | Crohn's disease | 1.57 | 8.85E-06 |
| Hs.114231 | C-type agglutinin receptor-2 | Crohn's disease | 1.57 | 8.79E-07 |
| Hs.12813 | DKFZP434J214 albumen | Crohn's disease | 1.52 | 1.21E-05 |
| Hs.2164 | Thromboblast basic protein (chemokine (C-X-C motif) part 7) | Crohn's disease | 1.51 | 2.90E-05 |
| Hs.300697 | Immunoglobulin heavy chain constant region γ 3 (G3m mark) | UC | 3.87 | 9.280E-13 |
| Hs.153261 | Immunoglobulin heavy chain constant region μ | UC | 2.60 | 2.72E-05 |
| n/a | Has the unknown EST of consensus sequence with immunoglobulin (Ig) κ orphon | UC | 2.42 | 5.66E-05 |
| Hs.406565 | Immunoglobulin (Ig) κ constant region | UC | 2.30 | 1.78E-06 |
| n/a | 28S ribosome-RNA(rRNA) 5 ' district | UC | 2.11 | 2.95E-07 |
| Hs.406565 | Immunoglobulin (Ig) κ constant region | UC | 2.08 | 2.88E-08 |
| Hs.406565 | Immunoglobulin (Ig) κ constant region | UC | 2.04 | 3.52E-07 |
| Hs.183125 | Killer cell agglutinin receptor subfamily F, the member 1 | UC | 2.02 | 5.45E-05 |
| Hs.411106 | Pore-forming protein 1 | UC | 1.98 | 7.07E-05 |
| Hs.153261 | Immunoglobulin heavy chain constant region μ | UC | 1.93 | 378E-05 |
| Hs.406565 | Immunoglobulin (Ig) κ constant region | UC | 1.88 | 2.19E-06 |
| Hs.406565 | Immunoglobulin (Ig) κ constant region | UC | 1.87 | 8.32E-09 |
| Hs.102950 | Coatmer albumen mixture, subunit γ, immunoglobulin (Ig) λ J3 | UC | 1.74 | 5.47E-05 |
| Hs.102950 | Coatmer albumen mixture, subunit γ, immunoglobulin (Ig) λ J3 | UC | 1.74 | 2.10E-05 |
| Hs.406565 | Immunoglobulin (Ig) κ constant region | UC | 1.72 | 2.57E-07 |
| Hs.355888 | Phospholipase C, β 2 | UC | 1.72 | 2.24E-05 |
| Hs.8272 | Prostaglandin d 2 synthase 21kDa (brain) | UC | 1.71 | 5.41E-05 |
| Hs.25338 | Proteolytic enzyme, Serine, 23 | UC | 1.68 | 7.72E-05 |
| Hs.381417 | Has the unknown EST of consensus sequence with the immunoglobulin kappa light chain variable region | UC | 1.67 | 3.86E-05 |
| Hs.75596 | The interleukin-22 acceptor, β | UC | 1.65 | 7.19E-05 |
| Hs.406565 | Immunoglobulin (Ig) κ constant region | UC | 1.64 | 2.48E-06 |
| Hs.102950 | Coatmer albumen mixture, subunit γ, immunoglobulin (Ig) λ J3 | UC | 1.64 | 4.13E-05 |
| Hs.406565 | Immunoglobulin (Ig) κ constant region | UC | 1.63 | 4.95E-06 |
| Hs.406565 | Immunoglobulin (Ig) κ constant region | UC | 1.61 | 3.75E-08 |
| Hs.380156 | The NK-acceptor, killer cell immunoglobulin-like receptor, two structural domains, long kytoplasm tail, 3 | UC | 1.60 | 3.16E-08 |
| Hs.405944 | Immunoglobulin (Ig) λ locus | UC | 1.59 | 1.55E-05 |
| Hs.3489356 | Immunoglobulin (Ig) λ sample polypeptide 1 | UC | 1.58 | 4.69E-05 |
| Hs.84 | The interleukin-22 acceptor, γ (severe combined immunodeficiency disease) | UC | 1.53 | 7.84E-05 |
| Hs.193128 | Beautiful nematode smu-1 homologue | UC | 1.52 | 5.16E-05 |
| Hs.238944 | Putative protein FLJ10631 | UC | 1.52 | 3.72E-05 |
The PBMC-and the IBD-relevant biomarkers of table 5:V group; The classification biomarker
| The sorter gene | Classification | Title | The single- |
| 1 | Crohn's disease | NGAL 2 (oncogene 24p3) | Hs.204238 |
| 2 | Crohn's disease | MutL homologue 3 (intestinal bacteria) | Hs.279843 |
| 3 | Crohn's disease | Serum is deprived response protein (phosphatidylserine is conjugated protein) | Hs.26530 |
| 4 | Crohn's disease | The H2B histone family, member Q | Hs.2178 |
| 5 | Crohn's disease | The H3 histone family, member K | Hs.70937 |
| 6 | Crohn's disease | Chemokine (C-X-C motif) |
Hs.89174 |
| 7 | Crohn's disease | Integrin β 3 (platelet glycoprotein IIIa, antigens c D61) | Hs.87149 |
| 8 | UC | (the G3m mark, IgHg3), this paper is also referred to as immunoglobulin heavy chain constant region γ 1 to immunoglobulin heavy chain |
Hs.300697 |
| 9 | UC | Immunoglobulin (Ig) κ constant region | Hs.406565 |
| 10 | UC | M27830 people 28S ribosomal RNA gene 5 ' district | n/a |
| 11 | UC | C type Protein-tyrosine-phosphatase receptor associated protein(RAP) | Hs.155975 |
| 12 | UC | Granzyme K (serine protease, |
Hs.3066 |
| 13 | UC | Immunoglobulin (Ig) κ constant region | Hs.406565 |
| 14 | UC | Immunoglobulin (Ig) κ constant region | Hs.406565 |
The source of PBMC-and IBD-relevant biomarkers
[0029] polynucleotide of PBMC-of the present invention and IBD-relevant biomarkers and polypeptide are separable from any experimenter's tissue or the cell of expressing PBMC-and IBD-relevant biomarkers.In a preferred non-limiting embodiments, described tissue comes autoblood (perhaps for example serum, blood plasma, hemocyte), lymphoglandula, saliva, stomach or intestines.The tissue sample itself that contains one or more PBMC-and IBD-relevant biomarkers can be used for the inventive method, and those skilled in the art can know can be by its conventional method that obtains, stores and/or preserve this class sample.But, it will be apparent for a person skilled in the art that, blood, PBMC specifically, the preferred source that should in the method for the diagnosis that provides, prognosis and/or monitoring IBD (being CD or UC) progress, express with judge PBMC-of the present invention and IBD-relevant biomarkers.
Isolating PBMC-and IBD-relevant biomarkers polynucleotide
[0030] the invention provides isolating polynucleotide and polypeptide as PBMC-and IBD-relevant biomarkers.Preferred nucleotide sequence of the present invention comprises the nucleotide sequence of genome, cDNA, mRNA, siRNA and chemosynthesis.
[0031] exemplary PBMC-of the present invention and IBD-relevant biomarkers are listed in table 1-5.The present invention comprises and lists in the table PBMC-of 1-5 and the polynucleotide sequence of IBD-relevant biomarkers.Polynucleotide of the present invention also are included under the stringent condition polynucleotide of polypeptide that keep the essential biologically active (being active fragments) of the PBMC-that lists in table 1-5 and IBD-relevant biomarkers with the polynucleotide sequence of PBMC-that lists in table 1-5 and IBD-relevant biomarkers or its complementary sequence hybridization and/or coding.Polynucleotide of the present invention also comprise the sequential portion of the polynucleotide sequence of listing in the table PBMC-of 1-5 and IBD-relevant biomarkers, and these sequential portions comprise at least 21 continuous nucleotides.
[0032] the present invention also comprises and lists in the table PBMC-of 1-5 and the polypeptide of IBD-relevant biomarkers.Polypeptide of the present invention also comprises the sequential portion of the polypeptide of listing in the table PBMC-of 1-5 and IBD-relevant biomarkers, and these sequential portions comprise at least 7 continuous amino acids.The preferred embodiments of the invention comprise any sequential portion of any polypeptide that is selected from the PBMC-of those biomarkers that lists in table 1-5 and IBD-relevant biomarkers, and this sequential portion keeps the essential biologically active of selected polypeptide.
[0033] the present invention only also comprise since well-known genetic code degeneracy and with the PBMC-that lists in table 1-5 and the different polynucleotide molecule of polynucleotide sequence of IBD-relevant biomarkers, so its encoded protein is with to list in the PBMC-that shows 1-5 identical with IBD-relevant biomarkers encoded protein.
[0034] polynucleotide that the present invention includes can be used as hybridization probe and primer, to identify and to separate the nucleic acid that has with the same or analogous sequence of encoding sequence of disclosed polynucleotide.Be used to identify that the hybridizing method with isolating nucleic acid comprises polymerase chain reaction (PCR), DNA hybridization, in situ hybridization and RNA hybridization, these methods all are that those skilled in the art are well-known.
[0035] can under the condition of different severity, carry out hybridization.The severity of hybridization comprises any two difficulty that nucleic acid molecule is hybridized each other.The present invention also comprises can be under the stringency that reduces, more preferably under the stringent condition and most preferably under high stringent condition with the polynucleotide of multi-nucleotide hybrid described herein.The example of stringency is shown in following table 6: high stringent condition is the same with for example condition A-F at least those strict conditions; Stringent condition is the same with for example condition G-L at least strict condition; The stringent condition that reduces is the same with for example condition M-R at least strict condition.
Table 6. stringent condition
| Stringent condition | Multi-nucleotide hybrid | Hybridization length (bp) 1 | Hybridization temperature and damping fluid 2 | Wash temperature and damping fluid 2 |
| A | DNA:DNA | ＞50 | 65 ℃; 1 * SSC or 42 ℃; 1 * SSC, 50% methane amide | 65℃；0.3×SSC |
| B | DNA:DNA | ＜50 | T B *；1×SSC | T B *；1×SSC |
| C | DNA:RNA | ＞50 | 67 ℃; 1 * SSC or 45 ℃; 1 * SSC, 50% methane amide | 67℃；0.3×SSC |
| D | DNA:RNA | ＜50 | T D *；1×SSC | T D *；1×SSC |
| E | RNA:RNA | ＞50 | 70 ℃; 1 * SSC or 50 ℃; 1 * SSC, 50% methane amide | 70℃；0.3×SSC |
| F | RNA:RNA | ＜50 | T F *；1×SSC | T F *；1×SSC |
| G | DNA:DNA | ＞50 | 65 ℃; 4 * SSC or 42 ℃; 4 * SSC, 50% methane amide | 65℃；1×SSC |
| H | DNA:DNA | ＜50 | T H *；4×SSC | T H *；4×SSC |
| I | DNA:RNA | ＞50 | 67 ℃; 4 * SSC or 45 ℃; 4 * SSC, 50% methane amide | 67℃；1×SSC |
| J | DNA:RNA | ＜50 | T J *；4×SSC | T J *；4×SSC |
| K | RNA:RNA | ＞50 | 70 ℃; 4 * SSC or 50 ℃; 4 * SSC, 50% methane amide | 67℃；1×SSC |
| L | RNA:RNA | ＜50 | T L *；2×SSC | T L *；2×SSC |
| M | DNA:DNA | ＞50 | 50 ℃; 4 * SSC or 40 ℃; 6 * SSC, 50% methane amide | 50℃；2×SSC |
| N | DNA:DNA | ＜50 | T N *；6×SSC | T N *；6×SSC |
| O | DNA:RNA | ＞50 | 55 ℃; 4 * SSC or | 55℃；2×SSC |
| 42 ℃; 6 * SSC, 50% methane amide | ||||
| P | DNA:RNA | ＜50 | T P *；6×SSC | T P *；6×SSC |
| Q | RNA:RNA | ＞50 | 60 ℃; 4 * SSC or 45 ℃; 6 * SSC, 50% methane amide | 60℃；2×SSC |
| R | RNA:RNA | ＜50 | T R *；4×SSC | T R *；4×SSC |
1: hybridization length is the length of hybridization region of the hybridization polynucleotide of expection.When the target polynucleotide of polynucleotide and unknown nucleotide sequence was hybridized, hybridization length was assumed that the length of hybridization polynucleotide.When the multi-nucleotide hybrid of known array, can and identify that the complementary district of one or more optimal sequences determines to hybridize length by the comparison polynucleotide sequence.
(1 * SSPE is 0.15MNaCl, 10mMNaH to 2:SSPE
2PO
4And 1.25mMEDTA, pH7.4) alternative SSC (1 * SSC is 0.15MNaCl and 15mM Trisodium Citrate) in hybridization and lavation buffer solution can carry out 15 minutes rinsing after hybridization is finished.
T
B *-T
R *: estimate that length should be than the melting temperature(Tm) (T of heterozygote less than the hybridization temperature of the heterozygote of 50 base pairs
m) low 5-10 ℃, T wherein
mObtain according to following formula.For the heterozygote of length less than 18 base pairs, T
m(℃)=2 (A+T base number)+4 (G+C base number).For the heterozygote of length between 18-49 base pair, T
m(℃)=81.5+16.6 (log
10Na
+)+0.41 (%G+C)-(600/N), wherein N is the base number in the heterozygote, Na
+Be in the hybridization buffer Na ion concentration (among 1 * SSC, Na
+=0.165M).
Other example of the stringent condition of multi-nucleotide hybrid is referring to Sambrook, J., E.F.Fritsch, and T.Maniatis, 1989, Molecular Cloning:A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY, the 9th and 11 chapters, and Current Protocols inMolecular Biology, 1995, editors such as F.M.Ausubel, John Wiley ﹠amp; Sons, Inc., the 2.10th and 6.3-6.4 joint, these documents are hereby incorporated by.
[0036] polynucleotide of the present invention also can be used as hybridization probe and primer, identifying and to separate the homology polynucleotide, the sequence encoding that promptly has polypeptide of the present invention and/or with the nucleic acid of the polypeptide of disclosed homologous peptide.These homologues are by species isolating polynucleotide and the polypeptide different with the species of disclosed polynucleotide and polypeptide, or polynucleotide in same species and polypeptide, but have significant sequence similarity with disclosed polynucleotide and polypeptide.Preferably, polynucleotide homologue and disclosed polynucleotide have at least 60% sequence identity and (more preferably have at least 75% identity; Most preferably have at least 90% identity), and having at least 30% sequence identity, homologous peptide thing and disclosed polypeptide (more preferably have at least 45% identity; Most preferably has at least 60% identity).Preferably, the homologue of disclosed polynucleotide and polypeptide separates from mammalian species, most preferably separates from the people.
[0037] polynucleotide of the present invention can be used as hybridization probe and primer, having the DNA of sequence of allele variant that coding is listed in the polynucleotide sequence of the PBMC-that shows 1-5 and IBD-relevant biomarkers to identify with separating.Allele variant is the natural variable form of listing in the polynucleotide sequence of the table PBMC-of 1-5 and IBD-relevant biomarkers, and its encoded polypeptides is identical with the polypeptide of listing in the genes encoding of showing 1-5 or have a remarkable similarity.Preferably, allele variant and disclosed polynucleotide have at least 90% sequence identity and (more preferably have at least 95% identity; Most preferably has at least 99% identity).
[0038] therefore, except the polynucleotide sequence of listing in table 1-5, the present invention also comprises and lists in the table PBMC-of 1-5 and the homologue and the allele variant of IBD-relevant biomarkers.
[0039] polynucleotide of the present invention also can be used as hybridization probe and primer, the condition of expressing with the cell of the polypeptide of identify expressing PBMC-of the present invention and IBD-relevant biomarkers and tissue and their.
[0040] in addition, polynucleotide of the present invention can be used for changing in (promptly regulating (for example strengthen, reduce or modify)) cell or the biology expression of gene corresponding to PBMC-of the present invention and IBD-relevant biomarkers.These corresponding genes are genomic dna sequences of the present invention, and they produce mRNA through transcribing, and obtain PBMC-of the present invention and IBD-relevant biomarkers polypeptide by these mRNA.
[0041] can be by using various inhibitory polynucleotides, for example antisense polynucleotides, combination and/or cutting are by the ribozyme of the mRNA of genetic transcription of the present invention, the oligonucleotide that forms triplex of target gene regulatory region and the short interfering rna that causes the degraded of said target mrna sequence-specific, in cell or biology, realize expression (Galderisi etc. for example, (1999) J.Cell.Physiol.181:251-57 of the change of PBMC-that the present invention comprises and IBD-relevant biomarkers; Sioud (2001) Curr.Mol.Med.1:575-88; Knauert and Glazer (2001) Hum.Mol.Genet.10:2243-51; Bass (200) Nature 411:428-29).
[0042] inhibition antisense polynucleotides of the present invention or ribozyme polynucleotide can with the complete coding strand complementation of gene of the present invention, or only complementary with its part.Perhaps, inhibitory polynucleotide can with the non-coding region complementation of genes encoding chain of the present invention.Can use method well-known in the art, use chemosynthesis and/or enzyme ligation to make up inhibitory polynucleotide of the present invention.Can modify the nucleosides key of the polynucleotide of chemosynthesis, resist the ability of nuclease-mediated degraded and increase its sequence-specific to strengthen it.Such key modify include but not limited to phosphorothioate bond, methyl-phosphorous acid ester bond, phosphoramidic acid ester bond, borine phosphoric acid ester bond, morpholino key and peptide nucleic acid(PNA) (PNA) key (Galderisi etc., ibid; Heasman (2002) Dev.Biol.243:209-14; Mickelfield (2001) Curr.Med.Chem.8:1157-79).Perhaps, can use polynucleotide of the present invention to produce antisense molecule with the expression vector that antisense (promptly reverse) direction subclone is gone into wherein with biological method.
[0043] in another embodiment, antisense polynucleotides molecule of the present invention is a α-different polynucleotide molecule.α-different polynucleotide molecule and complementary RNA form the specific double-strand heterozygote, in described complementary RNA, compare with common β unit, and chain is arranged in parallel with each other.According to technology well-known in the art, the antisense polynucleotides molecule can also comprise 2 '-o-methyl ribonucleotides or chimeric RNA-DNA analogue.
[0044] oligonucleotide (TFO) of the formation triplex of the inhibition that comprises of the present invention is with high specific and the affinity major groove (Knauert and Glazer, ibid) in conjunction with double-stranded DNA.Can prevent the triple-helix structure of genetic transcription with formation by regulatory region (being promotor and/or enhancer sequence) the complementary TFO of target and gene, thereby suppress expression of gene of the present invention.
[0045] in one embodiment of the invention, inhibitory polynucleotide of the present invention is short RNA interfering (siRNA) molecule.These siRNA molecules are short (preferred 19-25 Nucleotide; More preferably 19 or 21 Nucleotide) double stranded rna molecule causes the sequence-specific degraded of said target mrna.This degraded is called as RNA and disturbs (RNAi) (for example Bass (2001) Nature 411:428-29).The initial RNAi that identifies in unicellular lower eukaryote successfully is applied to mammalian cell, shows also that recently it prevents fulminant hepatitis (Song etc., (2003) Nature Med.9:347-51) in the mouse with the siRNA molecular therapy of target Fas mRNA.In addition, report recently that the siRNA that transmits in the sheath blocks pain reaction (Dorn etc., (2004) Nucleic Acids Res.32 (5): e49) in two rat models (pain model of agonist induction and neuropathic pain model).
[0046] can be by (Fire etc. together that two complementary single stranded RNA molecules (one of them mates a part of said target mrna) are annealed, United States Patent (USP) the 6th, 506, No. 559), or by using at the single hairpin RNA molecule (Yu etc. that folding up the double-stranded part that needs with generation certainly on one's body, (2002) Proc.Natl.Acad.Sci.USA 99:6047-52), produce siRNA molecule of the present invention.But siRNA molecule chemosynthesis (Elbashir etc., (2001) Nature 411:494-98), or use the single stranded DNA template by in-vitro transcription production (Yu etc., ibid).Perhaps, can use the expression vector that contains justice and antisense siRNA sequence, and instantaneous ground (Yu etc., ibid; Sui etc., (2002) Proc.Natl.Acad.Sci.USA 99:5515-20) or stably (Paddison etc., (2002) Proc.Natl Acad.Sci.USA 99:1443-48) produces the siRNA molecule with biological method.Recently, the adenovirus carrier of use expressing the hairpin RNA that is further processed into siRNA confirmed in former generation human body cell the said target mrna level with effectively and the sequence-specific mode reduce (Arts etc., (2003) Genome Res.13:2325-32).
[0047] can be based on the siRNA molecule of standard design target well-known in the art polynucleotide of the present invention (for example Elbashir etc., (2001) EMBO J.20:6877-88).For example, the target sections of said target mrna should begin with AA (preferably), TA, GA or CA; The GC ratio of siRNA molecule should be 45-55%; The siRNA molecule should not comprise into 3 identical Nucleotide of a row; The siRNA molecule should not comprise into 7 miscellaneous G/C of a row; The target sections should be in the ORF district of said target mrna, and should be after initial ATG 75bp at least before 75bp and the terminator codon at least.Use aforesaid standards or other known standard (for example Reynolds etc., (2004) Nat.Biotechnol.22:326-30), those of ordinary skills can design the siRNA molecule of target polynucleotide of the present invention.
[0048] also can import the expression that non-human transgenic animal in its genome realizes the change of gene in cell or biology of PBMC-of the present invention and IBD-relevant biomarkers by creating polynucleotide of the present invention.Such transgenic animal comprise the animal of the gene of the present invention (being transgenosis) with a plurality of copies.Tissue specificity is regulated sequence can effectively be connected to transgenosis, so that polypeptide expression of the present invention is led specific cells or specific growth period.In another embodiment, can produce transgenic nonhuman animal, it comprises the selecting system that allows transgene expression to be regulated.The cre/loxP recombinase system that an example of this system known in the art is phage P1.The method that produces transgenic animal (the especially animal of mouse and so on) through embryo operation and microinjection has become conventional, well-known in this area (for example Bockamp etc., (2002) Physiol.Genomics 11:115-32).In a preferred embodiment of the invention, the non-human transgenic animal comprises at least a PBMC-and IBD-relevant biomarkers.
[0049] also can ruined animal (being knock-out animal) be realized the expression of the change of gene of the present invention in cell or biology by insertion exogenous polynucleotide sequence corresponding to the native gene of polynucleotide of the present invention by creating it.The coding region of native gene can be destroyed, produces no function albumen thus.Perhaps, the upstream regulation district of native gene can be destroyed, or substitute with the different adjustment element, produces to express the albumen that still has function that changes.The method that produces knock-out animal comprises homologous recombination, well-known in this area (for example Wolfer etc., (2002) Trends Neurosci.25:336-40).
Isolating PBMC-and IBD-relevant biomarkers polypeptide
[0050] several aspect of the present invention relates to isolating PBMC-and IBD-relevant biomarkers albumen, its biologically-active moiety and polypeptide fragment, and they are suitable for use as the immunogen that produces anti-PBMC-and IBD-relevant biomarkers antibody.In one embodiment, the standard protein purification technology be can use, natural PBMC-and IBD-relevant biomarkers albumen isolated by the cell or tissue source by suitable purifying flow process.In another embodiment, PBMC-and IBD-relevant biomarkers albumen are by recombinant DNA technology production.As to recombinant expressed substituting, can use standard peptide synthetic technology chemosynthesis PBMC-and IBD-relevant biomarkers albumen or polypeptide.
[0051] can effectively be connected to expression control sequenc (for example pMT2 and pED expression vector) by listing in the table PBMC-of 1-5 and the polynucleotide sequence of IBD-relevant biomarkers, recombinant production is listed in PBMC-and the IBD-relevant biomarkers albumen among the table 1-5.The universal expression method of recombinant protein is well-known in this area.
[0052] numerous clones can be used as the host cell that is suitable for recombinant expressed PBMC-of the present invention and IBD-relevant biomarkers polypeptide.Mammalian host cell line comprise for example COS cell, Chinese hamster ovary celI, 293T cell, A431 cell, 3T3 cell, CV-1 cell, HeLa cell, L cell, BHK21 cell, HL-60 cell, U937 cell, HaK cell, Jurkat cell, normal diploid cell and derive from prior structure and former generation tissue block the cell strain of vitro culture thing.
[0053] or, might be in such as zymic lower eukaryotes or prokaryotic organism recombinant production polypeptide of the present invention.Potential suitable yeast strain comprises yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), schizosaccharomyces pombe (Schizosaccharomycespombe), kluyveromyces yeast strains (Kluyveromyces strain) and candida bacterial strain (Candida strain).Potential suitable bacterial isolates comprises intestinal bacteria (Escherichia coli), subtilis (Bacillus subtilis) and Salmonella typhimurium (Salmonellatyphimurium).If polypeptide of the present invention prepares with yeast or bacterium, then may modify it, so that obtain functional by for example phosphorylation or the suitable site of glycosylation.Covalently bound well-known chemical method or the enzyme process of using like this finished.
[0054] in another embodiment of the invention, also can be by PBMC-of the present invention and IBD-relevant biomarkers polynucleotide effectively being connected to the suitable control sequence in one or more insect expression vectors (for example baculovirus vector), and the use insect cell expression system, recombinant production PBMC-of the present invention and IBD-relevant biomarkers polypeptide.The materials and methods that is used for baculovirus/Sf9 expression system can kit form buy (MAXBAC_ test kit for example, Invitrogen, Carlsbad, CA).
[0055] in suitable host cells recombinant expressed after, then can use well-known purification process, example gel filters and ion exchange chromatography, is purified into polypeptide of the present invention from substratum or cell extract.Purifying also can comprise the affinity chromatography that adopts known material in conjunction with polypeptide of the present invention.These purification process also can be used for from natural origin purifying polypeptide of the present invention.
[0056] or, PBMC-of the present invention and IBD-relevant biomarkers polypeptide can also help identifying, the mode of purifying and/or detection is recombinant expressed.For example, polypeptide can be used as with the proteic syzygy of maltose binding protein (MBP), glutathione-S-transferase (GST) or Trx (TRX) and so on and expresses.Be used to express with the test kit of this fusion rotein of purifying can be respectively by New England BioLabs (Beverly, MA), Pharmacia (Piscataway, NJ) and Invitrogen (Carlsbad CA) is purchased.The also available little epi-position mark of polypeptide of the present invention uses subsequently at the specific antibody of this epi-position and identifies or purifying.Preferred epi-position is the FLAG epi-position, can (New Haven CT) is purchased by Eastman Kodak.
[0057] the signal sequence secretion that can be used for promoting secreted polypeptide or other target protein with separate.Signal sequence is a characteristic feature with hydrophobic amino acid nuclear, is generally come by cut on the maturation protein with one or more cutting incidents in secretion process.This signal peptide comprises the processing site, and these processing sites allow signal sequence to be scaled off on by maturation protein during through Secretory Pathway at their.Therefore, the present invention relates to described polypeptide, and signal sequence is by wherein by the polypeptide of proteolytic cleavage (being cleaved products) with signal sequence.In one embodiment, the polynucleotide sequence of coded signal sequence can effectively be connected to target protein in expression vector, is not the albumen of secretor type usually for example or otherwise is difficult to isolating albumen.The secretion of signal sequence pilot protein, for example the eucaryon host that is transformed into wherein by expression vector is secreted, and signal sequence is cut subsequently or simultaneously.Then can be easily by means commonly known in the art by the outer substratum purifying protein of born of the same parents.Perhaps, can use the sequence that helps purifying, for example have the sequence of GST structural domain, signal sequence is connected to target protein.
[0058] except the PBMC-and IBD-relevant biomarkers polypeptide and allele variant and homologue that list in table 1-5, the present invention also comprises such polypeptide: it structurally is different from the polypeptide (for example have a little change sequence) of listing in table 1-5, but has and the essentially identical biochemical characteristic of disclosed polypeptide (for example only on the function non-essential amino acid residue change).This quasi-molecule includes but not limited to contain change, displacement, replacement, insert or the variant of having a mind to engineered mistake of disappearance.These change, replace, replace, insert or the technology and the test kit of disappearance are that those skilled in the art are well-known.
[0059] the present invention also comprises PBMC-of the present invention and the proteic variant of IBD-relevant biomarkers, and it plays PBMC-and proteic agonist of IBD-relevant biomarkers or antagonist.In certain embodiments, PBMC-and the proteic agonist of IBD-relevant biomarkers can keep the PBMC-and the proteic essentially identical biological activity of IBD-relevant biomarkers of natural form, or one part biological activity, perhaps can strengthen PBMC-and the proteic activity of IBD-relevant biomarkers.In certain embodiments, PBMC-and the proteic antagonist of IBD-relevant biomarkers can be by proteic active PBMC-and proteic one or more activity of IBD-relevant biomarkers that suppress natural form of for example competitive PBMC-of adjusting and IBD-relevant biomarkers.Therefore, can handle the particular biological effect that excites by variant with limited function.In one embodiment, less to experimenter's side effect with having the bioactive variant treatment of the proteic part of natural form experimenter with respect to PBMC-and IBD-relevant biomarkers protein for treatment with natural form.In a further preferred embodiment, raise still downward modulation according to IBD treatment needs specific objective PBMC-and IBD-relevant biomarkers albumen, some conditioning agent can be used as PBMC-of the present invention and proteic agonist of IBD-relevant biomarkers or antagonist.
[0060] PBMC-and the proteic variant of IBD-relevant biomarkers can produce by mutagenesis, for example proteic point of discontinuity sudden change of PBMC-and IBD-relevant biomarkers or brachymemma.Perhaps, can be by screening mutant combinatorial library, for example screen the agonist or the antagonistic activity of PBMC-and the proteic truncated mutant of IBD-relevant biomarkers, identify PBMC-and IBD-relevant biomarkers protein variant as PBMC-and IBD-relevant biomarkers protein agonist or PBMC-and IBD-relevant biomarkers protein antagonist.In one embodiment, the diversified library (variegated library) of PBMC-and IBD-relevant biomarkers protein variant is passed through to produce in the mutagenesis of polynucleotide horizontal combination, and by diversified gene library coding.In certain embodiments, for example can use these albumen as human cytokines of the present invention.Can be by for example the mixture enzymatic of synthetic oligonucleotide being connected in the gene order, make the degeneracy group of potential PBMC-and IBD-relevant biomarkers protein sequence can be expressed as single polypeptide, perhaps be expressed as one group wherein contain PBMC-and IBD-relevant biomarkers protein sequence group than larger fusion protein (for example being used for phage display), produce the diverse libraries of PBMC-and IBD-relevant biomarkers protein variant.There is several different methods to can be used for producing the library of potential PBMC-and IBD-relevant biomarkers protein variant by degenerate oligonucleotide sequence.The chemosynthesis of degeneracy gene order can be carried out in the automatization dna synthesizer, then synthetic gene is connected in the suitable expression vector.The genomic application of degeneracy allows to provide the potential PBMC-of coding expectation and all sequences of IBD-relevant biomarkers protein sequence group in a mixture.The synthetic method of degenerate oligonucleotide is known in the art.
[0061] polypeptide of the present invention also can be produced by known conventional chemical synthesis method.The method that is used for chemosynthesis polypeptide of the present invention is that those skilled in the art are well-known.Such chemically synthesized polypeptide can have the biological nature identical with natural purified polypeptide, therefore can be used as the biological activity surrogate or the immune surrogate of native peptides.
The antibody of anti-PBMC-and IBD-relevant biomarkers
[0062] on the other hand, the present invention relates to the antibody to protein-specific, described albumen is corresponding to PBMC-of the present invention and IBD-relevant biomarkers, or by PBMC-of the present invention and IBD-relevant biomarkers coding.Preferably, described antibody is monoclonal antibody, and most preferably, described antibody is humanized antibody, sees below.
[0063] antibody molecule of anti-PBMC-of the present invention and IBD-relevant biomarkers can (antibiont traget antibody) can pass through the well-known method production of those skilled in the art.For example, monoclonal antibody can be produced by producing hybridoma according to currently known methods.Use standard method then, for example enzyme-linked immunosorbent assay (ELISA), the hybridoma that screening forms in this way is to identify one or more hybridomas of producing with polypeptid specificity bonded antibody of the present invention.Full-length polypeptide of the present invention can be used as immunogen, perhaps can use the antigenic peptide fragment of polypeptide.The antigenic peptide of polypeptide of the present invention comprises at least 7 continuous amino acid residues, and comprises epi-position, makes the antibody and the polypeptide that produce at peptide form the specific immunity mixture.Preferably, antigenic peptide comprises at least 10 amino-acid residues, more preferably comprises at least 15 amino-acid residues, more preferably comprises at least 20 amino-acid residues again, most preferably comprises at least 30 amino-acid residues.
[0064] substituting as the hybridoma for preparing secrete monoclonal antibody, can be by making up immunoglobulin (Ig) library (for example antibody phage display libraries) with PBMC-of the present invention and IBD-relevant biomarkers polypeptide screening reorganization, identify and the monoclonal antibody of separating, isolate immunoglobulin library member thus in conjunction with PBMC-and IBD-relevant biomarkers at PBMC-of the present invention and IBD-relevant biomarkers.The technology and the commercial kit that are used to produce and screen phage display library are that those skilled in the art are well-known, and the method and the reagent that are specially adapted to produce and screen the antibody display libraries also are like this.
[0065] can produce polyclonal serum and antibody by with the suitable experimenter of polypeptide immune of the present invention.Can utilize standard technique, for example use the ELSIA of immobilization biological labelled protein, monitor the antibody titer among the time dependent immune experimenter.If needs are arranged, can isolate antibody molecule by experimenter or substratum at polypeptide of the present invention, and by being further purified such as well-known technology such as A protein chromatographics, to obtain the IgG fraction.
[0066] in addition, can use the reorganization antibiont traget antibody of standard recombinant dna technology preparation, for example contain the chimeric Humanized single chain antibody of people and inhuman part, belong to the scope of the invention.Humanized antibody also can use transgenic mice production, described transgenic mice be beyond expression endogenous heavy chain immunoglobulin and light chain gene, but but expressing human heavy chain and light chain gene.Perhaps, can use the technology that is called the guiding selection to produce the humanized antibody of the selected epi-position of identification.In the method, Xuan Ding non-human monoclonal antibodies (for example murine antibody) is used to lead and selects the humanized antibody of the same epi-position of identification.
[0067] can produce chimeric antibody by recombinant DNA technology known in the art, comprise the gomphosis immunoglobulin chain.For example, with the gene of the Fc constant region of Restriction Enzyme digestion coding mouse (or other species) monoclonal antibody molecule, removing mouse Fc coding region, and be equal to the part replacement (referring to PCT/US86/02269 with the gene of coding people Fc constant region; EP 184,187; EP 171,496; EP 173,494; WO 86/01533; United States Patent (USP) the 4th, 816, No. 567; EP 125,023; Better etc., (1988) Science 240:1041-43; Liu etc., (1987) Proc.Natl.Acad.Sci.U.S.A.84:3439-43; Liu etc., (1987) J.Immunol.139:3521-26; Sun etc., (1987) Proc.Natl.Acad.Sci.U.S.A.84:214-18; Nishimura etc., (1987) Canc.Res.47:999-1005; Wood etc., (1985) Nature314:446-49; With Shaw etc., (1988) J.Natl.Cancer Inst.80:1553-59).
[0068], can carry out humanization by methods known in the art antagonist or immunoglobulin chain if needs are arranged.Can produce humanized antibody by the sequence that sequence replaces not participating in directly antigen bonded Fv variable region that is equal to of personnel selection Fv variable region, comprise the Humanized immunoglobulin chain.Be used to produce the universal method of humanized antibody referring to Morrison (1985) Science229:1202-07; Oi etc., (1986) BioTechniques 4:214-21; With United States Patent (USP) the 5th, 585,089,5,693,761 and 5,693, No. 762, the equal integral body of all these documents is hereby incorporated by.These methods comprise the encode nucleotide sequence of all or part of IgF v variable region of at least a separation, operation and the expression from heavy chain or light chain.The source of this nucleic acid is that those skilled in the art are well-known, for example can be obtained by the hybridoma of the antibody that produces anti-pre-determined target.Coding humanized antibody or its segmental recombinant DNA can be cloned in the suitable expression vector then.
[0069] humanization or CDR grafted antibody molecule or immunoglobulin (Ig) can be transplanted or the CDR displacement produces by CDR, wherein 1 of immunoglobulin chain, 2 or all CDR all can be replaced.Referring to No. the 5th, 225,539, United States Patent (USP) for example; Jones etc., (1986) Nature321:522-25; Verhoeyan etc., (1988) Science 239:1534-36; With Beidler etc., (1988) J.Immunol.141:4053-60, all these documents all integral body are hereby incorporated by.United States Patent (USP) the 5th, 225, the CDR implantation method of having described to can be used for preparing humanized antibody of the present invention for No. 539 (in addition referring to GB 2188638A).The inhuman CDR displacement of all available at least a portion of all CDR of specific people's antibody, perhaps only number of C DR can replace with inhuman CDR.It is essential to have only humanized antibody is combined desired number with predetermined antigens CDR displacement to be only.
[0070] by other parts that for example lack, add or replace antibody such as monoclonal antibody, chimeric antibody and the humanized antibody that constant region is modified, also belongs to scope of the present invention.For example, antibody can followingly be modified: (i) disappearance constant region; (ii) constant region is replaced by another constant region, for example is intended to increase the constant region of transformation period, stability or the affinity of antibody, or from another species or other constant region of antibody class; And/or (iii) modify one or more amino acid in the constant region, with Change Example such as glycosylation site number, effector cell function, Fc acceptor (FcR) in conjunction with, complement in conjunction with etc.
[0071] method of change antibody constant region is known in the art.Can be by replace at least one amino-acid residue in the antibody constant region part with different residues, production has the antibody affinity of the change of the C1 component of effector part (as the FcR on the cell) or complement (for example at) of the function of change (referring to for example EP 388,15lA1, United States Patent (USP) the 5th, 624,821 and 5,648, No. 260, all these documents all integral body are hereby incorporated by).Also the change of similar type can be applied to the immunoglobulin (Ig) of rat immune globulin and other species.For example, replace specific residue by being used in the residue that has appropriate functional group on its side chain, or by importing charged functional groups (for example L-glutamic acid or aspartic acid) or aromatic series non-polar residue (for example phenylalanine, tyrosine, tryptophane or L-Ala), might change antibody (IgG for example, human IgG for example) the Fc district to FcR (for example Fc γ R1) or to C1q bonded affinity (referring to for example United States Patent (USP) the 5th, 624, No. 821).
[0072] in addition, can use the people antibody of transgenic nonhuman animal production at PBMC-of the present invention and IBD-relevant biomarkers, described transgenic nonhuman animal is modified, produces human antibody so that response antigen is attacked, rather than the animal endogenous antibody.Referring to for example PCT publication No. WO 94/02602.Make coding among the non-human host heavy chain and the native gene inactivation of light chain immunoglobulin chain, and in the active gene seat of will encode people's heavy chain and the light chain immunoglobulin (Ig) insertion host genome.For example use the yeast artificial chromosome who contains essential people DNA sections to mix people's gene.Contain the filial generation animal that the middle transgenic animal that are less than the quota modification obtain to provide whole expections modifications by cross-breeding then.This non-human animal's a embodiment is a mouse, is called XENOMOUSE
TM, it is disclosed in PCT publication No. WO 96/33735 and WO 96/34096.This animal produces the B cell of secretion total man immunoglobulin (Ig).Antibody can be by directly obtaining with the animal after the target immunogen immune, and as for example polyclonal antibody prepared product, perhaps origin comes from the immortalization B cell of this animal as producing the hybridoma acquisition of monoclonal antibody.In addition, recyclable and expression coding has the gene of the immunoglobulin (Ig) of people variable region, with direct acquisition antibody, perhaps can further modify, to obtain antibody analog, for example strand Fv molecule.
[0073] binding ability of antibody of the present invention can detect by the following method: Biacore analysis, enzyme-linked immunosorbent assay (ELISA), X-ray crystallography, sequential analysis and scanning mutagenesis and other method known in the art.
[0074] also can use other protein binding molecule to regulate the activity of PBMC-and IBD-relevant biomarkers.This protein binding molecule comprises little module immune drug (SMIP
TM) (Trubion Pharmaceuticals, Seattle, WA).SMIP is by the single chain polypeptide of forming in conjunction with territory, the hinge area polypeptide with 0-1 cysteine residues and immunoglobulin (Ig) CH2 and CH3 structural domain (other is referring to www.trubion.com) of connection structure (for example antigen, counter receptor etc.).SMIP and use with application and for example be disclosed in U.S.'s published number 2003/0118592,2003/0133939,2004/0058445,2005/0136049,2005/0175614,2005/0180970,2005/0186216,2005/0202012,2005/0202023,2005/0202028,2005/0202534 and 2005/0238646 and relevant patent families member, all these documents all integral body are hereby incorporated by.
[0075] can produce the fragment of antibiont traget antibody by cutting antibody according to method well-known in the art.For example, can produce immunocompetent F (ab ') and F (ab ') by using such as pepsic enzyme processing antibody
2Fragment.
[0076] antibiont traget antibody of the present invention also can be used for separating, purifying and/or detect in supernatant liquor, the cell lysate or cell surface on PBMC-and IBD-relevant biomarkers polypeptide.Disclosed in the present invention antibody diagnosticability ground is used to monitor PBMC-and the proteic level of IBD-relevant biomarkers, as the integral part of clinical testing procedure, perhaps be used for therapeutic regulation agent target is contained the antigenic cell or tissue of antibiont traget antibody.For example, curative of the present invention includes but not limited to small molecules, can be connected with the antibiont traget antibody, so that with curative target PBMC-and IBD-relevant biomarkers.
The detection of PBMC-and IBD-relevant biomarkers
[0077] the present invention is by detecting the unconventionality expression or the activity level of intravital PBMC-of experimenter and IBD-relevant biomarkers, diagnosis, prognosis are provided and monitor PBMC-and the unconventionality expression of IBD-relevant biomarkers or the method for IBD (for example Crohn's disease or the ulcerative colitis) progress that activity level directly or indirectly causes thus, include but not limited to these methods are applied to the human experimenter.For example, these methods can be implemented by using wrapped diagnostic kit, described diagnostic kit comprises antibody and the PBMC-as described herein and the conditioning agent of IBD-relevant biomarkers polynucleotide and/or polypeptide of at least a following composition: PBMC-and IBD-relevant biomarkers polynucleotide and fragment, PBMC-and IBD-relevant biomarkers polypeptide and derivative, anti-PBMC-and IBD-relevant biomarkers, described diagnostic kit for example can be in clinical setting conventional the use.In addition, those skilled in the art will appreciate that the expression of one or more PBMC-and IBD-relevant biomarkers or the change of activity level also can detect by the well-known method beyond this paper described method.
[0078] PBMC-and the IBD-relevant biomarkers gene product that comprises in detection and the quantitative biological sample measured in diagnosis of the present invention, prognosis and monitoring.PBMC-and IBD-relevant biomarkers gene product include but not limited to PBMC-and IBD-relevant biomarkers mRNA, cDNA and genomic dna, and PBMC-and IBD-relevant biomarkers polypeptide; These gene products can use the well-known method of those skilled in the art to detect.
[0079] for example, can use the mensuration based on hybridization, for example RNA hybridization, in situ hybridization, Dot blot and slot blot and oligonucleotide arrays directly detect the mRNA with quantitative PBMC-and IBD-relevant biomarkers.Be meant the mensuration that wherein probe nucleic acid and target nucleic acid are hybridized based on the mensuration of hybridization.Under some form, target, probe or these two are immobilized.Immobilized nucleic acids can be DNA, RNA or other oligonucleotide or polynucleotide, and can comprise natural or non-natural nucleotide, nucleotide analog or main chain.Being used for the nucleotide sequence of the system of selection of nucleic acid probe sequence of the present invention based on PBMC-and IBD-relevant biomarkers, is well-known in the art.
[0080] or, detect and quantitatively before can the increase mRNA of PBMC-and IBD-relevant biomarkers.It is well-known that this class amplification type is determined at this area, comprises polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), PCR-enzyme-linked immunosorbent assay (PCR-ELISA), ligase chain reaction (LCR), self-sustained sequence replication, transcription amplification system, Q-β replicative enzyme or other polynucleotide amplification method arbitrarily.Those skilled in the art can be based on listing in the table PBMC-of 1-5 and the nucleotide sequence of IBD-relevant biomarkers, need not too much experiment and just can easily design and produce and be used to produce and detect the PBMC-of amplification and the primer and the probe of IBD-relevant biomarkers gene product.For example can be by gel electrophoresis; By hybridizing with probe nucleic acid; By order-checking; By detecting fluorescence, phosphorescence or radiated signal; Or by in numerous well-known methods any, the PBMC-and the IBD-genes involved product of direct analysis amplification.In addition, those skilled in the art know the method that is used to increase the signal that produces by the target nucleic acid sequence amplification.Those of skill in the art will recognize that no matter use which kind of amplification method,, then all can use numerous quantivative approach known in the art (quantitative PCR (Q-PCR) for example if expect quantitative PBMC-and IBD-genes involved product; This paper is also referred to as " PCR in real time ", " quantitatively PCR in real time ", " quantitatively real-time reverse transcriptase-polymerase chain reaction ", " quantitatively real-time RT-PCR " etc.)).
[0081] can use the well-known immunologic assay of the above-mentioned antibiont traget antibody of numerous uses to detect PBMC-of the present invention and IBD-relevant biomarkers polypeptide (or its fragment).Immunologic assay is meant and utilizes the mensuration of specificity in conjunction with the antibody (for example polyclonal antibody, monoclonal antibody, chimeric antibody, humanized antibody, scFv and fragment thereof) of PBMC-and IBD-related polypeptide (or its fragment).Be suitable for cell sorting (FACS) and western blotting that these well-known immunologic assays of the invention process comprise ELISA, ria-determination (RIA), immunoprecipitation, immunofluorescence, fluorescent activation.In addition, available its can come the antibody of mark antibiont mark by the radiolabeled biotin mark that the standard imaging technique detects in intravital existence of experimenter and location.
[0082] every kind of PBMC-and IBD-relevant biomarkers all can independently be investigated, but provide the combination of two or more PBMC-and IBD-relevant biomarkers to belong to the scope of the invention, and these combinations are used for the inventive method and composition, to increase the degree of confidence of analyzing.In one embodiment, the invention provides PBMC-of the present invention and IBD-relevant biomarkers a plurality of groups, for example model.One group of biomarker can comprise 2-5,5-15,15-35,35-50,50-100 or the PBMC-more than 100 kinds and IBD-relevant biomarkers and/or be made up of it basically.In one embodiment, one group of biomarker of the present invention comprises at least 2 kinds of PBMC-and IBD-relevant biomarkers and/or is made up of it basically.In one embodiment, one group of biomarker of the present invention comprises at least 3 kinds of PBMC-and IBD-relevant biomarkers and/or is made up of it basically.In one embodiment, one group of biomarker of the present invention comprises at least 4 kinds of PBMC-and IBD-relevant biomarkers and/or is made up of it basically.In one embodiment, one group of biomarker of the present invention comprises at least 5 kinds of PBMC-and IBD-relevant biomarkers and/or is made up of it basically.In one embodiment, one group of biomarker of the present invention comprises at least 6 kinds of PBMC-and IBD-relevant biomarkers and/or is made up of it basically.In one embodiment, one group of biomarker of the present invention comprises at least 7 kinds of PBMC-and IBD-relevant biomarkers and/or is made up of it basically.In one embodiment, one group of biomarker of the present invention comprises at least 8 kinds of PBMC-and IBD-relevant biomarkers and/or is made up of it basically.In one embodiment, one group of biomarker of the present invention comprises at least 9 kinds of PBMC-and IBD-relevant biomarkers and/or is made up of it basically.In one embodiment, one group of biomarker of the present invention comprises at least 10 kinds of PBMC-and IBD-relevant biomarkers and/or is made up of it basically.In one embodiment, one group of biomarker of the present invention comprises at least 11 kinds of PBMC-and IBD-relevant biomarkers and/or is made up of it basically.In one embodiment, one group of biomarker of the present invention comprises at least 12 kinds of PBMC-and IBD-relevant biomarkers and/or is made up of it basically.
[0083] in another embodiment, select many group PBMC-and IBD-relevant biomarkers, make that the biomarker in arbitrary group all has some common trait.For example, compare amount or the active increase that all can show at least 1.5 times of first group biomarker in inflammatory bowel (being Crohn's disease or ulcerative colitis) patient's PBMC with the experimenter's who does not have IBD substantially PBMC.Perhaps, compare with first group, second group biomarker all can show difference and regulate.Equally, different biomarker groups can be made up of the difference in functionality classification (be proteolysis, signal transduction, transcribe etc.) or the biomarker of sample (being blood, kidney, spleen, lymphoglandula, brain, intestines, colon, heart, urine etc.), perhaps can select different biomarker groups, to represent the different steps of inflammatory bowel (being Crohn's disease or ulcerative colitis).In a preferred embodiment, of the present inventionly respectively organize the biomarker that biomarker comprises blood (PBMC specifically).Can by selection sort be the biomarker of I group biomarker, the biomarker that is categorized as II group biomarker, the biomarker that is categorized as III group biomarker, the biomarker that is categorized as the biomarker of IV group biomarker and/or is categorized as V group biomarker as one group of biomarker, obtain PBMC-of the present invention and IBD-relevant biomarkers group.Also can be by selecting biomarker to obtain respectively to organize biomarker independently by the biomarker that is categorized as I group, II group, III group, IV group and/or V group.In a preferred embodiment, one group of biomarker comprises PBMC-and the IBD-relevant biomarkers group that is categorized as V group biomarker and/or is made up of it basically.The technician will recognize that also one group of biomarker of the present invention can comprise PBMC-of the present invention and IBD-relevant biomarkers, the V especially of the present invention group biomarker of any amount and arbitrary combination and/or be made up of it basically.For example, one group of non-limiting biomarker of the present invention can comprise immunoglobulin heavy chain constant region γ 1 and immunoglobulin (Ig) κ constant region PBMC-and IBD-relevant biomarkers and/or be made up of it basically.Of the present invention another organized non-limiting biomarker and can be comprised people 28S ribosome-RNA(rRNA) 5 ' district, C type Protein-tyrosine-phosphatase receptor associated protein(RAP), H3 histone family member K, integrin β 3 (platelet glycoprotein IIIa, antigens c D61) and H2B histone family member Q PBMC-and IBD-relevant biomarkers and/or be made up of it basically.Of the present invention another organized non-limiting biomarker and can be comprised immunoglobulin heavy chain constant region γ 1, granzyme K, mutL homologue 3, NGAL 2, CXCL5, serum and deprive and reply phosphatidylserine conjugated protein and H3 histone family member K PBMC-and IBD-relevant biomarkers and/or be made up of it basically.In one embodiment, determining whether the patient suffers from the IBD of (1) Crohn's disease or ulcerative colitis form, (2) Crohn's disease, and/or (3) ulcerative colitis aspect, and/or suffer from Crohn's disease or suffer from aspect the ulcerative colitis distinguishing IBD patient, one group of biomarker of the present invention provides at least 70% tolerance range (more preferably at least 80% tolerance range, most preferably at least 90% tolerance range).
[0084] except many group PBMC-and IBD-relevant biomarkers are provided, also provide one group of PBMC-and IBD-relevant biomarkers that is coupled to solid support expediently also to belong to the scope of the invention.For example, use method well-known in the art, PBMC-of the present invention and IBD-relevant biomarkers polynucleotide can be coupled to array (biochip that for example is used for hybridization analysis), resin (for example can be packed into column chromatography with the resin in the pillar) or matrix (the nitrocellulose matrix that for example is used for rna blot analysis).The preparation of these arrays and using method comprise and use array and computer readable medium (comprising PBMC-of the present invention and IBD-relevant biomarkers) and/or database (for example relational database), and be well-known in this area.
[0085], but there are situation or an active discrete analysis at selected various PBMC-of this group and IBD-relevant biomarkers in the test sample by this support is provided.For example, in array, can use method well-known in the art, will be connected to independently on the different known location on the array with each member's complementary polynucleotide of one group of PBMC-and IBD-relevant biomarkers.This array for example can with the multi-nucleotide hybrid that extracts by experimenter's blood sample (preferred PBMC sample).Therefore but the hybridization of the polynucleotide of test sample and array optional position on array, that can determine PBMC-and IBD-relevant biomarkers in the sample exists situation or amount.Therefore, not only the tissue specificity of one group of IBD biomarker can be determined, and its expression level in tissue also can be determined.In a preferred embodiment, use is based on the array of biochip.Equally, can carry out elisa assay to the specific immobilized antibody of not homopolypeptide biomarker with the hybridization of experimenter's protein sample.
[0086] in another embodiment, the expression of operation report detection of nucleic acids one or more PBMC-of the present invention and IBD-relevant biomarkers.This report nucleic acid can be used for the material that high flux screening changes the peripheral blood lymphocytes express spectra.Structure and use that this reporter molecules is measured are well-known.
[0087] for example, the structure of reporter molecules that is used for the transcriptional regulatory of PBMC-of the present invention and IBD-relevant biomarkers generally needs the adjusting sequence of PBMC-and IBD-relevant biomarkers, typically is promotor.Can obtain promotor by numerous ordinary methods.For example, genomic library can be hybridized with the label probe of being made up of the nucleic acid encoding district, contains the genomic library clone of promoter sequence with evaluation.Can check order to isolating clone, with the sequence of upstream, identification code district.Another kind method is to use the amplified reaction of the primer of the coding region 5 ' end that is annealed to PBMC-and IBD-relevant biomarkers polynucleotide.Amplification template for example can be the restriction gene group nucleic acid that is connected with anchor blister connector (anchor bubble adaptor).
[0088] for making up reporter molecules, the promotor of selected PBMC-and IBD-relevant biomarkers effectively can be connected with report nucleic acid, for example not use the open reading-frame (ORF) of selected PBMC-and IBD-relevant biomarkers polynucleotide.By transfection method the nucleic acid construct thing is transformed in tissue culture cells such as the peripheral blood lymphocytes, to produce the report cell.
[0089] can use many well-known report nucleic acid.In one embodiment, report nucleic acid is green fluorescent protein.In second embodiment, reporter molecules is a beta-galactosidase enzymes.In other embodiments, report nucleic acid is alkaline phosphatase, β-Nei Xiananmei, luciferase, E.C. 2.3.1.28 or other report nucleic acid known in the art.For example can remain on the episome by report nucleic acid construct thing, or be inserted in the karyomit(e) by use target homologous recombination.The preparation and the using method of these report nucleic acid are well-known.
Adopt the analysis of I-V group biomarker
[0090] can be categorized as 5 different groups although those of skill in the art will recognize that PBMC-of the present invention and IBD-relevant biomarkers, each independent biomarker is PBMC-of the present invention and IBD-relevant biomarkers.In addition, the technician will appreciate that biomarker only is to be classified as these groups in order to characterize purpose.For example, determined already that the PBMC-and the IBD-relevant biomarkers of I group were the biomarkers of differential expression in CD patient's PBMC, also are like this in UC patient's PBMC.Therefore, these common biomarkers are sorted in together, can be expediently one be used from the mensuration of screening IBD treatment with test compound to circulate a notice of them, perhaps can one be used from diagnosis, prognosis and/or monitoring IBD, no matter IBD is CD form or UC form.The technician will appreciate that also the PBMC-and the IBD-relevant biomarkers that are categorized as II group, III group, IV group and/or V group also can be used for screening the test compound that diagnosis, prognosis and/or monitoring IBD use, no matter IBD is CD form or UC form.But, it is to be noted that II organizes biomarker, promptly be included in the biomarker in the CD biomarker group, may be the best group that when IBD is the CD form, is used to screen the method for the test compound that diagnosis, prognosis and/or monitoring IBD use.By contrast, III organizes biomarker, promptly is included in the biomarker in the UC biomarker group, may be the best group that is used to screen the method for the test compound that diagnosis, prognosis and/or monitoring IBD use when IBD is the UC form.In addition, IV organizes biomarker, promptly be included in the biomarker in the CDvUC biomarker group, especially V organizes biomarker, promptly being included in the biomarker in the classification biomarker group, may be to distinguish the best group that CD patient and UC patient are used to screen the method for the test compound that diagnosis, prognosis and/or monitoring IBD use when very important.
Screening
[0091] except the method for diagnosis, prognosis and monitoring IBD progress, PBMC-of the present invention and IBD-relevant biomarkers polynucleotide and polypeptide also can be used for screening assay, can regulate the active of PBMC-and IBD-relevant biomarkers and therefore can suppress or alleviate IBD (being Crohn's disease or ulcerative colitis) medicine of symptom or the lead compound of medicine potentially to identify.These screening assay comprise high-throughput screening method, and are well-known in this area.For example, make diagnosis suffer from experimenter's sample of IBD or the doubtful IBD of suffering from or contain PBMC-and the sample of IBD-relevant biomarkers (or natural or reorganization) can with multiple test compound (organic molecule for example, biotechnological formulation) a kind of contact in, each is handled the expression of PBMC-in sample and IBD-relevant biomarkers or activity level can or contact PBMC-in the sample of different tests compound and the expression or the activity level of IBD-relevant biomarkers compared with untreated samples, to determine whether any test compound provides: 1) expression or the activity level of at least a PBMC-and IBD-relevant biomarkers significantly descend, indicate the activity inhibitor of at least a PBMC-and IBD-relevant biomarkers thus, or 2) expression or the activity level of at least a PBMC-and IBD-relevant biomarkers significantly increase, and indication increases at least a PBMC-and the active toughener of IBD-relevant biomarkers thus.In a preferred embodiment, use high flux screening to measure, for example by BIACORE
_(Biacore International AB, Uppsala, Sweden) high flux screening that provides is measured, or BRET (transfer of noclilucence resonance energy) and FRET (FRET (fluorescence resonance energy transfer)) mensuration, and ELISA and based on the mensuration of cell, to regulating at least a PBMC-and the active test compound of IBD-relevant biomarkers is identified.
[0092] in addition, the invention still further relates to a kind of screening and can regulate the method for the test compound of PBMC-and IBD-relevant biomarkers and binding partner binds, this method is following carries out: test compound, PBMC-and IBD-associated protein are mixed mutually with binding partners, and whether definite binding partners combine with PBMC-and IBD-associated protein, and this is in conjunction with just adjusting that how to be subjected to test compound or negative the adjusting.
[0093] as mentioned above, can regulate PBMC-and the active conditioning agent of IBD-relevant biomarkers and can be in numerous natural or synthetic compound, biomolecules, protein, peptide, oligopeptides, polysaccharide, Nucleotide or the polynucleotide any.Test compound can be for example small molecules or biotechnological formulation.As described below, test compound can be provided by numerous libraries well-known in the art.
[0094] test compound of the present invention can be obtained by any available source, comprises the system library of natural and/or synthetic compound.Test compound also can be by any acquisition in the numerous schemes in combinatorial library method known in the art, comprise: biological library, class peptide library (have the functional group of peptide but have the molecular library of new non-peptide main chain, its antienzyme degraded, but still retains biological activity; Referring to for example Zuckermann etc., (1994) J.Med.Chem.37:2678-85); Parallel solid phase of space addressable or liquid phase library; The synthetic library method that needs deconvolution (deconvolution); " pearl one compound " library method; And the synthetic library method of using affinity chromatography to select.Biological library and class peptide library method are limited to peptide library, and other 4 kinds of methods can be applicable to peptide, non-peptide oligomer or micromolecular compound library (generally referring to for example Lam (1997) Anticancer Drug Des.12 (3): 145-67).
The method of diagnosis, prognosis and monitoring inflammatory bowel progress
[0095] " diagnosis " or " diagnosis " refer to differentiate the existence of pathologic condition or do not exist.Diagnostic method comprises (the being unusual) PBMC-that following detection is significantly regulated and the expression of IBD-relevant biomarkers: measure PBMC-in experimenter's (people or non-human mammal) biological sample and IBD-relevant biomarkers gene product (for example mRNA, cDNA or polypeptide, comprise its fragment) test volume, and the normal amount of compare test amount and PBMC-and IBD-relevant biomarkers gene product or scope (being the amount or the scope of the individuality of the known IBD of not suffering from).
[0096] in one embodiment, the PBMC-in two samples of comparison and the level of IBD-relevant biomarkers, one or more PBMC-in the specimen and the explanation of the unconventionality expression of IBD-relevant biomarkers suffer from IBD.In other embodiments, 2,3,4 or the unconventionality expression of more kinds of biomarkers explanation case suffer from serious IBD.In another embodiment, the unconventionality expression of one or more biomarkers explanation has the possibility of suffering from IBD, 2,3,4 or the unconventionality expression indication of the more kinds of biomarkers possibility of suffering from IBD increase.On the other hand, the invention provides the biomarker that the differing appearance of its amount or active and IBD or seriousness or type are associated.For example, as shown, the unconventionality expression of PBMC-in the table 5 and IBD-relevant biomarkers may be associated with the diagnostic result of Crohn's disease or ulcerative colitis.Also expression level subsequently can be compared with the different express spectras of disease different steps, with the express spectra that confirms whether the experimenter has coupling.Although it is concrete diagnostic method may not provide clear and definite IBD diagnostic result, just enough if this method provides the positive indication that helps diagnose.
[0097] the present invention also provides unconventionality expression by detecting at least a PBMC-and IBD-relevant biomarkers or the method for activity level prognosis IBD." prognosis " or " prognosis " refers to predict may developing and/or seriousness of pathologic condition.Method of prognosis comprises the test volume of measuring at least a PBMC-and IBD-relevant biomarkers gene product in experimenter's biological sample, and the prognosis amount of compare test amount and PBMC-and IBD-relevant biomarkers gene product or scope (being the amount or the scope of the individuality of IBD seriousness change).The various amounts of PBMC-and IBD-relevant biomarkers and IBD are that some prognosis of Crohn's disease and/or ulcerative colitis is consistent in the specimen.The amount that detects PBMC-and IBD-relevant biomarkers gene product with specific prognostic level is that the experimenter provides prognosis.In one of the present invention embodiment that relates to IBD (or IBD of particular form), the expression of one or more PBMC-and IBD-relevant biomarkers or active significantly the rise usually are associated with unusual increase.Relate in the embodiment of IBD (or IBD of particular form) at another, the expression of one or more PBMC-and IBD-relevant biomarkers or activity are significantly down-regulated usually and are associated with unusual the reduction.
[0098] in addition, but prognosis as herein described measure can be used for determining the experimenter whether administering therapeutic or prevention with unusual PBMC-with the IBD-relevant biomarkers is expressed or curative or the prophylactic agent (for example agonist, antagonist, peptide mimics, protein, peptide, polynucleotide, small molecules or other drug candidate) of the IBD that activity is relevant.Therefore, regulating PBMC-and IBD-relevant biomarkers such as PAI-2 can make IBD be improved to normal level (for example being similar to or being similar to substantially the level of the tissue that does not have IBD substantially).
[0099] about the gastroenterology field, can design prognosis and measure, whether long-term surviving or progression of disease are had bad prospect with the experimenter who determines this disease treatment of experience.In a preferred embodiment, can be after diagnosis immediately (being in several days) determine prognosis.IBD (for example Crohn's disease or ulcerative colitis) by setting up IBD or particular form can present a kind of express spectra that the particular expression spectrum is associated with the possibility of prognosis mala increase by showing effect to the express spectra of the different steps of acute illness.Prognosis can be used for designing positive therapeutic program more then, with the possibility of avoiding chronic IBD and improving long-term surviving and maintain a good state.
[0100] in a preferred embodiment of the invention, biological sample is used disclosed molecule and method, to detect in PBMC-and the IBD-relevant biomarkers gene situation that exists of one or more known hereditary changes that will cause PBMC-and IBD-relevant biomarkers unconventionality expression.This detection can be used for determining the seriousness of IBD, or prognosis is owing to the expression or the active IBD potentiality of being regulated of PBMC-and IBD-relevant biomarkers.In another specific embodiments, one or more hereditary changes are associated to prognosis or the susceptibility of IBD with the experimenter.Can include but not limited to sequencing reaction, electrophoretic mobility mensuration and oligonucleotide hybridization by the hereditary change of PBMC-and IBD-relevant biomarkers gene in the method discriminating sample well-known in the art.
[0101] the present invention also provides the expression by monitoring PBMC-and IBD-relevant biomarkers or active to monitor IBD be the progress of Crohn's disease and/or ulcerative colitis or the method for the course of disease.Monitoring method comprises to be measured for the first time and the PBMC-from the biological sample that the experimenter takes out and the test volume of IBD-relevant biomarkers gene product for the second time, and this tittle relatively.The change of one or more changes indication IBD courses of disease of the amount of PBMC-and IBD-relevant biomarkers between the first time and the second time.These monitoring experiments also can be used for estimating the effect (for example clinical experiment in the middle of) of particular treatment intervention to the patient, promptly estimate the PBMC-of response curative provided herein and the regulating effect of IBD-relevant biomarkers.
What [0102] will recognize is, measuring method of the present invention not necessarily needs to detect the absolute value of PBMC-and IBD-relevant biomarkers gene product, and it is enough because relative value is concerning many application of these methods.What also will recognize is; except the amount or abundance of PBMC-and IBD-relevant biomarkers gene product, can be by relatively differentiating PBMC-change or unusual and IBD-relevant biomarkers gene product or its expression pattern (polypeptide of for example suddenly change transcript, brachymemma) with normal gene product and express spectra.
Methods of treatment
[0103] the invention provides treatment experimenter's prevention and methods of treatment, described experimenter to IBD be that Crohn's disease and/or ulcerative colitis are risky, susceptible or made a definite diagnosis.For example can measure or, susceptible risky to IBD or the experimenter who has made a definite diagnosis are differentiated in its combination by any diagnosis described herein or prognosis, described IBD is expressed by unusual PBMC-and IBD-relevant biomarkers or activity causes or bring out.In one aspect, regulate PBMC-and IBD-relevant biomarkers protein expression or active PBMC-and IBD-relevant biomarkers albumen or conditioning agent by giving the experimenter, the invention provides that the experimenter and unusual PBMC-of prevention and IBD-relevant biomarkers are expressed or the prevention method of active relevant IBD.Show be the symptom of feature with differentiation PBMC-and IBD-relevant biomarkers protein expression before, can give prophylactic agent, make that IBD is prevented, perhaps optionally postpone its development.Another aspect of the present invention is related to therapeutic purpose and regulates the expression of PBMC-and IBD-relevant biomarkers or the methods of treatment of activity level.Therefore, in exemplary, this control method of the present invention comprises makes cell (for example PBMC) contact with expression level or one or more the active conditioning agents of regulating PBMC-and IBD-relevant biomarkers.
[0104] expression of PBMC-and IBD-relevant biomarkers or the conditioning agent of activity level, it is the conditioning agent of at least a PBMC-and IBD-relevant biomarkers, can be conditioning agent as herein described, for example PBMC-and IBD-relevant biomarkers polynucleotide (comprising relevant PBMC-and IBD-dependency biomarker polynucleotide (for example inhibitory polynucleotide)), PBMC-and IBD-relevant biomarkers albumen, the proteic natural target molecule of PBMC-and IBD-relevant biomarkers (for example PBMC-and IBD-relevant biomarkers protein substrate), the antibiont traget antibody, PBMC-and IBD-relevant biomarkers agonist, PBMC-and IBD-relevant biomarkers antagonist or other small molecules.Suitable conditioning agent can be determined based on screening assay described herein.
[0105] these control methods can perhaps carry out (for example by giving the experimenter with conditioning agent) in vivo external carrying out (for example by cultivate PBMC in the presence of conditioning agent).In one embodiment, described method comprises the PBMC-and IBD-relevant biomarkers albumen or polynucleotide molecule or the relevant agonist with IBD-of PBMC-that gives as curative, with compensatory remarkable minimizing or unusual PBMC-and IBD-relevant biomarkers protein expression or activity.PBMC-and IBD-relevant biomarkers albumen are significantly down-regulated and/or the PBMC-and the IBD-relevant biomarkers activity that wherein increase may have under the situation of beneficial effect therein, stimulate or raise PBMC-and IBD-relevant biomarkers activity caters to the need.
[0106] in another embodiment, described method comprises inhibitory polynucleotide or the polypeptide that gives as curative, with compensatory remarkable increase or unusual PBMC-and IBD-relevant biomarkers expression or active.PBMC-and IBD-relevant biomarkers expression therein or active PBMC-and the IBD-relevant biomarkers activity that is significantly raised and/or wherein reduce may have under the situation of beneficial effect, and inhibition or downward modulation PBMC-and IBD-relevant biomarkers activity cater to the need.
[0107] several pharmacogenomics methods that determine whether to give the conditioning agent of at least a PBMC-and IBD-relevant biomarkers of imagining are that those skilled in the art are well-known, comprise full genome association, candidate gene method and gene expression spectrum analysis.Pharmaceutical composition of the present invention prepare with its expection route of administration fit (for example oral compositions generally comprises inert diluent or edible carrier).Other limiting examples of route of administration comprises parenteral (for example intravenously, subcutaneous, intramuscular), oral (for example suck), rectum, through skin (part) and mucosal.With the matched pharmaceutical composition of each expecting way be that this area is known.
[0108] conditioning agent of at least a PBMC-and IBD-relevant biomarkers useful as drug composition when mixing with pharmaceutically acceptable carrier.Except the conditioning agent and carrier of at least a PBMC-and IBD-relevant biomarkers, this composition also can comprise various thinners, weighting agent, salt, buffer reagent, stablizer, solubilizing agent and other material well-known in the art.Term " pharmaceutically acceptable " is meant the not non-toxic substance of the bioactive validity of interferon activity composition.The feature of carrier depends on route of administration.
[0109] pharmaceutical composition of the present invention also can comprise cytokine, lymphokine or other Hemopoietic factor, for example M-CSF, GM-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-14, IL-15, G-CSF, STEM CELL FACTOR and erythropoietin.Pharmaceutical composition also can comprise anti-cytokine antibodies, thrombus dissolving or the antithrombotic factor (for example plasminogen activator and the VIII factor) and/or other anti-inflammatory drug.The factor that these are extra and/or conditioning agent can be included in the pharmaceutical composition, produce synergy with the conditioning agent with at least a PBMC-and IBD-relevant biomarkers, and the side effect of conditioning agent is minimized.In addition, composition of the present invention can comprise that also (except the conditioning agent of at least a PBMC-of the present invention and IBD-relevant biomarkers) is used for the treatment of the known drug of IBD, for example sulfasalazine, 5-ASA, steroid etc.By contrast, the conditioning agent of at least a PBMC-and IBD-relevant biomarkers can be included in the preparation of the specific cells factor, lymphokine, other Hemopoietic factor, thrombus dissolving or the antithrombotic factor or anti-inflammatory drug, causes the side effect of cytokine, lymphokine, other Hemopoietic factor, thrombus dissolving or the antithrombotic factor or anti-inflammatory drug to minimize.
[0110] pharmaceutical composition of the present invention can be the liposome form, wherein except other pharmaceutically acceptable carrier, the conditioning agent of at least a PBMC-and IBD-relevant biomarkers also can mix with the amphiphilic substance such as lipid, and these amphiphilic substances exist with the micella in aqueous solution, insoluble monolayer, liquid crystal or lamellose aggregated forms.The lipid that is applicable to Liposomal formulation includes but not limited to monoglyceride, triglyceride, sulfatide, lysolecithin, phosphatide, saponin(e, bile acide etc.The preparation of this lipoid plastid preparation is that those skilled in the art are well-known.
[0111] term used herein " treatment significant quantity " be meant be enough in pharmaceutical composition or the method to demonstrate significant patients ' interest (for example improve the IBD relative disease symptom, cure the IBD relative disease or increase the curative ratio etc. of IBD relative disease) the total amount of each activeconstituents.When each activeconstituents list time spent, this term is meant the composition that this is independent.When each activeconstituents coupling, this term is meant the combined amount of the activeconstituents that produces curative effect, no matter is drug combination, sequentially drug using or medication simultaneously.
[0112] when implementing treatment of the present invention or using method, gives the experimenter with at least a PBMC-of treatment significant quantity and the conditioning agent of IBD-relevant biomarkers, for example Mammals (for example people).Give conditioning agent, can according to the inventive method single with or with the other therapies coupling, described other therapies is for example for using the treatment of cytokine, lymphokine or other Hemopoietic factor or anti-inflammatory drug.When giving jointly with one or more medicines, the conditioning agent of at least a PBMC-and IBD-relevant biomarkers can with other medicines simultaneously or the medication of sequential ground.If sequentially drug using, then the attending doctor will determine to give the conditioning agent of for example at least a PBMC-and IBD-relevant biomarkers and other conditioning agent or other conditioning agent of at least a PBMC-and IBD-relevant biomarkers by suitable order.
[0113] in one embodiment, the conditioning agent of at least a PBMC-of the present invention and IBD-relevant biomarkers, its pharmaceutical composition for example, use with conjoint therapy, promptly, for example can be used for treating the curative of pathologic conditions or obstacle (for example immunity and/or inflammatory diseases) with other conditioning agent combination.Term herein " coupling " is meant or gives substantially simultaneously simultaneously or sequentially conditioning agent.If sequentially drug using is then when beginning to give second kind of compound, preferably at therapentic part or still can detect in two kinds of compounds of effective concentration first kind to the experimenter.
Combination therapy
[0114] combination therapy for example can comprise and the common preparation of at least a additional procedures medicine and/or the PBMC-of common medication and the conditioning agent of IBD-relevant biomarkers.The additional adjustment agent can comprise as at least a in greater detail cytokine inhibitor, growth factor receptor inhibitors, immunosuppressor, anti-inflammatory agent, metabolic poison, enzyme inhibitors, cytotoxic agent or cytostatics hereinafter.These conjoint therapies can advantageously utilize the administering therapeutic medicine than low dosage, have avoided possible toxicity or the complication relevant with various monotherapies thus.And curative disclosed herein acts on and the different approach of IBD injury approach, therefore expects the effect of conditioning agent of its enhancing and/or at least a PBMC-of synergy and IBD-relevant biomarkers.
[0115] curative with the conditioning agent coupling of PBMC-and IBD-relevant biomarkers can be those medicines that disturb inflammatory reaction in different steps.In one embodiment, the conditioning agent of at least a PBMC-as herein described and IBD-relevant biomarkers can be prepared and/or common medication jointly with at least a cytokine and/or growth factor antagonist.Cytokine and/or growth factor antagonist can comprise soluble receptors, inhibitor peptides, small molecules, part fusions, antibody (it is in conjunction with cytokine or somatomedin or its acceptor or other cell surface molecule) and " anti-inflammatory cytokines " and agonist thereof.
[0116] can include but not limited to the antagonist of at least a interleukin (for example IL-1, IL-2, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21 and IL-22), cytokine (for example TNF α, LT, EMAP-II and GM-CSF) or somatomedin (for example FGF and PDGF) with the limiting examples of the medicine of the conditioning agent coupling of PBMC-described herein and IBD-relevant biomarkers.Described medicine also can include but not limited to the antagonist of at least a acceptor of interleukin, cytokine and somatomedin.The conditioning agent of PBMC-and IBD-relevant biomarkers also can with inhibitor (for example antibody) coupling of cell surface molecule, described cell surface molecule for example is CD2, CD3, CD4, CD8, CD20 (CD20 inhibitor Rituximab (RITUXAN for example
_), CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90 or its part, comprise CD154 (gp39 or CD40L, or LFA-1/ICAM-1 and VLA-4/VCAM-1 (Yusuf-Makagiansar etc., (2002) Med.Res.Rev.22:146-67).The antagonist that can comprise IL-1, IL-12, TNF α, IL-15, IL-17, IL-18, IL-21 and IL-22 acceptor with other compound of the conditioning agent coupling of PBMC-described herein and IBD-relevant biomarkers.
[0117] exemplary drugs that can be used from conjoint therapy with the conditioning agent one of PBMC-and IBD-relevant biomarkers comprises IL-12 antagonist (for example combining the antibody (referring to for example WO 00/56772) of IL-12), IL-12 acceptor inhibitor (antibody of for example anti-IL-12 acceptor) and solubility IL-12 acceptor and fragment thereof.The example of IL-15 antagonist comprises that the soluble fragments and the IL-15 of antibody, IL-15 acceptor of anti-IL-15 or its acceptor is conjugated protein.The example of IL-18 antagonist comprises the antibody of anti-IL-18, the soluble fragments and the IL-18 conjugated protein (IL-18BP, Mallat etc., (2001) Circ.Res.89:E41-45) of IL-18 acceptor.The example of IL-1 antagonist comprises il-1-saccharase (ICE) inhibitor (for example Vx740), IL-1 antagonist (IL-1RA (Kineret (KINERET for example
TM), Amgen)), sIL-1RII (Immunex) and anti-IL-1 receptor antibody.
[0118] example of TNF antagonist comprises the antibody of anti-TNF (for example human TNF alpha), for example D2E7 (people's anti-TNF alpha antibody, United States Patent (USP) the 6th, 258, No. 562, HUMIRA
TM, Abbott Labs), CDP-571/CDP-870/BAY-10-3356 (the humanization anti-TNF alpha antibodies, Celltech/Pharmacia), cA2 (chimeric anti-TNF alpha antibodies, REMICADE
TM, Centocor) with anti-TNF antibodies fragment (for example CPD870).Other example comprises soluble TNF acceptor (for example people p55 or p75) fragment and derivative, for example p55kD TNFR-IgG (55kD TNF acceptor-IgG fusion rotein, LENERCEPT
TM) and 75kd TNFR-IgG (75kD TNF acceptor-IgG fusion rotein, ENBREL
TM(etanercept-Immunex)).Referring to for example vander Poll etc., (1997) Blood.89:3727-34; Mori etc., (1996) J.Immunol.157:3178-82.Other example comprises enzyme antagonist (TNF α converting enzyme inhibitor (TACE) for example, for example (GW 3333 for alpha sulfonyl hydroxamic acid derivatives (WO 01/55112) or N-formyl hydroxy amine inhibitors,-005 or-022, GlaxoSmithKline) and TNF-bp/s-TNFR (soluble TNF is conjugated protein, referring to for example Lantz etc., (1991) J.Clin.Invest.88:2026-31; Kapadia etc., (1995) Amer.J.Physiol.Heart Circ.Phys.268:H517-25).The TNF antagonist can be soluble TNF acceptor (for example people p55 or p75) fragment and derivative, for example 75kd TNFR-IgG and TNF α saccharase (TACE) inhibitor.
[0119] in other embodiments, the conditioning agent of PBMC-as herein described and IBD-relevant biomarkers can with at least a following medication combined medication: IL-13 antagonist, for example solubility IL-13 acceptor and/or anti-il-13 antibody; With the IL-2 antagonist, for example the IL-2 fusion rotein is (for example by the DAB 486-IL-2 and/or the DAB 389-IL-2 of Seragen preparation, referring to for example Sewell etc., Arthritis Rheum.36:1223-33) and anti-IL-2R antibody (for example anti-Tac-H humanized antibody (1993), Protein Design Labs, referring to Junghans etc., (1990) Cancer Res.50:1495-502).Another kind of combination comprises conditioning agent and the anti-CD4 inhibitor of non-removing such as IDEC-CE9.1/SB 210396 (anti-CD 4 antibodies, combination GlaxoSmithKline) of PBMC-and IBD-relevant biomarkers.Other combination comprises the conditioning agent of PBMC-and IBD-relevant biomarkers and CD80 (B7.1) and CD86 (B7.2) again stimulates pathway antagonists (for example antibody, soluble receptors or antagonism part), palatelet-selectin glycoprotein ligand (PSGL), PSGL-1 inhibitor (antibody and the micromolecular inhibitor of for example anti-PSGL and/or PSGL-1), T cell and B cell scavenging agent (for example anti-CD4 or anti-CD22 antibody) and anti-inflammatory cytokines and agonist (for example antibody) thereof altogether.Anti-inflammatory cytokines can comprise IL-4 (for example Schering-Plough Biopharma), (for example SCH 52000 for IL-10, recombinant il-10, Schering-Plough Biopharma), IL-11, IL-13 and TGF β or its agonist (for example agonist antibody).
[0120] in other embodiments, the conditioning agent of at least a PBMC-and IBD-relevant biomarkers can be prepared with at least a antiphlogiston, immunosuppressor, metabolic poison and enzyme inhibitors and/or give altogether altogether.Can unite the medicine of use with the conditioning agent of PBMC-as herein described and IBD-relevant biomarkers or the limiting examples of inhibitor includes but not limited at least a following medicine: NSAID (non-steroidal anti-inflammatory drug) (NSAID) (includes but not limited to acetylsalicylic acid, salsalate, two fluorine Buddhist nun willows, Ibuprofen BP/EP, Ketoprofen, nabumetone, piroxicam, Naproxen Base, diclofenac, indomethacin, sulindac, tolmetin, R-ETODOLAC, ketorolac, the Ao Shapu piperazine, tenidap, meloxicam, piroxicam, Aceclofenac, tolmetin, tiaprofenic acid, nimesulide etc.), sulfasalazine, reflunomide (for example prednisolone), cell factor inhibiting anti-inflammatory agent (CSAID), Nucleotide biosynthesis inhibitor (purine biosynthesis inhibitor (antifol for example for example, and the pyrimidine biosynthesis inhibitor methotrexate for example)), dihydroorate dehydrogenase (DHODH) inhibitor for example, leflunomide (referring to for example Kraan etc., (2004) Ann.Rheum.Dis.63:1056-61) for example.Be used for to comprise one or more NSAID, CSAID, DHODH inhibitor (for example leflunomide) and antifol (for example methotrexate) with the curative of the conditioning agent coupling of at least a PBMC-and IBD-relevant biomarkers.
[0121] can comprise at least a following medicine with the example of other conditioning agent of the conditioning agent coupling of PBMC-and IBD-relevant biomarkers: reflunomide (oral, suction and local injection); Immunosuppressive drug (for example S-Neoral and tacrolimus (FK-506)); (for example CCI-779 is (referring to for example Elit (2002) Curr.Opin.Investig.Drugs 3:1249-53 for for example sirolimus (rapamycin) or forms of rapamycin analogs and/or derivative, rapamycin ester derivative for example for the mTOR inhibitor; Huang etc., (2002) Curr.Opin.Investig.Drugs3:295-304)); Disturb the medicine (for example IRAK, NIK, IKK, p38 or map kinase inhibitor) of pro-inflammatory cytokine (for example TNF α and IL-1) signal transduction; TPL-2, Mk-2 and NF κ b inhibitor; Cox 2 inhibitor (for example celecoxib, rofecoxib etc. and variant thereof); Phosphodiesterase inhibitor (for example rolipram); Inhibitor of phospholipase enzymes (for example cPLA2 2 (cPLA2) inhibitor, for example fluoroform keto analog (United States Patent (USP) the 6th, 350, No. 892)); Vascular endothelial growth factor (VEGF) inhibitor; The vegf receptor inhibitor; Angiogenesis inhibitor; RAGE and solubility RAGE; Erss (ERB) agonist, ERB-NF κ b antagonist; Interferon-beta (for example IFN β-1a and IFN β-1b); Ke Pasong (copaxone); And reflunomide.
[0122] can comprise with other useful curative of the conditioning agent coupling of one or more PBMC-and IBD-relevant biomarkers: budesonide (budenoside); Urogastron; Aminosalicylate; Ismipur; Azathioprine; Metronidazole; Lipoxidase inhibitor; Mesalazine; Olsalazine; Balsalazide; Antioxidant; The thromboxane inhibitor; Somatomedin; Elastase inhibitor; Pyridine-imidazolium compounds; The prodrug of prednisolone, dexamethasone or the budesonide of glucuronide or dextran-put together; (ISIS 2302 for ICAM-1 antisense phosphorothioate ester oligodeoxynucleotide; Isis Pharmaceuticals, Inc.); Soluble complement acceptor 1 (TP10; T Cell Sciences, Inc.); The slow release type mesalazine; Platelet activation factor (PAF) antagonist; Ciprofloxacin; Lignocaine; Cyclosporin A; Oxychloroquine (PLAQUENIL
TM); Minocycline HCl (MINOCIN
TM); And Kineret (KINERET
TM).
[0123] selects to be used for to depend on to a great extent such as factors such as concrete experimenter, required target and selected treatment times with the concrete curative of the conditioning agent coupling of PBMC-of the present invention and IBD-relevant biomarkers.These decisions belong to those skilled in the art's technology and ken fully.
[0124] can comprise one or more following medicines with other example of the curative of the conditioning agent coupling of PBMC-and IBD-relevant biomarkers: Ismipur (6-MP), azathioprine, sulfasalazine, mesalazine, olsalazine, chloroquine, Oxychloroquine (PLAQUENIL_), Trolovol; Gold sulphur fourth salt (aurothiornalate) (intramuscular and oral), azathioprine, colchicine (colchicine), beta-2-adrenoceptor agonist (salbutamol, terbutaline, Salmeterol (salmeteral)), xanthine (theophylline, aminophylline), cromoglycate, nedocromil, ketotifen, the different third atropic ammonium and oxygen holder ammonium, mycophenolate mofetil, adenosine agonists, antithrombotic, complement inhibitor and adrenergic agent.
[0125] in one embodiment, the conditioning agent of PBMC-and IBD-relevant biomarkers can with one or more antibody couplings, described antibody is at other target of participate in regulating immunne response.Can unite with the conditioning agent of PBMC-of the present invention and IBD-relevant biomarkers and be used for the treatment of or the limiting examples of the medicine that epidemic prevention is replied comprises following medicine:, include but not limited to CD25 (IL-2 acceptor-a), CD11a (LFA-1), CD54 (ICAM-1), CD4, CD45, CD28, CTLA4, ICOSL, ICOS, CD80 (B7.1) and/or CD86 (B7.2) at the antibody of other cell surface molecule.In another embodiment, the conditioning agent of PBMC-and IBD-relevant biomarkers and one or more general immunosuppressor coupling, for example cyclosporin A or FK506.In another embodiment, the conditioning agent of PBMC-and IBD-relevant biomarkers and the coupling of CTLA4 agonist, for example CTLA4Ig-A Baxipu (abatacept, ORENCIA
_).
[0126] pharmaceutical composition of the present invention is mixed with route of administration fit predetermined with it.The method that realizes administration is that those of ordinary skills are known.Also might obtain can part or liquid preparations for oral administration, maybe can see through the composition of mucosal delivery.The administration of the conditioning agent of the present invention that uses at the pharmaceutical composition that is used for implementing the inventive method can be carried out by multiple usual manner, for example orally ingestible method, inhalation, endermosis, subcutaneous drug regimen, intravenous injection method, rectum enema, suppository interpolation or the like.
[0127] solution or the suspension that is used for intracutaneous or subcutaneous application generally includes one or more following components: sterile diluent, for example water for injection, salt brine solution, fixed oil, polyoxyethylene glycol, glycerine, propylene glycol or other synthetic; Antibacterial agent, for example phenylcarbinol or nipagin; Antioxidant, for example xitix or sodium bisulfite; Sequestrant, for example ethylenediamine tetraacetic acid (EDTA); Buffer reagent, for example acetate, Citrate trianion or phosphoric acid salt; And tension regulator, for example sodium-chlor or glucose.PH usable acid or alkali are regulated, for example hydrochloric acid or sodium hydroxide.These preparations can be encapsulated in ampoule, disposable syringe or the multiple dose vials of being made by glass or plastics.
[0128] pharmaceutical composition that is suitable for injecting comprises aseptic aqueous solution agent or dispersion liquid and is used to face the sterile powder injection that the time spent is mixed with aseptic parenteral solution or dispersion liquid.For intravenous administration, suitable carrier comprises physiological saline, bacteriostatic water, CREMAPHORE
TMEL (BASF, Parsippany, NJ) or phosphate buffered saline(PBS) (PBS).In all cases, composition must be aseptic, and answer fluidization to the degree with easy injectivity, must be stable under production and storage condition, must be anticorrosion, and to resist such as microbiological contamination effects such as bacterium and fungies.Carrier can be solvent or the dispersion medium that contains water for example, ethanol, polyvalent alcohol (for example glycerine, propylene glycol and liquid macrogol etc.) and suitable mixture thereof.By using such as the coating material of Yelkin TTS, the granularity that with regard to dispersion liquid, needs and passing through to use tensio-active agent, can keep suitable flowability by maintenance.Can pass through various antibacterial agents and anti-mycotic agent, for example p-Hydroxybenzoate, trichloro-butyl alcohol, phenol, xitix, Thiomersalate etc. are realized the prevention to microbial process.In many cases, preferably in composition, comprise isotonic agent, for example sugar, polyvalent alcohol (for example N.F,USP MANNITOL, sorbyl alcohol) and sodium-chlor.Comprise the absorption that the reagent (for example monostearate aluminium and gelatin) that postpone to absorb can obtain prolonging composition for injection in the composition.
[0129] when the conditioning agent of oral at least a PBMC-that treats significant quantity and IBD-relevant biomarkers, tackiness agent is tablet, capsule, pulvis, solution or elixir form.When with the tablet form administration, pharmaceutical composition of the present invention also can comprise solid carrier in addition, for example gelatin or assistant agent.Tablet, capsule and pulvis comprise the tackiness agent of about 5-95%, preferably comprise the tackiness agent of about 25-90%.When with the liquid form administration, can add liquid vehicle, for example the oil in water, oil, animal or plant source (for example peanut oil (but will sincerely remember the allergic frequency of peanut in the colony)), mineral oil, soybean oil or sesame oil or synthetic oil.The pharmaceutical composition of liquid form also can comprise normal saline solution, glucose or other sugar soln, or dibasic alcohol, for example ethylene glycol, propylene glycol or polyoxyethylene glycol.When with the liquid form administration, pharmaceutical composition comprises the tackiness agent of about 0.5-90% (weight), preferably comprises the tackiness agent of about 1-50% (weight).
[0130] when the conditioning agent of at least a PBMC-that treats significant quantity by intravenously, skin, subcutaneous injection and IBD-relevant biomarkers, conditioning agent can be accepted the aqueous solution agent for the parenteral of no thermal source form.This parenteral can be accepted the preparation of protein solution to relevant requirements such as pH, isotonicity, stability, and said preparation belongs to those skilled in the art's ken.Except the conditioning agent of at least a PBMC-and IBD-relevant biomarkers, be used for intravenously, skin or hypodermic preferred pharmaceutical compositions and vadose solution matchmaker, for example sodium chloride injection, Ringer injection liquid, glucose injection, dextrose ﹠ sodium chloride injection, lactic acid salt Ringer injection liquid or other solvent known in the art such as also should comprise.Pharmaceutical composition of the present invention also can comprise stablizer, sanitas, buffer reagent, antioxidant or other additive well known by persons skilled in the art.
[0131] amount of the conditioning agent of at least a PBMC-and IBD-relevant biomarkers depends on the character and the seriousness of disease to be treated in the pharmaceutical composition of the present invention, and the character of the previous treatment that stood of patient.At last, the attending doctor will determine the amount of the conditioning agent of treatment at least a PBMC-of each individual patient and IBD-relevant biomarkers.At first, the attending doctor gives at least a PBMC-of low dosage and the conditioning agent of IBD-relevant biomarkers, and observes patient's reaction.Can give the heavy dose of at least a PBMC-and the conditioning agent of IBD-relevant biomarkers, until the patient is obtained optimum curative effect, dosage generally no longer increases at that time.The various pharmaceutical compositions that expection is used to implement the inventive method should comprise about 0.1 μ g to about 100mg/kg body weight.
[0132] it is variable to use pharmaceutical composition of the present invention to carry out the time-histories of intravenously (i.v.) treatment, depends on the disease and the potential specific reaction of severity of disease to be treated and each individual patient.Expect that the time-histories of the each administration of conditioning agent of at least a PBMC-and IBD-relevant biomarkers can be in the scope of 12-24 hour continuous intravenous administration, perhaps some other suitable time section.Curatives such as subcutaneous (s.c) that use pharmaceutical composition of the present invention, suppository have also been imagined.But these curative medications every day are once, weekly medication once, perhaps more preferably per two the week or medication in every month once.The conditioning agent that also is expected at least a PBMC-and IBD-relevant biomarkers is under the micromolecular situation, and curative can be once a day, twice on the one, three times on the one ... medication.At last, when using pharmaceutical composition of the present invention, the attending doctor will determine the suitable course of treatment or use the micromolecular suitable course of treatment, and the time of administering therapeutic medicine.
Medicine box
[0133] the present invention also is provided for determining patients with inflammatory bowel long-term surviving or the prognosis medicine box that maintains a good state, and described medicine box comprises the reagent of the expression that is used to estimate biomarker of the present invention.Also imagined and be used to the medicine box diagnosing and monitor.Preferably, described reagent can comprise one or more antibiont traget antibodies or its fragment, and wherein said antibody or its fragments specific are in conjunction with the albumen corresponding to PBMC-and IBD-relevant biomarkers.Optional described medicine box can comprise a kind of polynucleotide probes, and wherein said probe specificity is in conjunction with the polynucleotide of transcribing corresponding to PBMC-that lists in table 1-5 and IBD-relevant biomarkers.Described medicine box also can comprise one group of PBMC-and IBD-relevant biomarkers, and they can be used as arrayed at biochip for example on the GENECHIP_.
[0134] the present invention also is provided for estimating the adaptive medicine box of the inflammatory bowel of each the inhibition experimenter in the multiple compound.These medicine boxs comprise multiple testing compound, and the reagent (be the corresponding protein specific antibody, or corresponding polynucleotide specific probe or primer) that is used to estimate the expression of listing in the table PBMC-of 1-5 and IBD-relevant biomarkers.
[0135] the establishing criteria technology it will be apparent to those skilled in the art that the modification of the invention described above composition and method, also should comprise in the present invention.
[0136] following examples have further been set forth the present invention, should not regard these embodiment as restrictive.The content of all reference, patent and the patent application of mentioning in whole the application all is hereby incorporated by.
Embodiment
[0137] embodiment of following statement helps to understand the present invention, but is not intended to and should be counted as limiting the scope of the invention by any way.Embodiment does not comprise the detailed description of ordinary method, for example by healthy volunteer and patient's separating periphery blood monocytic cell of suffering from inflammatory bowel.These methods are that those of ordinary skills are well-known.
Materials and methods
Embodiment 1.1: patient information and clinical evaluation
[0138] collects by 42 apparent healthy individual, 59 CD patients and 26 UC patients altogether in North America and European clinical place and be used for the blood sample that pharmacogenomics is analyzed.The ethics examination board of mechanism in each clinical place or the ethics council have all ratified this research, implementation procedure not before the informed consent that obtains every patient.
[0139] individual demographic characteristics's comparative result sees Table 7 in this research.
Table 7: the IBD experimenter's of normal anosis individuality (C) and Crohn's disease (CD) or ulcerative colitis (UC) form demographic characteristics
| Type | CD | UC | C | CvIBD p value | CDvVC p value |
| Sample number | 59 | 26 | 42 | ||
| Age (on average) | 41.3 | 46.7 | 44.1 | 0.96 1 | 0.055 1 |
| Sex | 38 women of 21 |
18 women of 8 |
18 women of 24 male sex | 0.014 2 | 0.66 2 |
| The |
1 Spaniard of 7 Black people of 51 |
1 Spaniard of 3 Black people of 22 |
1 Indians of 1 Aisa people of 40 white men | 0.09 3 | 0.82 3 |
1: utilization sided t check and based on the p value of the t statistical computation of ANOVA estimation of error.
2: the p value that the male sex in the group calculates the likelihood ratio chi square test of women's frequency is compared in utilization.
3: the p value that the white man in the group calculates the likelihood ratio chi square test of non-white man's frequency is compared in utilization.
[0140] health volunteer (24 male sex, 18 women) mainly is white man, and the age is in the 25-60 scope in year.CD patient (21 male sex, 38 women) mainly is white man, the age in the scope in 20-65 year, Crohn's disease activity index scoring (CDAI) between 220-400, stomachache grading 〉=25 and/or diarrhoea grading 〉=25.Add that by radioactivity research, splanchnoscopy histological examination or surgical pathology confirm at least 6 months CD diagnostic result; If confirm diagnostic result by biopsy, then comprise the patient who is diagnosed as Crohn's disease.UC patient (8 male sex, 18 women) mainly be white man, age is in the 25-73 scope in year, according to Mayo ulcerative colitis points-scoring system (Mayo Ulcerative Colitis Scoring System, MUCSS) doctor's total appraisal scoring is slightly being marked to moderate (scoring is 1 or 2) scope.Except the standard clinical standard, also add that by splanchnoscopy biopsy provides the diagnostic result of left side UC.
[0141] women is significantly different between health population and IBD colony to the male sex's ratio, but does not have difference between two IBD colonies; All there is not significant difference (being p＜0.05 level) race (white man is to non-white man) between health population and the IBD colony or between two IBD colonies and age.Study concomitant drugs between two IBD colonies and use and to show, 5-ASA and any other medicine that seldom uses that is reported as concomitant drugs are not all obscured the comparison in this research.
Embodiment 1.2: blood sample collection and processing
[0142], gathers everyone blood (8ml), the Vacutainer cell preparation of packing into pipe (CPT in clinical place; Becton Dickinson, Franklin Lakes, NJ) in, and deliver to the center processing laboratory according to manufacturer's suggestion all through the night and carry out PBMC and separate.All PBMC that analyze in this research are getting behind the blood and are handling in 24 hours.Before the RNA purifying, use ABX Pentra 60C+ cellanalyzer (Irvine, CA) PBMC to purifying carries out total cell count, with the absolute number and the percentage of record neutrophilic granulocyte, lymphocyte, monocyte, eosinophilic granulocyte and basophilic granulocyte.1 PBMC sample of UC patient does not carry out cell counting, so this express spectra is excluded outside following A NCOVA analyzes.The expression data that when exploitation and check predictive model, comprises this patient.(Qiagen, Valencia is CA) by the total RNA of PBMC purifying to use the small-sized post scheme of RNeasy.
Embodiment 1.3: oligonucleotide arrays hybridization and data reduction
[0143] (Affymetrix, Santa Clara CA) change total RNA (2 μ g) into biotinylation cRNA according to the Affymetrix method.With mark cRNA (10 μ g) fragmentation, and prepare for foregoing hybridization (Twine etc., ibid).As the description in the Affymetrix technical manual, make biotinylation cRNA and Affymetrix HG-U133A people GENECHIP
_Hybridization array.With abundance at 1: 300,000 (3ppm) mixes in each sample to 11 biotinylation control transcripts in 1: 1000 (1000ppm) scope, hybridization then is to be used as typical curve (Hill etc., (2001) Genome Biol.2 (12): research0055.1-0055.13).Use GENECHIP
_MAS 5.0 softwares are estimated specific hybrid intensity, calculate the signal value of each probe groups, and make and do not have/exist judgement.Then by the reference standard curve, the signal value of each probe groups is converted into represents 10
6The frequency values of the transcript number that exists in the individual transcript (Hill etc., ibid).Estimate each transcript, if it satisfies two following non-strict standards then includes research in: be judged as ' existence ' and be at least one sample (healthy person, UC or CD) therein or be higher than frequency values 10 (10ppm).7,908 sequences satisfy these filter criterias, are used for analyzing.
Embodiment 1.4: variance analysis (ANOVA) and covariance analysis (ANCOVA)
[0144] during the average differential expression in check disease group, (analysis of covariance, ANCOVA) method is adjusted the difference that the PBMC cell type is formed to use covariance analysis.Use to the frequency of number conversion as the response measurement result, to the independent ANCOVA of each transcript operation.The ANCOVA model comprises disease group, sex, neutrophilic granulocyte percentage, monocyte percentage and these projects of eosinophilic granulocyte percentage.In ANCOVA, for each cell type, calculate the slope of describing the linear relationship between cell type percentage and the specific gene expression level, carry out the t check, to determine that whether this slope has notable difference (wherein, not having linear relationship between slope 0 expression cell type percentage and the expression level) with 0.
[0145] selection of including the cell type in the ANCOVA model in is limited by following Consideration: the 1) correlation degree between the cell type, 2) each cell type distributes between the disease type difference degree and 3) the percentage size of every kind of cell type.Covariance among the ANCOVA should be each other height correlation not.Lymphocyte percentage is strong negative correlation with monocyte percentage and neutrophilic granulocyte percentage, does not include ANCOVA in for this reason.
[0146] except overall inspection to treatment group difference and cell type regression effect, also use the sided t check to carrying out paired comparison at the adjusted disease group mean value of cell type percentage difference, the denominator of t-statistics is obtained by ANCOVA error term.At last, because being distributed in relatively between the disease group of the women and the male sex is also obviously different, so include sex in ANCOVA.
[0147] the original p value that is drawn by above-mentioned analysis is adjusted, so that a large amount of statistical test of being carried out to be described.Use multiple change filter (1.5 times) to reduce the incidence of false positive measurement result together with the conservative significance level of α=0.0001.
Embodiment 1.5: use the microarray expression data to carry out gene Selection and the prediction of supervision classification
[0148] use was before described (Golub etc., (1999) Science 286:531-37) and can be carried out gene Selection in the GeneCluster2.0 version that www.broad.mit.edu/cancer/software/software.html obtains and predict with the supervision classification.In these are analyzed, only use 4228 transcripts that satisfy strict data reduction strainer (at least 50% existence calling is arranged, and at least 50% Crohn's disease or UC sample have the frequency greater than 10ppm) in Crohn's disease and UC sample.Sample in selecting at random respectively to organize is as the member in the data set (data set MB), and this data set is made up of the training set (75%) or the test set (25%) of express spectra.Use the sample training set to carry out gene Selection, in training set, show the highest classification distribution sorter resultnat accuracy, that have minimum gene by staying one and 4 times of cross validation to differentiate.To the prediction of the sample evaluation in test set disaggregated model, the classification of sample is distributed resultnat accuracy in the report test set then.
[0149] for gene Selection, all expression datas in training set and the test set all carry out the logarithm conversion before analysis.In the data training set, use bilateral method (characteristic number in of all categories equates) to set up the model that contains the feature (transcript sequence) of accelerating, described bilateral method adopts uses mean value to carry out the S2N similarity measurement that classification is inferred.Use the binary method to compare CD patient and UC patient's PBMC express spectra.By staying the predicted gene sorter that contains 2-200 gene in one and 4 times of cross validation evaluation procedure 2, produce the minimum predictive model that the most accurate classification is distributed to identify.Use weighted voting algorithm to carry out classification member's prediction.
Embodiment 1.6:Ingenuity path analysis
[0150] (Ingenuity, Mountain View CA) are used to annotate IBD genes involved, CD specific gene and the UC specific gene that derives from the ANCOVA analysis to Ingenuity path analysis (IPA) instrument.The classical pathway of these gene lists and the annotation of functional category are obtained by Gene-By-Gene View, and/or use retrieval IPKB (Ingenuity approach knowledge base) feature to obtain.
Embodiment 1.7: quantitatively real-time polymerase chain reaction (Q-PCR) confirms microarray results
[0151] analyzes two 96 orifice plates that contain from 59 Crohn's disease (CD) patients and 26 ulcerative colitiss (UC) peripheral blood of patients monocytes (PBMC) RNA sample by Q-PCR.The various PBMC RNA of 45ng sample altogether is transferred in 96 orifice plates in the mode that keeps the original order of sample.2 CD patients and 1 UC patient's PBMC RNA sample does not contain the RNA of capacity; Thereafter, these patients' sample is excluded outside Q-PCR analyzes.Use large vol cDNA storehouse test kit (Applied Biosystems, San Diego CA) that every kind of RNA sample is carried out reverse transcription in 100 μ l reactants.Reactant is in 25 ℃ of incubations 10 minutes, then in 37 ℃ of incubations 2 hours, and is stored in-80 ℃, until amplification.Use the gene specific TAQMAN_ probe of design in advance and primer sets (TAQMAN_ determination of gene expression, Applied Biosystems) (to increase and relative expression's level of quantitative classification biomarker corresponding to the GENBANK accession number (being visible single-gene identification number in the above table 5) of the gene in the 12-gene sorter.The expression level of amplification and 4 kinds of house-keeping genes of quantitative every kind of RNA sample also: (1) B2M (β 2M), (2) beta-actin, (3) 18S ribosome-RNA(rRNA) (18S) and (4) glyceraldehyde-3-phosphate dehydrogenase (GAPDH).Be used for the TAQMAN_ probe of each target transcript and Applied Biosystems (ABI) ID of primer and see Table 8.
| Table 8: the TAQMAN probe of each target gene and the ABI ID of primer | ||||
| ABI ID | The gene title | Symbol | The single-gene identification number | |
| Hs99999901_s1 | 18SrRNA | 18S | ||
| Hs00194353_m1 | NGAL 2 (oncogene 24p3) | LCN2 | Hs.204238 | |
| Hs00271778_m1 | MutL homologue 3 (intestinal bacteria) | MLH3 | Hs.279843 | |
| Hs00190538_ml | Serum is deprived and is replied (phosphatidylserine is conjugated protein) | SDPR | Hs.26530 | |
| | Histone | 2, H2be | HIST2H2BE | Hs.2178 |
| | Histone | 1, H3h | HISTlH3H | Hs.70937 |
| Hs00171085_m1 | Chemokine (C-X-C motif) |
CXCL5 | Hs.89714 | |
| Hs00173978_m1 | Integrin β 3 (platelet glycoprotein IIIa, antigens c D61) | ITGB3 | Hs.87149 | |
| Hs00415042_m1 | Immunoglobulin (Ig) κ constant region | IGKC | Hs.406565 | |
| Hs00174778_m1 | C type Protein-tyrosine-phosphatase receptor associated protein(RAP) | PTPRCAP | Hs.155979 | |
| Hs00157878_m1 | Granzyme K (serine protease, | GZMK | Hs.3066 | |
| |
|||
| Hs00187842_m1 | Beta-2-microglobulin | B2M | |
| Hs99999903_m1 | Actin muscle, β | ACTB | |
| Hs99999905_m1 | Glyceraldehyde-3-phosphate dehydrogenase | GAPDH | |
| Hs00413854_g1 | Immunoglobulin heavy chain constant region γ 1 (G1m mark) | IGHG1 | |
| Hs00203983_m1 | Mitochondrial ribosomal protein S28 | MRPS28 |
[0152] uses ABI 7900HT sequence detection system (Applied Biosystems, SanFrancisco, CA), in the light reaction plate that 96 holes are sealed fast, in 25 μ l reaction volumes (containing 1X TAQMAN_Fast Universal Master Mix, 1X TAQMAN_ determination of gene expression and 2.25ng cDNA), carry out the quantitative PCR in real time of every kind of target transcript.On each 96 orifice plate, include the only negative control sample of DEPC water (no template contrast in; NTC) and the positive control sample of people leukopack RNA, at each gene specific TAQMAN_ probe and primer sets.The acquiescence the quick closed circulation condition of ABI 7900HT as follows: 95 ℃ 20 seconds; 95 ℃ of 1 second and 60 ℃ of totally 40 circulations in 20 seconds.The classification biomarker of Ce Dinging is listed in table 9 in this way.
| The target gene that table 9. is measured | |
| IgHg1 | Immunoglobulin heavy chain constant region γ 1 (being also referred to as IgHg3) |
| IgKc | Immunoglobulin (Ig) κ constant region |
| 28S | People 28S ribosome-RNA(rRNA) 5 ' district |
| PTP，C-assoc | C type Protein-tyrosine-phosphatase receptor associated protein(RAP) |
| Granzyme K | Granzyme K (serine protease, |
| |
MutL homologue 3 (intestinal bacteria) |
| NGAL-2 | NGAL 2 (oncogene 24p3) |
| CXCL5 | Chemokine (C-X-C motif) |
| Serum is deprived and is replied | Serum is deprived and is replied (phosphatidylserine is conjugated protein) |
| Histone 3K | The H3 histone family, member |
| Integrin β | |
| 3 | Integrin, β 3 (platelet glycoprotein IIIa, antigens c D61) |
| Histone 2BQ | The H3 histone family, member Q |
[0153] acceptable standard is: increase for the gene specific of each target primer to detectable leukopack RNA positive control sample to undetectable NTC sample amplification and (2) for each target primer (1).For in each classification biomarker and 4 house-keeping genes each, write down circulation threshold (Ct) value of each amplified reaction.Being stdn, calculating in target gene in each PBMC sample and 4 house-keeping genes poor (Δ Ct) between each the cycle threshold, and press formula aptly and calculate the average multiple that UC and CD express between expressing and change: average multiple is poor=and 2
(Δ CtUC-Δ CtCD)Or 2
(Δ CtCD-Δ CtUC)
Embodiment 1.8: use the classification prediction that exercises supervision of Q-PCR expression values
[0154] with the parameter (linearity) of discriminatory analysis, distribution free k=3 nearest-neighbor and distribution free k=10 nearest-neighbor classification apportion design be applied to classify in 3 data sets (two alternative random assignments (data set 1 and data set 2) of data set MB (in above embodiment 1.5, describing) and data identical with the test subclass) of biomarker expression level (being cycle threshold) each with the training subclass, each data set all is divided into training subclass and test subclass, each stdn in 4 house-keeping genes that obtained by Q-PCR (describing in above embodiment 1.7) of described classification biomarker expression level.Concentrate in whole 3 data, analyze the rna expression level (43 CD add 19 UC samples and are used for training, and 14 CD add 6 UC samples and are used for test) of 57 Crohn's disease (CD) patients and 25 ulcerative colitiss (UC) patient's PBMC sample.Equally, 3 data sets with the standardized classification biomarker of house-keeping gene 18S expression level are carried out full model, backward, forward and progressively method logic analysis, significance level is made as p=0.05 or p=0.15.Use SAS_8.2 (Gary, NC) and SPOTFIRE_DECISIONSITE
TM8.0 (Somerville, MA) training set of computed in software and match stop and test set tolerance range, sensitivity, specificity, positive desired value (PPV) and negative desired value (NPV).At last, relatively use Δ Ct (at 12 classification biomarkers, cycle threshold stdn by house-keeping gene 18S) ((each all comprises 43 CD that are used to train and adds 19 UC samples the sorter tolerance range that linear discriminant analysis produces at 20 data sets with using identical Δ Ct, and 14 CD that are used to test add 6 UC samples), these 20 data sets are training subclass and test subclass by random assignment) the sorter tolerance range that draws of logic analysis.
Embodiment 2.1: the cell of the purifying PBMC sample of health volunteer, cd patient and patients of ulcerative colitis is formed
[0155] before the expression pattern analysis part of research, detect the sedimentary cell of purifying PBMC of experimenter in whole 3 groups (health volunteer, CD patient and UC patients) and form, carry out RNA afterwards and separate.Table 10 has shown the percentage of basophilic granulocyte, eosinophilic granulocyte, lymphocyte, monocyte and neutrophilic granulocyte in the PBMC sample.
Table 10: IBD patient and the sample number of normal anosis individuality (C) and the mean percentage (%) of cells in sample type of taking from Crohn's disease (CD) or ulcerative colitis (UC) form
| Type | CD | UC | C | C is to IBD p value 1 | CD is to UC p value 1 |
| Sample number | 59 | 25 | 42 | ||
| Basophilic granulocyte (%) | 0.33 | 0.30 | 1.05 | 0.012 | 0.93 |
| Eosinophilic granulocyte (%) | 1.10 | 0.91 | 0.37 | 0.0003 | 0.37 |
| Lymphocyte (%) | 52.20 | 59.58 | 78.90 | ＜0.0001 | 0.056 |
| Monocyte (%) | 29.41 | 27.63 | 14.65 | ＜0.0001 | 0.52 |
| Neutrophilic granulocyte (%) | 14.96 | 10.58 | 5.00 | ＜0.0001 | 0.035 |
1: use the sided t check and based on the p value of the t statistical computation of ANOVA estimation of error.
[0156] when healthier experimenter's PBMC and IBD patient's PBMC, the cell of PBMC sample is formed significantly different (p＜0.05).Basophilic granulocyte and lymphocytic total percentage are significantly lower in IBD patient's PBMC, and the percentage of eosinophilic granulocyte, monocyte and neutrophilic granulocyte significantly promotes in IBD patient's PBMC.Previous research has pointed out that neutrophilic granulocyte promotes through similar purge process, as if this lifting is owing to the change of settled density, and this changes and the neutrophilic granulocyte change relevant (Schmielau and Finn (2001) Cancer Res.61:4756-60) of active state in cancer patients's peripheral blood late.It may be the disease-related activation incident that is captured by the PBMC sepn process based on CPT that the selectivity of eosinophilic granulocyte, monocyte and neutrophilic granulocyte promotes, and does not have significant difference (not produce data) because the cell of whole blood is formed between each group.
[0157] by contrast, basophilic granulocyte, eosinophilic granulocyte and monocyte ratio between CD and UC PBMC sample not remarkable different (p＜0.05).When two IBD groups relatively, have only neutrophilic granulocyte significantly different (11% pair 15%, p=0.035).
Embodiment 2.2: all IBD patients are with respect to the PBMC expression level difference of normal healthy controls
[0158] forms the incoherent disease related gene of obvious difference for differentiating, use covariance analysis (ANCOVA) to differentiate the transcript of differential expression, consider the difference that cell is formed between the PBMC sample simultaneously with cell.To 7908 transcript operation ANCOVA that express horizon-lai filters by standard, the percentage of including eosinophilic granulocyte, monocyte and neutrophilic granulocyte in is as the co-variation amount.
[0159] select which kind of cell type to include in partly by following true decision: the co-variation amount among the ANCOVA is height correlation not each other.Lymphocyte percentage and monocyte percentage and the strong negative correlation of neutrophilic granulocyte percentage are not included among the ANCOVA for this reason.To every kind of cell type, calculate the slope of describing the linear relationship between cell type percentage and the specific gene expression level, and carry out t check, whether notable difference (wherein, not having linear relationship between slope 0 expression cell type percentage and the expression level) is arranged to determine slope with 0.At last, because being distributed in relatively between the disease group of the women and the male sex is also obviously different,, appear as sex-specific rather than the transcript relevant with the patient's condition with evaluation so include sex in ANCOVA.
[0160] analyzes by ANCOVA, the level of 220 transcripts has the difference above 1.5 times between Crohn's disease and healthy PBMC, and in paired comparison, has a unadjusted p value less than 0.0001 based on ANCOVA, use as above identical standard, there were significant differences between UC and healthy PBMC for the level of 120 transcripts.These sequences have 45 to compare with health level in UC and the different expression of CD PBMC mean deviation, and these common PBMC-and IBD-associated retroviral thing change (going up table 1) with equidirectional in these two kinds of diseases.
[0161] several groups of genes of remainder is used a kind of other strainer, to identify the only PBMC transcript of variant expression in a kind of illness.In 220 relevant transcripts of CD (〉=1.5 times of variations, p＜0.0001), always have 67 sequences and significantly change (p＞0.05) in to the comparison of healthy person at UC PBMC, it is specific therefore to be counted as CD.67 CD specificity PBMC sequences, promptly the CD biomarker is listed in above table 2.In the relevant transcript of 120 UC (〉=1.5 times of variations, p＜0.0001), always have 22 sequences and significantly change (p＞0.05) in to the comparison of healthy person at CDPBMC, it is specific therefore to be counted as UC.22 UC specificity PBMC sequences, promptly the UC biomarker is listed in above table 3.
[0162] in Figure 1A, summed up the classical gene approach of maximum likelihood with remarkable overexpression at every kind of contrast.In this is analyzed, relate to the remarkable overexpression CD of the transcript gene label of the classical classification of prostaglandin metabolism, and coding relates to the proteic transcript of the classical classification of apoptosis and B cell signalling and seems overexpression UC gene label.Figure 1B has summed up the difference in functionality classification that comprises with respect to normal healthy controls transcript of differential expression in Crohn's disease.The major function classification that is raised in CD PBMC comprises enzyme, transcriptional and the transmembrane receptor that participates in prostaglandin metabolism, comprises several integrin isotypes.At last, Fig. 1 C has summed up the overexpression that enriches of constant region for immunoglobulin, and this overexpression is that the UCPBMC gene expression label is distinctive.
Embodiment 2.3: differentiate the gene label of distinguishing Crohn's disease and ulcerative colitis
[0163] because the major objective of this research is to determine gene expression pattern among CD and UC patient's the PBMC different must being enough to can be based on the classification of the gene expression profile among the independent PBMC, so the gene expression label between these two kinds of diseases is directly contrasted.The ANCOVA that CD composes UC PBMC relatively identifies 49 transcripts and have significant difference level (〉=1.5 times of differences, p＜0.0001) between CD and UC patient's PBMC.These CdvUC biomarkers are listed in above table 4.
[0164], use supervision classification prediction procedure to differentiate the minimal information sequence set that to carry out the disease specific classification based on the ANCOVA result of the direct correlated significant difference of indicating CD and UC PBMC gene label.Be the training set formed by 44 CD and 22 UC spectrums with IBD patient's PBMC sample randomization and compose the test sets of forming by 15 CD and 6 UC.Increase big or small gene sorter to one group and measured relative resultnat accuracy, CD nicety of grading and UC nicety of grading (Fig. 2 A).As shown in Fig. 2 A, according to the evaluation of 4 times of cross validations, one group that is made up of two gene sorters (being NGAL 2 and IgHg3) provides 64% tolerance range (Fig. 2 A).According to the evaluation (Fig. 2 A) of 4 times of cross validations of training set, the minimum predictive model with the highest overall accuracy (91%) of distinguishing UC and CD PBMC spectrum is 14-sequence (12-gene) sorter (above table 5).According to the evaluation of staying a cross validation, 14-sequence sorter has 94% overall accuracy (not produce data).Gene sorter in the table 5 is arranged with the descending order of signal to noise ratio; Promptly in cd patient, raised and be listed in the classification biomarker of table 5, NGAL 2 (the 1st sorter gene) has highest signal to noise ratio, integrin β-3 (the 7th sorter gene) has lowest signal-to-noise, in patients of ulcerative colitis, raised and be listed in the classification biomarker of table 5, IgHg1 (the 8th sorter gene) has highest signal to noise ratio, and IgKc (the 14th sorter gene) has lowest signal-to-noise.The size that increases set of classifiers does not increase to tolerance range this more than level (Fig. 2 A).This 12-gene sorter is used for that the classification member is distributed to 14 CD spectrums and 6 UC of keeping to test set and composes (Fig. 2 B).Use this predictive model, all samples in the test set all as the situation of clinical diagnosis quilt correctly classify.Use in every group of this sorter and have only the degree of confidence of 1 individuality to be lower than 0.2, show with high relatively degree of confidence and make these judgements by weighted voting algorithm.These results show that the potential practicality of use PBMC express spectra helps the molecular diagnosis of CD and UC.
Embodiment 2.4: quantitatively reverse transcriptase-polymerase chain reaction (Q-PCR) confirms the microarray observed result in real time
[0165] no matter by the set of classifiers tolerance range of the expression level data centralization that obtains in the microarray analysis based on the distribution of nearest-neighbor classification, the average multiple of transcript changes relatively low in the CD/UC sorter.Therefore, carry out quantitative PCR in real time (Q-PCR), with the relative expression who confirms in this research CD and UC sample to be observed by the Affymetrix microarray technology.Use 4 independent house-keeping genes that are used for the stdn target gene: B2M (β 2M), beta-actin, GAPDH and 18S ribosome-RNA(rRNA) (18S).All CD in the research use identical reverse transcription mixture and method to change cDNA into UC RNA sample.The average multiple that uses B2M to calculate by microarray and PCR in real time changes relatively is shown in Fig. 3, under each situation as the stdn thing in using 4 house-keeping genes, the relative multiple of whole 12 branch genoids changes very consistent (table 11).
| Table 11: the relative multiple after the stdn changes | |||||||||||
| In UC, raise (with respect to CD) | |||||||||||
| Check | Stdn | IgHg1 | IgKc | 28S | PTP， C-assoc | Granzyme K | |||||
| Affy | The frequency of amplifying | 3.87 | 2.30 | 2.11 | 1.43 | 1.35 | |||||
| Q-PCR | β2M | 3.11 | 2.04 | 1.32 | 1.47 | 1.85 | |||||
| Beta-actin | 3.05 | 2.00 | 1.30 | 1.44 | 1.81 | ||||||
| GAPDH | 2.83 | 1.86 | 1.21 | 1.34 | 1.69 | ||||||
| 18S | 2.68 | 1.98 | 1.31 | 1.40 | 1.86 | ||||||
| In CD, raise (with respect to UC) | |||||||||||
| Check | Stdn | mutL3 | NGAL-2 | CX CL5 | Serum is deprived and is replied | Histone 3K | Integrin β-3 | Histone 2BQ | |||
| Affy | The frequency of amplifying | 2.01 | 1.75 | 1.85 | 1.66 | 1.65 | 1.62 | 1.57 | |||
| Q-PCR | β2M | 1.93 | 1.84 | 2.28 | 1.49 | 1.32 | 1.49 | 1.27 | |||
| Beta-actin | 1.97 | 1.88 | 2.33 | 1.52 | 1.35 | 1.52 | 1.29 | ||||
| GAPDH | 2.12 | 2.02 | 2.50 | 1.64 | 1.45 | 1.64 | 1.39 | ||||
| 18S | 2.13 | 1.96 | 2.44 | 1.59 | 1.47 | 1.61 | 1.36 | ||||
[0166], distinguished in the transcript of gene (biomarker of promptly classifying) to have only 28S rRNA fragment to seem significantly to be over-evaluated for CD/UC by discriminating at first by microarray hybridization at 12 based on these results.
Embodiment 2.5: use the expression values that obtains by Q-PCR to carry out accurate classification prediction
[0167] will be with 4 house-keeping gene (β 2M, beta-actin, GAPDH and 18S) in the linear discriminant analysis (LDA) and 3 data set (data set MB of Δ Ct of each standardized 12 classification biomarker (see Table 5 and table 8), data set 1, data set 2) the k-NN discriminatory analysis of identical Δ Ct is compared, these 3 data sets comprise in the training set from isolating 43 the PBMC RNA samples of CD patient with from isolating 19 the PBMCRNA samples of UC patient, and in the test set from isolating 14 the PBMC RNA samples of CD patient with from isolating 6 the PBMC RNA samples of UC patient.
[0168] table 13 has shown tolerance range, sensitivity, specificity, PPV and the NPV detected result to the classification performance of parameter (linearity), distribution free k=3 nearest-neighbor or the distribution free k=10 nearest-neighbor discriminatory analysis method of 3 data sets (data set MB, data set 1 and data set 2) of the expression level of each the standardized classification biomarker in 4 special (B2M (β 2M), beta-actin, GAPDH and 18S ribosome-RNA(rRNA) (18S)) genes.
| Table 13: | |||||||
| House-keeping gene | Data set | Method | Tolerance range | Sensitivity | Specificity | PPV | NPV |
| β2M | Data set MB | Parameter | 0.833 | 1.000 | 0.667 | 0.875 | 1.000 |
| β2M | Data set MB | K-NN k＝3 | 0.762 | 0.857 | 0.667 | 0.857 | 0.667 |
| β2M | Data set MB | K-NN k＝10 | 0.750 | 1.000 | 0.500 | 0.824 | 1.000 |
| Beta-actin | Data set MB | Parameter | 0.833 | 1.000 | 0.667 | 0.875 | 1.000 |
| Beta-actin | Data set MB | K-NN k＝3 | 0.798 | 0.929 | 0.667 | 0.867 | 0.800 |
| Beta-actin | Data set MB | K-NN k＝10 | 0.750 | 1.000 | 0.500 | 0.824 | 1.000 |
| GAPDH | Data set MB | Parameter | 0.750 | 1.000 | 0.500 | 0.824 | 1.000 |
| GAPDH | Data set MB | K-NN k＝3 | 0.762 | 0.857 | 0.667 | 0.857 | 0.667 |
| GAPDH | Data set MB | K-NN k＝10 | 0.750 | 1.000 | 0.500 | 0.824 | 1.000 |
| 18S | Data set MB | Parameter | 0.917 | 1.000 | 0.833 | 0.933 | 1.000 |
| 18S | Data set MB | K-NN k＝3 | 0.845 | 0.857 | 0.833 | 0.923 | 0.714 |
| 18S | Data set MB | K-NN k＝10 | 0.917 | 1.000 | 0.833 | 0.933 | 1.000 |
| β2M | Data set 1 | Parameter | 0.964 | 0.929 | 1.000 | 1.000 | 0.857 |
| β2M | Data set 1 | K-NN k＝3 | 0.821 | 0.643 | 1.000 | 1.000 | 0.545 |
| β2M | Data set 1 | K-NN k＝10 | 0.798 | 0.929 | 0.667 | 0.867 | 0.800 |
| Beta-actin | Data set 1 | Parameter | 0.964 | 0.929 | 1.000 | 1.000 | 0.857 |
| Beta-actin | Data set 1 | K-NN k＝3 | 0.821 | 0.643 | 1.000 | 1.000 | 0.545 |
| Beta-actin | Data set 1 | K-NN k＝10 | 0.845 | 0.857 | 0.833 | 0.923 | 0.714 |
| GAPDH | Data set 1 | Parameter | 0.929 | 0.857 | 1.000 | 1.000 | 0.750 |
| GAPDH | Data set 1 | K-NN k＝3 | 0.786 | 0.571 | 1.000 | 1.000 | 0.500 |
| GAPDH | Data set 1 | K-NN k＝10 | 0.845 | 0.857 | 0.833 | 0.923 | 0.714 |
| 18S | Data set 1 | Parameter | 0.845 | 0.857 | 0.833 | 0.923 | 0.714 |
| 18S | Data set 1 | K-NN k＝3 | 0.810 | 0.786 | 0.833 | 0.917 | 0.625 |
| 18S | Data set 1 | K-NN k＝10 | 0.845 | 0.857 | 0.833 | 0.923 | 0.714 |
| β2M | Data set 2 | Parameter | 0.774 | 0.714 | 0.833 | 0.909 | 0.556 |
| β2M | Data set 2 | K-NN k＝3 | 0.738 | 0.643 | 0.667 | 0.818 | 0.444 |
| β2M | Data set 2 | K-NN k＝10 | 0.881 | 0.929 | 0.833 | 0.929 | 0.833 |
| Beta-actin | Data set 2 | Parameter | 0774 | 0.714 | 0.833 | 0.909 | 0.556 |
| Beta-actin | Data set 2 | K-NN k＝3 | 0.702 | 0.571 | 0.833 | 0.889 | 0.455 |
| Beta-actin | Data set 2 | K-NN k＝10 | 0.762 | 0.857 | 0.667 | 0.857 | 0.667 |
| GAPDH | Data set 2 | Parameter | 0.810 | 0.786 | 0.833 | 0.917 | 0.625 |
| GAPDH | Data set 2 | K-NN k＝3 | 0.667 | 0.500 | 0.833 | 0.875 | 0.417 |
| GAPDH | Data set 2 | K-NN k＝10 | 0.810 | 0.786 | 0.833 | 0.917 | 0.625 |
| 18S | Data set 2 | Parameter | 0.845 | 0.857 | 0.833 | 0.923 | 0.714 |
| 18S | Data set 2 | K-NN k＝3 | 0.810 | 0.786 | 0.833 | 0.917 | 0.625 |
| 18S | Data set 2 | K-NN k＝10 | 0.845 | 0.857 | 0.833 | 0.923 | 0.714 |
[0169] differentiate that classification is the most successful to data set MB, irrelevant with the method for using, point out the performance of every kind of method and each data set relevant (table 13) of analysis.Parameter is similar with distribution free k=10 nearest-neighbor method effect, but on an average these two kinds than distribution free k=3 nearest-neighbor method more successful (table 13).
[0170] goes out system's difference of performance 3 data centralized displaying with the standardized classification biomarker of 18S.18S is better than other house-keeping gene to a certain extent; Analysis with the standardized classification biomarker of 18S has higher value and less mutability (table 13) all the time aspect tolerance range, sensitivity and the specificity.
[0171], so the expression level with the standardized classification biomarker of 18S is only carried out logic analysis because 18S seems to be better than the house-keeping gene of other test.At the full model of logic analysis, backward, forward or progressively select in the back-and-forth method to almost not influence (not produce data) of classification.For data set MB and data set 1, the full model method is finished to such an extent that be equal to or is better than other simplifying model (not produce data).With regard to data set 2, select with the significance level that is set at p=0.05 by method forward or progressively, adopt the model of 18S standardized 2 or 3 kind of classification biomarker (comprise MLH3 and IgKC these two) to have classification prediction (not produce data) preferably.This possibility of result is owing to the following fact: the most of biomarker with atypia expression level is included in the test set of data set 2, and forward or the model of progressively selecting do not contain the biomarker of those unconventionality expressions.Being included in this variation in the biomarker, can also to be interpreted as what full model be not best actuating logic method, and why carry out discriminatory analysis with the model that comprises whole 12 sorters and produce relatively poor classification.Tolerance range, sensitivity and the specificity of the different choice method of logic analysis shows that the Different Logic system of selection similarly finishes (not produce data).For example, if the test set that a kind of method is compared data centralization to training set demonstrates better classification, vice versa, and then the result with whole 3 kinds of other methods demonstrates identical characteristic.
[0172] then, the classification that will be undertaken by the logic analysis of using full model compares with the classification that linear discriminant analysis by the operation parameter method carries out.Although observe the data set difference between these two kinds of methods, the classification prediction is all accurately carried out in these two kinds of analyses.Use data set MB, data set 1 and data set 2 carry out the tolerance range of classification prediction generating 0.833-0.917, the specificity (table 14) of the sensitivity of 0.786-1.000 and 0.667-1.000.But, when 20 data sets relatively between these two kinds of methods as if variant (Fig. 4).Logic analysis with full model is finished better to cross validation, and discriminatory analysis is done better to test set classification.
Table 14
| Data set | Analytical procedure | Sensitivity | Specificity | PPV | NPV | Tolerance range |
| MB | Differentiate | 0.786 | 1.000 | 1.000 | 0.667 | 0.893 |
| MB | Logic | 1.000 | 0.667 | 0.875 | 1.000 | 0.833 |
| |
Differentiate | 0.857 | 0.833 | 0.923 | 0.714 | 0.845 |
| |
Logic | 1.000 | 0.833 | 0.933 | 1.000 | 0.917 |
| |
Differentiate | 0.786 | 1.000 | 1.000 | 0.667 | 0.893 |
| |
Logic | 0.857 | 0.833 | 0.923 | 0.714 | 0.845 |
Discuss
[0173] focus of this research is that the versatility and the specificity of attempting the gene expression pattern among definite (1) PBMC relevant with CD and UC expressed the molecular diagnosis whether label can help disease with (2) disease specific.Compare with health volunteer's express spectra, have the dozens of gene to seem differential expression in CD and UC patient's the express spectra.Many coding nucleoprotein are arranged, for example transcriptional, and most of the downward modulation in these genes.Example comprises NFKB2, RNA-binding factor CUGBP1 and CUGBP2, COPEB and ELK3.UC and CD common imbalance inflammatory process may be the active results that regulate of these transcriptional.
The gene of the high expression level that [0174] usually is raised in these two kinds of inflammatory bowels is proteinase inhibitor SERPINB2 (also being called PAI, Type 1 plasminogen activator inhibitor, II type).Reported the plasminogen activator level (deBruin etc., (1988) Thromb.Haemost.60:262-66) that increases in IBD patient's mucous membrane focus, the PAI-1 of increase is found in IBD patient's blood plasma.Although different with PAI-1, PAI-2 and u-PA have enzyme spcificity, and with the total enzyme spcificity of t-TA than low degree, in the rheumatoid arthritis synovia, reported the PAI-2 level (Kruithof etc., (1995) Blood 86:4007-24) that promotes.These results suggest, the change of the component of haemolysis fibre and condensed system may impel the thromboembolic complication risk to increase, and may facilitate visible colitis and hemorrhage (de Jong etc., (1989) Gut 30:188-94) in IBD patient.Also do not report the effect of PAI-2 in IBD, but this research prompting, the PAI-2RNA level and the disease-related that promote among the PBMC.
[0175] transcript of multiple functional category seems to be raised by specificity in CD patient's PBMC, comprises prostaglandin metabolism enzyme, chemokine and transcriptional.CD specificity PBMC gene profile shows unconspicuous short scorching gene expression profile in UC specificity PBMC gene profile.Participate in the metabolic gene of Prostaglandins and Leukotrienes, for example Arachidonate 12-lipoxygenase (ALOX12) and prostaglandin endoperoxide synthase 1 (PTGS1, cyclo-oxygenase 1), in CD patient's PBMC, significantly increase, and prostaglandin d 2 synthase (PTGDS) descends.Expection should cause arachidonic acid to increase to the transformation of selective prostaglandins to these effects of prostaglandin(PG) route of synthesis.Although the prostaglandin(PG) content in IBD patient's focus promotes (Schmidt etc., (1996) Hepatogastroenterology 43:1508-12), evidence prompting recently, in fact at least a prostaglandin(PG) (PGE
2) level in CD patient's monocyte, descend (Trebble etc., (2004) Clin.Nutr.23:647-55).Whether the relative lifting of the transcript of the not clear arachidonic acid metabolism enzyme of encoding in CD patient's PBMC is relevant with this observations on function, but PGE
2Being turned out to be by file is that a kind of origin comes from the important instrumentality of cytokine (Barrera etc., (1996) J.Cell.Physiol 166:130-37) that GI T lymphocyte discharges.The rise of the PG pathways metabolism in the cycle P BMC of cd patient may be illustrated in this disease/go out the change of the cell of intestinal lamina propria.
[0176] several chemokines ( C-X-C part 4 and 7, platelet factor 4 variant 1) are raised in CD.Generally speaking, in organizing, the CD of this paper PBMC differentiated to be that transcript that raises and those transcripts that are reported as rise in 7 CD patients of Mannick and colleague (Mannick etc., ibid) analysis almost do not have overlapping astoundingly.Do not know that this is whether bigger owing to patient's number of this paper research number gene bigger, inquiry, between unnamed gene difference or these research some obscure factor.But, Mannick and colleague report in CD by transcript coding transforming growth factor (the TGF)-beta induced type transcript (Mannick etc., ibid) of strong rise.At this, the beta induced type transcript of a kind of different TGF-TSC-22 is also differentiated to be raised in CD PBMC.As if these observationss show that the rise of TGF-signal transduction is obvious in CD PBMC.The composing type of this approach promotes and can cause the downward modulation of Smad-dependent pathway, and this can cause the activity inhibited of TGF-β subsequently, with the termination immunne response, and plays cause of disease effect (Mannick etc., ibid) again in the CD morbidity.
[0177] a part of Crohn's disease relative disease label gene profile might be platelet-derived.Nearest evidence shows that thrombocyte can participate in chronic intestinal inflammations (Danese etc., (2004) Am.J.Gastroenterol.99:938-45), this research in thrombocyte with from the isolating PBMC of CD patient by copurification to higher degree (not produce data).Therefore, the copurification platelet levels that the detected result of platelet factor 4 and platelet factor 4 variant 1 promotes in may be owing to isolating PBMC in CD genes involved label.But, other transcript (Gnatenko etc. in preceding 10 non-plastosome transcripts of in thrombocyte, reporting, (2003) Blood 101:2285-93) do not appear in the CD associated retroviral thing tabulation of this paper, pointing out these akaryotic levels is not unique source of these transcripts.All transcripts in before related with the thrombocyte CD disease label are also expressed (not produce data) with conspicuous level in the T of purifying cell, B cell and/or monocyte, the previous transcript relevant with thrombocyte of prompting can be derived from the monocyte that separates and carry out expression pattern analysis in this research.
[0178] UC specific gene group becomes dominance by the overexpression immunoglobulin coding sequence, makes the people associate observed active IgG plasmocyte component (Farrell etc., (2002) Lancet 359:331-40) in UC patient.This result is with consistent at the research of B-cell receptor gene purposes, and this research has shown that the lymphocyte infiltration in the UC mucous membrane is the periphery source, rather than (Dunn-Walters etc., (1999) the Gut 44:382-86 in mucous membrane source; Thoree etc., (2002) Gut 51:44-50).IgG1 and IgG4 antibody have superiority in UC, and IgG2 antibody increases (Kett and Brandtzaeg (1987) Gut 28:1013-21) in CD.Studied the popular of IgG1 type recently, shown that it is that UC is specific, (Furrie etc., (2004) Gut 53:91-98) kept in the mucous membrane bacterium opsonization that generation is bigger in UC and the feedforward of polymorphonuclear leukocyte respiratory burst.In this research, in UC PBMC, annotated for constant region for immunoglobulin γ 3 (IgHG3) by wherein a kind of transcript of the most remarkable lifting.(promptly sharing 100% Nucleotide identity according to BLAST) mapped to the several sequences that belong to immunoglobulin heavy chain constant region γ 1 (G1m mark) in the zone that this IgHG3 eligible comprises on the Affymetrix chip in fact, and differentiated for the mark of inflammation UC gastrointestinal epithelial (Warner and Dieckgraefe, ibid; Lawrance etc., ibid).The analysis of the IgHG3 transcript expression level in individual patient peripheral blood spectrum can be used as the distinctiveness biomarker (not produce data) between UC and the CD.But, these results also with UC patient in serum IgG 1 level with respect to the previous observations consistent (Gouni-Berthold etc., (1999) Hepatogastroenterology 46:1720-23) of the remarkable increase of 1 level of the serum IgG among the CD patient.
[0179] quite a few patient who suffers from inflammatory bowel can not classify according to current methods, constitutes " determining type IBD " case (Winther etc., (1998) Drugs Today (Bare) .34:935-42; Bentley etc., (2002) J.Clin.Pathol55:955-60; Guindi and Riddell (2004) J.Clin.Pathol.57:1233-44).Therefore, this research main purpose is to determine PBMC spectrum among UC and the CD patient different must being enough to can be with these classifications of diseases.The result of classification forecast analysis shows that the gene label among the PBMC can accurately be distinguished UC and CD sample.Transcriptional differences is not to form because of cell, seems quite similar because the cell of patient's PBMC is formed.
[0180] although should carry out preceding checking in than large group, the disease specific pattern that the present invention differentiates can provide the molecular diagnosis basis of UC and CD, and can help to be categorized as the diagnosis that trouble is not determined the patient of type IBD.Very possiblely be, the Th1 of CD of Ti Chuing and UC and Th2 character are to cause the major cause of difference in this research respectively, the similar label of those labels that other inflammatory diseases based on Th1 and Th2 may have and CD and UC are differentiated.However, as if the PBMC spectrum that this paper differentiates still has clinical application in IBD, because described gene sorter can distinguish that these often are difficult to the closely-related disease of distinguishing and can't distinguishing sometimes.
Originally studies show that [0181] with regard to IBD, transcribing in circulating monocytic cell, T cell and the B cell composed the sensitivity monitoring thing that can be used as the biological physiology state.Because these cells come and go in various tissues and move, so the cell response integral part at microenvironment is a responsive transcription; This reaction can be quantitative by analyzing express spectra.Expression pattern can reflect at the primary of disease physiopathology or the disease mechanisms of secondary response.Because PBMC runs through body transportation, so it can be used as the alternative monitoring thing that easily obtains that is difficult for the tissue measured and system (for example IBD involve tissue and system).
Claims (50)
1. method of diagnosing patient's inflammatory bowel said method comprising the steps of:
A. from patient's sample separation; With
B. the normal or unconventionality expression of at least a PBMC relevant biomarkers and IBD relevant biomarkers in the test sample,
The unconventionality expression of wherein at least a PBMC relevant biomarkers and IBD relevant biomarkers illustrates that this patient might suffer from inflammatory bowel.
2. the process of claim 1 wherein that described sample is the gleanings of peripheral blood lymphocytes.
3. the process of claim 1 wherein described detection step use based on hybridization mensuration carry out.
4. the process of claim 1 wherein that described detection step carries out with immunologic assay.
5. the process of claim 1 wherein that described detection step carries out with polymerase chain reaction.
6. the method for claim 5, wherein said polymerase chain reaction is a quantitative polyase chain reaction.
7. the process of claim 1 wherein that described detection step detects the expression of one group of PBMC relevant biomarkers and IBD relevant biomarkers.
8. the method for claim 7, wherein said one group of PBMC relevant biomarkers and IBD relevant biomarkers comprise at least a common biomarker.
9. the method for claim 8, wherein said one group of PBMC relevant biomarkers and IBD relevant biomarkers comprise at least a I group biomarker.
10. the method for claim 9, wherein said one group of PBMC relevant biomarkers and IBD relevant biomarkers comprise PAI-2.
11. the method for claim 7, wherein said one group of PBMC relevant biomarkers and IBD relevant biomarkers comprise at least a CD biomarker.
12. the method for claim 11, wherein said one group of PBMC relevant biomarkers and IBD relevant biomarkers comprise at least a II group biomarker.
13. the method for claim 11, wherein said one group of PBMC relevant biomarkers and IBD relevant biomarkers comprise ALOX12.
14. the method for claim 11, wherein said one group of PBMC relevant biomarkers and IBD relevant biomarkers comprise PTGDS.
15. the method for claim 11, wherein said one group of PBMC relevant biomarkers and IBD relevant biomarkers comprise NGAL 2.
16. the method for claim 7, wherein said one group of PBMC relevant biomarkers and IBD relevant biomarkers comprise at least a UC biomarker.
17. the method for claim 16, wherein said one group of PBMC relevant biomarkers and IBD relevant biomarkers comprise at least a III group biomarker.
18. the method for claim 17, wherein said one group of PBMC relevant biomarkers and IBD relevant biomarkers comprise IgHG3.
19. the method for claim 7, wherein said one group of PBMC relevant biomarkers and IBD relevant biomarkers comprise at least a CDvUC biomarker.
20. the method for claim 19, wherein said one group of PBMC relevant biomarkers and IBD relevant biomarkers comprise at least a IV group biomarker.
21. the method for claim 7, wherein said one group of PBMC relevant biomarkers and IBD relevant biomarkers comprise at least a classification biomarker.
22. the method for claim 21, wherein said one group of PBMC relevant biomarkers and IBD relevant biomarkers comprise at least a V group biomarker.
23. comprising, the method for claim 7, wherein said one group of PBMC relevant biomarkers and IBD relevant biomarkers be selected from following biomarker group: I group biomarker group, II group biomarker group, III group biomarker group, IV group biomarker group and V group biomarker group.
24. a method of diagnosing patient's ulcerative colitis said method comprising the steps of:
A. from patient's sample separation; With
B. the normal or unconventionality expression of at least a ulcerative colitis relevant biomarkers in the test sample,
The unconventionality expression of wherein at least a ulcerative colitis relevant biomarkers illustrates that this patient might suffer from ulcerative colitis.
25. the method for claim 24, wherein said sample are the gleanings of peripheral blood lymphocytes.
26. the method for claim 24, wherein at least a ulcerative colitis relevant biomarkers are selected from PBMC relevant biomarkers and the IBD relevant biomarkers that is categorized as III group biomarker.
27. the method for claim 26, wherein at least a ulcerative colitis relevant biomarkers comprises IgHG3.
28. using based on the mensuration of hybridization, the method for claim 24, wherein said detection step carry out.
29. the method for claim 24, wherein said detection step is carried out with immunologic assay.
30. the method for claim 24, wherein said detection step is carried out with polymerase chain reaction.
31. the method for claim 30, wherein said polymerase chain reaction is a quantitative polyase chain reaction.
32. the method for claim 24, wherein said detection step detects the expression of one group of PBMC relevant biomarkers and IBD relevant biomarkers.
33. a method of diagnosing patient's Crohn's disease said method comprising the steps of:
A. from patient's sample separation; With
B. the normal or unconventionality expression of at least a Crohn's disease relevant biomarkers in the test sample,
The unconventionality expression of wherein at least a Crohn's disease relevant biomarkers illustrates that this patient might suffer from Crohn's disease.
34. the method for claim 33, wherein said sample are the gleanings of peripheral blood lymphocytes.
35. the method for claim 33, wherein at least a Crohn's disease relevant biomarkers are selected from PBMC relevant biomarkers and the IBD relevant biomarkers that is categorized as II group biomarker.
36. using based on the mensuration of hybridization, the method for claim 33, wherein said detection step carry out.
37. the method for claim 33, wherein said detection step is carried out with immunologic assay.
38. the method for claim 33, wherein said detection step is carried out with polymerase chain reaction.
39. the method for claim 38, wherein said polymerase chain reaction is a quantitative polyase chain reaction.
40. the method for claim 33, wherein said detection step detects the expression of one group of PBMC relevant biomarkers and IBD relevant biomarkers.
41. a method of distinguishing patient's ulcerative colitis diagnostic result and Crohn's disease diagnostic result said method comprising the steps of:
A. from patient's sample separation; With
B. the normal or unconventionality expression of at least a classification biomarker in the test sample,
Wherein at least aly illustrate that with the unconventionality expression of differentiating the classification biomarker that cd patient is relevant this patient might suffer from Crohn's disease, perhaps
Wherein at least aly illustrate that with the unconventionality expression of differentiating the classification biomarker that patients of ulcerative colitis is relevant this patient might suffer from ulcerative colitis.
42. the method for claim 41, wherein said sample are the gleanings of peripheral blood lymphocytes.
43. the method for claim 41, wherein at least a classification biomarker are selected from the classification biomarker that is categorized as V group biomarker.
44. using based on the mensuration of hybridization, the method for claim 41, wherein said detection step carry out.
45. the method for claim 41, wherein said detection step is carried out with immunologic assay.
46. the method for claim 41, wherein said detection step is carried out with polymerase chain reaction.
47. the method for claim 46, wherein said polymerase chain reaction is a quantitative polyase chain reaction.
48. the method for claim 41; wherein said detection step comprises the normal or unconventionality expression of a group categories biomarker in the test sample; a wherein said group categories biomarker comprises immunoglobulin heavy chain constant region γ 1; immunoglobulin (Ig) κ constant region; people 28S ribosome-RNA(rRNA) 5 ' district; C type Protein-tyrosine-phosphatase receptor associated protein(RAP); granzyme K; mutL homologue 3; NGAL 2; CXCL5; serum deprives that to reply phosphatidylserine conjugated protein; H3 histone family member K; integrin β 3 (platelet glycoprotein IIIa, antigens c D 61) and H2B histone family member Q biomarker.
49. the method for claim 41; wherein said detection step comprises the normal or unconventionality expression of a group categories biomarker in the test sample; a wherein said group categories biomarker comprises at least 2 kinds and is selected from following classification biomarker: immunoglobulin heavy chain constant region γ 1; immunoglobulin (Ig) κ constant region; people 28S ribosome-RNA(rRNA) 5 ' district; C type Protein-tyrosine-phosphatase receptor associated protein(RAP); granzyme K; mutL homologue 3; NGAL 2; CXCL5; serum deprives that to reply phosphatidylserine conjugated protein; H3 histone family member K; integrin β 3 (platelet glycoprotein IIIa, antigens c D 61) and H2B histone family member Q biomarker.
50. the method for claim 41; wherein said detection step comprises the normal or unconventionality expression of a group categories biomarker in the test sample; a wherein said group categories biomarker comprises at least 8 kinds and is selected from following classification biomarker: immunoglobulin heavy chain constant region γ 1; immunoglobulin (Ig) κ constant region; people 28S ribosome-RNA(rRNA) 5 ' district; C type Protein-tyrosine-phosphatase receptor associated protein(RAP); granzyme K; mutL homologue 3; NGAL 2; CXCL5; serum deprives that to reply phosphatidylserine conjugated protein; H3 histone family member K; integrin β 3 (platelet glycoprotein IIIa, antigens c D 61) and H2B histone family member Q biomarker.
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Cited By (5)
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| CN104583759A (en) * | 2012-03-22 | 2015-04-29 | 银诺维姆科学组织 | New method of dynamic spectroscopy under physiological conditions |
| CN107208159A (en) * | 2015-01-30 | 2017-09-26 | 恩特姆生物科学公司 | Host DNA as Crohn's disease biomarker |
| CN108780088A (en) * | 2015-11-06 | 2018-11-09 | 维蒂卡实验室公司 | The method for detecting marker of inflammation in companion animals and treating inflammatory conditions |
| CN112156111A (en) * | 2020-08-31 | 2021-01-01 | 中南大学湘雅三医院 | Application of cord blood platelet mitochondria in preparation of medicine for treating autoimmune disease |
| WO2024060326A1 (en) * | 2022-09-22 | 2024-03-28 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Antibody combination and system for detecting prognosis of nasopharyngeal cancer, and use |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104583759A (en) * | 2012-03-22 | 2015-04-29 | 银诺维姆科学组织 | New method of dynamic spectroscopy under physiological conditions |
| US9709486B2 (en) | 2012-03-22 | 2017-07-18 | Inoviem Scientific | Method of dynamic spectroscopy under physiological conditions |
| CN107208159A (en) * | 2015-01-30 | 2017-09-26 | 恩特姆生物科学公司 | Host DNA as Crohn's disease biomarker |
| CN108780088A (en) * | 2015-11-06 | 2018-11-09 | 维蒂卡实验室公司 | The method for detecting marker of inflammation in companion animals and treating inflammatory conditions |
| CN112156111A (en) * | 2020-08-31 | 2021-01-01 | 中南大学湘雅三医院 | Application of cord blood platelet mitochondria in preparation of medicine for treating autoimmune disease |
| CN112156111B (en) * | 2020-08-31 | 2022-10-18 | 中南大学湘雅三医院 | Application of cord blood platelet mitochondria in preparation of medicine for treating autoimmune disease |
| WO2024060326A1 (en) * | 2022-09-22 | 2024-03-28 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Antibody combination and system for detecting prognosis of nasopharyngeal cancer, and use |
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