CN105353132B - Applications of the non B s IgG as ancestral cells mark - Google Patents
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- CN105353132B CN105353132B CN201510776518.9A CN201510776518A CN105353132B CN 105353132 B CN105353132 B CN 105353132B CN 201510776518 A CN201510776518 A CN 201510776518A CN 105353132 B CN105353132 B CN 105353132B
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
- G01N33/686—Anti-idiotype
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/5743—Specifically defined cancers of skin, e.g. melanoma
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/02—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates involving antibodies to sugar part of glycoproteins
Abstract
The invention discloses the sialylated IgG in non-B cell source（non B‑sIgG）As the application of ancestral cells mark, antineoplastic is prepared especially as tumour ancestral cells therapy target, sialylated glycosyl glycosylates not related with O on the IgG.As an application aspect, antibody, immunocyte, albumen and the small molecule in the specific recognition non B sIgG and its sialylated glycosyl site are capable of by preparing can easily detect the distribution of this kind of cell.Particularly tumour ancestral cells, design can effectively control the diagnosis, treatment and prognosis evaluation of this kind of tumour, clinically there is huge application value for the detection reagent or targeted drug of the specific molecular.
Description
Technical field
The present invention relates to biological immunology technical field, and in particular to the sialylated IgG and the IgG in non-B cell source
Application of the sialylated glycosyl site as ancestral cells mark.
Background technology
Traditional immunology concept thinks immunoglobulin（Immunogloubin, Ig）To be only limitted to B lymphs thin in source
Born of the same parents and thick liquid cell, due to the gene rearrangement of Ig gene functions does not occur in other body cells, therefore the generation of Ig molecules are not had.
However, Qiu Xiao men of virtue and ability seminar once at home and abroad reported that non-B cell, particularly tumour cell can also produce Ig first.At present
Clearly, in body except classical Ig molecules caused by B cell, also there is it is a kind of it is undiscovered so far, in source, knot
Structure and function etc. are different from classical Ig Ig gene outcomes, including IgG, IgA, IgM etc., referred to as non-B cell source Ig
（non B-Ig）.
Non- B cell source Ig（Non B-Ig）It can be produced by the non-B cell of a variety of different pedigrees, it is characterized in that：① non
B-Ig has more extensive expression, such as epithelial cell, nerve cell, reproduction cell, mesenchymal tissue and the hematopoiesis in medullary system source
Cell has the expression of Ig molecules.2. it is different from the Ig molecules variable region diversity in B cell source, non B-Ig light chains with again
Conserved sequence is presented in chain variable region sequence between different tissue sources, Different Individual；3. non B-Ig gene expression regulation machines
System is also not quite similar with B cell.4. high-level, high-frequency expression is presented in kinds of tumor cells in non B-Ig；non B-Ig
It is related to the growth and existence of tumour cell, even relates to the generation and transfer of tumour, but specific effect and molecular mechanism is always not
Answered.
Chinese invention patent application publication No. CN102901817A（Publication date 2013.01.30）Disclose a species specificity
Identify the IgG in non-B cell source（non B-Ig）The monoclonal antibody RP215 of weight chain variable district judge tumor cell proliferation,
Application in terms of migration, chemotherapeutic drug resistance and identification adult/tumour ancestral cells.The IgG for disclosing non-B cell expression exists
It is glycosylation modified variant with IgG in circulation.The epitope that RP215 is identified is exactly this kind of distinctive glycosyl of IgG weight chain variable districts
Class associated epitope.But do not know that what this distinctive glycosyl class associated epitope is specifically yet;In addition, only pass through experiment
It was found that monoclonal antibody RP215 can identify ancestral cells, and then it can be bred by RP215 for cell, migrated, tumour cell
Treat drug resistance etc. and be marked and detect etc. and apply.
RP215 is Lee seminars of University of British Columbia by the use of the albumen that ovarian cancer cell line extracts as exempting from
Mouse is immunized in epidemic focus, and the monoclonal antibody obtained is screened from 3000 strain of hybridoma.Produce the miscellaneous of monoclonal antibody RP215
Tumor cell strain is handed over to be preserved in Unite States Standard biology product collecting center, numbering ATCCHB10095.RP215 not only specific recognitions
Oophoroma, while the also cell of the other organization type tumours of specific recognition.But the antigen that the antibody is identified was not known at that time
What is, be named as " Cancer antigen, CA215 ", and think its can as " Pan-marker " of cancer cell, until
2007, they just had found that CA215 includes the IgG of tumor cells expression, and tentatively prompt the epitope of RP215 identifications may be with sugar
Baseization is related.Therefore cancer antigen（CA215）It is believed to " Pan-marker " as cancer cell.Although RP215 can be special
Opposite sex identification CA215, but the associated epitope of its identification is not known, therefore can not also enter traveling one for cancer antigen CA215
The development and application of step.
Chinese invention patent application publication No. CN102014955A（Publication date 2011.04.13）Disclose one kind and contain glycosyl
Wide spectrum cancer markers are used for Serologic detection, and specify that CA215 is in the nature immunoglobulin-like material, while with secreting type
Exist with two kinds of molecular forms of film combination type, and there is different molecular weight.CA215 is present in as undetermined aggregation
Cancer cell surfaces.With RP215 monoclonal antibodies specific immune response can occur for CA215 antigenic determinant, be identified by RP215
Antigenic determinant include glycosyl part, the glycosyl is not generally occur in Ig, the weight chain variable district of the glycosyl and Ig sample molecules
It is associated.Mannosylated antigen determinant includes a unique composition, and the composition is different from human IgG, is also different from and occurs therewith
The RP215 monoclonal antibodies of immune response.Saccharic composition analysis result finds that the CA215 includes about 1-3% trehaloses, 9-15%
GalNAc, 27-30%N- acerylglucosamines, 6-15% glucose, 47-51% mannoses, without N- acetyl group
Neuraminic acid and N- ethylene glycol neuraminic acids.But it is any structure by the antigenic determinant of RP215 identifications without explanation, that is,
The epitope of the IgG can not be determined by saying.
Although increasing evidence shows that the Ig in non-B cell source has huge application value, due to studying work
The limitation of many factors such as tool and method, the understanding for non B-Ig is still extremely limited, therefore limits its development and application.
The content of the invention
In order to further further investigate non-B cell source Ig structure and biological function, so as to develop its it is huge should
With value, the present invention has found by numerous studies analysis in the case where its corresponding research tool is extremely limited and demonstrates non-B
Cell derived IgG activity specific structure, it is by sialylated glycosyl site, based on this discovery, the present invention provides
Following technical scheme：
The invention provides non B-sIgG（The sialylated IgG in non-B cell source）As ancestral cells mark
Using sialylated glycosyl glycosylates not related with O- on the IgG.
Described ancestral cells refer to pluripotent stem cell, including embryonic stem cell, tumour ancestral cells, into soma/
Progenitor cells and hematopoietic stem/progenitor.
Different from previous result of study, it has been found that for the IgG in non-B cell source, specify that it is corresponded to
The specific epitope of RP215 monoclonal antibodies is sialylated glycosyl site, and this glycosylation is not closed with O- glycosylations
System, i.e., main component is N-acetylneuraminic acid and N- ethylene glycol neuraminic acids.In addition it was also found that this glycosylate simultaneously
It is not present in the Ig such as IgA, IgM in other non-B cell sources etc..
Sialic acid (Sialic acid, SA) is the general name of a kind of carboxylated monose acylated derivatives containing 9 carbon atoms,
It is mainly two kinds of N-acetyl-neuraminate and NeuGc ALPHA2-3Gal in the sialic acid of human body, is given birth to mostly by glucose metabolism
Into.
Preferably, in above-described application, the ancestral cells are adult ancestral cells.
Preferably, in above-described application, the ancestral cells are tumour ancestral cells.We study hair
It is existing, after non B-IgG albumen synthesis, under the control of enzyme, by the modification after translation, at weight chain variable district position
Affix acylneuraminate glycosyl, turn into the biomolecule with specific activity, can particularly promote the generation and transfer of tumour,
Therefore Tumor Stem/progenitor cell marker thing can be used as.Tumour ancestral cells are related to tumour generation, Epithelial and stromal conversion
(EMT), the biological property such as metastases and prognosis mala, all there is very big value for clinical diagnosis and the treatment of tumour.
Further, preferably, it is with non B-sIgG and/or IgG sialic acid in application described above
Change generation and/or transfer detection reagent that glycosyl site prepares tumour as target spot, prepare the medicine of oncotherapy, or prepare swollen
Knurl prognostic evaluation reagent.
Closer, in above-described application, the tumour is epithelial tumour.Epithelial tumour be by epithelium Lai
The malignant tumour that the cell carcinogenesis in source occurs, including breast cancer, carcinoma of urinary bladder, lung cancer, carcinoma of mouth, prostate cancer, cancer of pancreas, liver
Cancer, colorectal cancer（Including colon cancer and the carcinoma of the rectum）, stomach cancer, kidney etc..
Preferably, it is with non B-sIgG and/or comprising the IgG glycosyl epitopes in above-described application
Immunogen immune animal prepares antibody；Using non B-sIgG and/or comprising the IgG glycosyls site as target spot prepare immunocyte,
Albumen and/or small molecule.
Present invention also offers a kind of ancestral cells immunologic combined detection reagent kit, and it includes specific recognition non
The antibody of B-sIgG and/or IgG saliva acidizing sugar base table position；Or specific recognition non B-sIgG and/or the IgG saliva
The albumen and/or small molecule in liquid acidifying glycosyl site.
Closer, in above-mentioned ancestral cells immunologic combined detection reagent kit, specific agent formulations include：
Antigen retrieval buffers：It is by 10mM Tris（Trishydroxymethylaminomethane）With the Tris-EDTA of 1mM EDTA compositions
Buffer solution, pH 9.0；
Primary antibody：RP 215；
Mark secondary antibody：By the anti-mouse IgG of mark, the label is fluorescence or HRP；
PBS；
If label is HRP, in addition to 3,3- diaminobenzidine.
Antigen retrieval buffers in kit of the present invention are different from general commercial prod, and being free of in its buffer solution typically makes
Tween 20.
Certain density Tris and EDTA are common agents, and those skilled in the art can be bought by commercial sources,
Can also voluntarily it be prepared by prior art knowledge.
For example, 0.02M EDTA solution compound methods：Weigh 8g Na2H2Y·2H2O is placed in 250mL beakers plus water is micro-
After heat of solution, 1000mL is diluted to, 20 times of dilution is 1mM during use.10mM Tris compound methods：First prepare 1MTris-
HCl (mother liquor) 1L：121.1g Tris are weighed to be placed in 1000mL beakers；About 800mL deionized water is added, is sufficiently stirred molten
Solution；It is slowly added into HCl sides edged and surveys pH value（About 30mL）.100 times of dilution is 10mM during use.
RP 215 can be to ATCC（American Type Culture collection warehousing）Purchase.
The invention has the advantages that：
The present invention by the further research to non-B cell source IgG, draw non-B cell source sialylated IgG and
Its sialylated glycosyl site can as the mark and drug target of ancestral cells particularly tumour ancestral cells,
This kind of cell can easily be detected by being capable of the antibody in the sialylated glycosyl sites of specific recognition non B-IgG by preparing
Distribution, particularly tumour ancestral cells, design, can be effective for the detection reagent or targeted drug of the specific molecular
The diagnosis, treatment and prognosis evaluation of this kind of tumour are controlled, clinically there is huge application value.
Brief description of the drawings
Fig. 1 is to the IgG in the sialylated IgG (TIg) and people's venous blood in tumour cell source using different molecular
(sialylated IgG in IVIg) carries out specific recognition testing result.In A, lectin is the solidifying of specific recognition sialic acid
Collection element, as positive control；The band of merge marks shows that RP215 and lectin identification signal are completely overlapped；anti-IgG
All antibody for including sialylated and non-sialylated IgG components of identification, herein signal it is stronger show that it identifies be
All IgG components.B is the qualification result of the non B-sIgG with lectin affinity columns after purification, and input represents impure
Change, through represents the IgG components of uncombined affinity column, and elution represents the IgG combined with affinity column eluted
Component.C is the result for detecting non B-sIgG after being handled with sialidase with RP215, and 1 represents without processing, 2 expression O-
After glucosides ferment treatment, 3 expressions are handled with sialidase.
Fig. 2 is staining conditions of the non B-sIgG in the pulmonary adenocarcinoma of different differentiation degrees.Wherein, A figures are immune
Histochemical staining result；B figures are statistic analysis result.Scale is 50 μm.
Fig. 3 is staining conditions of the non B-sIgG in adenocarcinoma of lung primary tumor and transfer stove.Wherein, A figures are SABC
Coloration result；B figures are statistic analysis result.Scale is 50 μm.
Fig. 4 shown in lung adenocarcinoma cell line A549, is struck IgG using siRNA technologies after subtracting, is detected with Transwell
Method detects cell migration ability；Cell invasion ability is detected with Matrigel detection methods, control is nonsense siRNA.Wherein A
Figure is that siRNA strikes decreasing effect fruit（GAPDH is reference gene）；B figures are that cell migration ability compares and its statistic analysis result；C schemes
Compare for cell invasion ability and its statistic analysis result.
Fig. 5 shown in lung adenocarcinoma cell line A549, is struck IgG using siRNA technologies after subtracting, is detected with RT-PCR swollen
Tumor metastasis and the change of EMT correlation molecules.Wherein, Ig γ are IgG heavy chain genes, and E-cadherin is that calcium is glutinous plain, Vimentin
For vimentin, MMP-2 is Extracellular matrixproteins 2, and MMP-9 is Extracellular matrixproteins 9, Slug,
Twist and ZEB1/2 is the molecule for promoting EMT transcriptions, and GAPDH is reference gene；503 be siRNA1, and 279 be siRNA2, and NC is
Nonsense siRNA.
Fig. 6 is that RP215 dyes pathological score and the statistical analysis of survival of patients time and survival probability to non B-sIgG
As a result.
Fig. 7 is depression effect Vitro Experimental Results of the tumour IgG to T lymphocytes, and wherein ordinate is inhibiting rate,
Cancer IgG are the IgG of tumor tissues extraction, and IVIG is the IgG in people's venous blood.
Fig. 8 is that non B-sIgG promote to test inside tumour growth（Dynamic is observed）As a result, abscissa is the time, indulges and sits
It is designated as gross tumor volume.Display gross tumor volume increases with time lengthening.
Fig. 9 is volume statistical result measured after final taking-up tumour after kill mouse.
Embodiment
With reference to specific embodiment, the invention will be further described, to help present disclosure is understood.Following reality
The experimental method of unreceipted actual conditions in example is applied, is generally carried out according to this area conventional meanses.
The discovery in the non-B cell source IgG of embodiment 1 sialylated glycosyl site
First with the Ig extracted from tumour（TIg）And the Ig extracted from normal person's venous blood（The IVIG of commercialization）
As research object, a kind of agglutinin that can be combined with sialic acid is utilized respectively（Golden elder skin agglutinin, Sambucus
nigra agglutinin, SNA）As associated proteins, or with specific monoclonal antibody RP215（It is real by Canadian Dr. professors Lee
Room preparation is tested, it is existing in Peking University's Human disease gene research center）As primary antibody, detected and sent out by Western blot
Existing, the band that the band that RP215 is identified is identified with SNA almost fits like a glove, and its positive signal is only displayed in IgG groups
The slower part of mobility in point（Figure 1A）.Further, we are using in the agarose column affinity purification IVIG with reference to SNA
Sialylated IgG, detected and found by Western blot, compare outflow component, RP215 can preferably be identified and eluted
Glycoprotein, and its identification band still be shown in the part that mobility is slower in IgG components（Fig. 1 B）.This result is demonstrate,proved
Bright RP215 mainly identifies sialylated glycoprotein.
In order to further confirm that what RP215 identified is non B-IgG sialylated glycosyl site, we are with nerve
Propylhomoserin enzyme and O-glycosides ferment treatment IVIG, after finding the digestion of sialic acid site, the band that RP215 is identified almost disappears, and O-
After glycosylation site is processed, the band that RP215 is identified has almost no change（Fig. 1 C）.This result proves that RP215 can know
Other non B-IgG sialylated glycosyl site, and the sialylated glycosyl and O- glycosylations are not related, and may be with N-
Glycosylation is related.
Non B-IgG are identified in Western blot method, it has been found that RP215 is only capable of identification one
55kDa band, and this band can weaken after the IgG albumen in striking low cell；The albumen of RP215 specific recognitions is carried out
Mass spectral analysis, as a result show, RP215 specific recognitions are IgG.Further the glycosylation to the IgG of RP215 identifications is reflected
It is fixed, as a result find that RP215 simply identifies sialylated IgG.It has been found that Ig not only has different subclass, and due to sugar
The difference of base, the function that Ig is exercised are also different.Than being shown if any result of study, sialylated IVIG has anti-inflammatory effect
Should, and IVIG can cause pro-inflammatory effect after sialyltransferase either galactase processing.In addition, Lee seminars
The albumen obtained to RP215 affinity chromatographys carries out mass spectral analysis, as a result shows, except IgG, also by sub-fraction Ig superfamily eggs
In vain, such as CD147, CD54.Itd is proposed that what RP215 may be identified was Ig superfamilies in 2012；But it is contemplated that arrive Lee classes
Topic group includes RP215 identifications IgG interaction protein for the albumen of Mass Spectrometer Method, it is therefore believed that, RP215 identifications
Only IgG sialylated glycosyl site.
Distributions of the non B-sIgG of embodiment 2 in normal structure and tumor tissues
After it is determined that RP215 only identifies the sialylated glycosyl sites of non B-IgG, we have inquired into non B-sIgG and existed
Express spectra in normal structure and tumor tissues.
（1）The expression of non B-sIgG in the normal tissue
Selection includes different normal structure chips, uses SABC（IHC）Method, have detected the non that RP215 is identified
The expression of B-sIgG in the normal tissue.As a result find, in the normal tissue, non B-sIgG are in mainly in squamous cell
The existing positive；But the only other types of multilayer epithelium of small part（Mammary gland, sweat gland, prostate, bronchus etc.）And simple epithelium（Bile duct
Epithelium, enteric epithelium, Weishang skin and thymus epithelial etc.）There is positive reaction.It will be appreciated, however, that the basal layer cell of multilayer epithelium
（With adult ancestral cells characteristic）Expressed in strong positive.And other tissues, such as muscle, connective tissue, lymphocyte have no
Positive reaction（Table 3）.
In view of non B-sIgG show that Strong positive signals are primarily targeted for having ancestral cells feature in multilayer epithelium
Basal cell, further we compare RP215 Strong positive signals in 19 kinds of normal epithelial derived tissues with IHC method
With the mark p63 of Normal adult stem cell signal distributions, distribution of both discoveries in normal epithelial tissues is consistent
, the basal cell being predominantly located in squamous cell and stratified epithelium cell.This is also prompting the high expression of non B-sIgG just
Normal cell has the feature of stem cell.
Express spectras of the non B-sIgG of table 1 in normal structure
| Type and pathology | Total number of samples | Non B-sIgG positive sample numbers | Dyeing scoring and positive rate explanation |
| Normal gastric mucosa | 5 | 3 | 75 points, only have a small amount of cell to be positive dyeing in mucomembranous gland |
| Normal cerebral tissue | 5 | 1 | 20 points, a small amount of positive staining is positioned at extracellular matrix |
| Normal colonic tissue | 3 | 1 | 25 points, a small amount of glandular epithelium is positive dyeing |
| The tumor tissues of nearly normal colonic tissue | 2 | 2 | 65 points, a small amount of glandular epithelium is positive dyeing |
| Normal bone marrow tissue | 5 | 2 | 0 point |
| The tumor tissues of nearly normal rectal tissue | 5 | 5 | 50 points, a small amount of glandular epithelium is positive dyeing |
| Normal cervical tissues | 3 | 3 | 110 points (basal cells);350 (other scaly epitheliums) |
| The tumor tissues of nearly normal cervical tissues | 2 | 1 | 110 points (basal cells);400 (other scaly epitheliums) |
| Normal small intestine tissue | 5 | 5 | 56 points, a small amount of glandular epithelium is positive dyeing |
| Normal ovarian tissue | 2 | 0 | 0 point |
| The tumor tissues of nearly normal ovarian tissue | 3 | 0 | 0 point |
| Normal esophageal tissue | 5 | 5 | 163 points (basal cells);320 (other scaly epitheliums) |
| Normal galactophore tissue | 1 | 1 | 200 points (basal cells);200 (layer epithelial cell) |
| The tumor tissues of nearly normal galactophore tissue | 4 | 1 | 200 points (basal cells);200 (layer epithelial cell) |
| Normal liver tissue | 5 | 4 | 83 points, positive staining is presented in a small amount of liver cell |
| Normal prostate tissue | 2 | 1 | 300 points (basal cells);0 (layer epithelial cell) |
| The tumor tissues of nearly normal prostate tissue | 3 | 3 | 187 points (basal cells);0 (layer epithelial cell) |
| Normal pancreatic tissue | 5 | 2 | 40 points, positive staining is presented in a small amount of pancreatic cell |
| Normal testis tissue | 2 | 1 | 100 points (androgones) |
| The tumor tissues of nearly normal testis tissue | 3 | 2 | 75 points (androgones) |
| Normal lung tissue | 5 | 1 | 50 points, positive staining is presented in a small amount of alveolar epithelial cells |
| Normal spleen tissue | 5 | 0 | 0 point |
| Normal kidney tissue | 4 | 2 | 75 points, positive staining is presented in a small amount of renal cells |
| The tumor tissues of nearly normal kidney tissue | 1 | 0 | 0 point |
| The tumor tissues of nearly normal lymphoid nodal tissue | 5 | 0 | 0 point |
| Normal skin tissue | 5 | 5 | 46 points (basal cells);270 (other squamous cells) |
| Normal thymus tissue | 2 | 1 | 100 points, positive staining is presented in thymic epithelial cells |
| The tumor tissues of nearly normal thymus tissue | 3 | 2 | 150 points, positive staining is presented in thymic epithelial cells |
Standards of grading：Positive staining intensity is divided into four standards (0,1+, 2+, and 3+), and 0 is completely negative, and 1+ is weak
The positive, 2+ are moderate positive, and 3+ is strong positive.Final score is the percentage organized according to shared by the positive strength of different stage
Calculate, 0-300 points of fraction range.
（2）Expression of the non B-sIgG in a variety of epithelial tumor tissues
Distribution characteristics of the non B-sIgG in kinds of tumors tissue is have detected with the method for SABC.It is a variety of have detected
After the tumour of epithelial origin, the tumour and lymthoma in a leaf source, it has been found that non B-sIgG are in almost all of epithelium
Positive signal can be detected in the tumour in source, and in a tumour in leaf source, except the sarcoma of epithelialization,
Positive signal can not be almost detected completely；In lymthoma, especially B cell lymphoma, positive letter also can not be all detected
Number（Table 2）, illustrate for tumor tissues, what RP215 mainly to be identified is the tumour of epithelial origin.
Identification for epithelial origin tumour, it is our surprising discovery that, after antibody concentration is reduced, RP215 strong positive
Signal is also to concentrate on sub-fraction cell, that is, the basaloid cells being distributed in tumor tissues, and the tumour of invasion and attack state
Cell.According to prompting is had been reported, these cells all have the feature of tumour ancestral cells.
Table 2
| Pathological diagnosis and type | Total number of samples | Non B-sIgG positive sample numbers | Positive sample is evaluated |
| Fibrolipoma (B) | 1 | 0 | |
| Lipoma (B) | 1 | 0 | |
| Fibroma (B) | 1 | 0 | |
| Mesenchymoma (B) | 2 | 0 | |
| Angioleiomyoma (B) | 1 | 0 | |
| Hemangioma (B) | 2 | 0 | |
| Liomyoma (B) | 4 | 0 | |
| Chondrosarcoma (M) | 4 | 0 | |
| Fibrosarcoma (M) | 8 | 0 | |
| Prms (M) | 2 | 0 | |
| Alveolar rhabdomyosarcoma (M) | 2 | 0 | |
| Synovial sarcoma (M) | 2 | 2 | Only positive staining is shown in epithelioid cell |
| Epithelioid sarcoma (M) | 1 | 1 | Only positive staining is shown in epithelioid cell |
| Leiomyosarcoma (M) | 6 | 0 | |
| Malignant stromal tumors (M) | 3 | 0 | |
| Celiothelioma (M) | 1 | 1 | Only positive staining is shown in epithelioid cell |
| Malignant Hemangiopericytoma (M) | 1 | 0 | |
| Malignant mesenchymoma (M) | 1 | 0 | |
| Embryonal-cell lipoma (M) | 2 | 0 | |
| MFH (M) | 3 | 0 |
B：It is benign；M：It is pernicious.
The non B-sIgG of embodiment 3 are used for the differentiation degree for assessing adenocarcinoma of lung tumour
Immunohistochemical staining is carried out to 232 adenocarcinoma of lung cases with RP215 monoclonal antibodies, coloration result is scored, with reference to
Clinical case data, statistical analysis is carried out, is shown in Table 3.
Table 3
| Pathological characters | Number of cases | RP215 dyeing scoring mean (SD) | P |
| Age | 0.6830 | ||
| <60 | 126 | 23.8(40.5) | |
| ≥60 | 106 | 25.7(38.3) | |
| Sex | 0.9926 | ||
| Female | 106 | 22.8(37.9) | |
| Man | 126 | 26.2(40.8) | |
| Smoking history | 0.2523 | ||
| Non-smoking | 28 | 15.7(40.1) | |
| Smoking | 33 | 9.2(26.2) | |
| Differentiation | 0.0159* | ||
| Middle and high differentiation | 134 | 17.6(29.1) | |
| Low differentiation | 94 | 34.4(49.6) | |
| TNM stagec | 0.9253 | ||
| I-II | 159 | 26.2(42.6) | |
| III-IV | 61 | 20.6(32.4) |
Mean, average value；SD, standard deviation；*, P<0.05, there is notable significant difference.
Showed by immune group result, dyeing of the monoclonal antibody RP215 in PD adenocarcinoma of lung case are significantly stronger than differentiated
With the case of medium differentiation（P=0.0159）, that prompts non B-IgG expresses the ripe differentiation degree positive correlation with adenocarcinoma of lung（Figure
2）.And the differentiation degree of tumour and the prognosis of cancer patient are closely related, the low differentiation of tumour, often represent malignancy of tumor degree compared with
Height, the prognosis of patient are often poor.
Therefore, using the kit in the present invention, can using the immunohistochemical staining situation of non B-sIgG molecules as
The index of adenocarcinoma of lung tumor differentiation degree is assessed, it is suitable for patients with lung adenocarcinoma selection for the antidiastole of clinically adenocarcinoma of lung
Therapeutic scheme, the prognosis of patient can be assessed to a certain extent, carry out early prevention and treatment.
The non B-sIgG of embodiment 4 are used to assess adenocarcinoma of lung transfer case
We carry out SABC dye with monoclonal antibody RP215 to the Metastatic Lymph Nodes of 42 adenocarcinoma of lung primary tumors and its pairing
Color, both RP215 staining conditions are contrasted.As a result show, RP215 is significantly stronger than primary in the staining power of transfer stove
Stove, P=0.0019（Fig. 3）.Prompt RP215 that dyeing is closely related with the transfer of adenocarcinoma of lung.
In order to further prove that the non B-sIgG of RP215 identifications are related to the transfer of adenocarcinoma of lung.We utilize siRNA pairs
IgG in lung adenocarcinoma cell line A549 strike subtracting（Fig. 4 .A）, and have detected the change of cell migration ability and invasive ability
Change.
As a result show, after IgG, which is struck, to be subtracted, A549 transfer ability and invasive ability significantly reduce.（Fig. 4 .B, C）Together
When, we are detected with the RT-PCR molecules related to some metastases, are as a result shown, after IgG, which is struck, to be subtracted, E calcium glues
It is known as and is raised, matrix metalloproteinase MMP-9, Epithelial and stromal conversion（EMT）Associated transcription factor Twist, ZEB1/2 are aobvious
Write and lower, the migration and invasive ability that imply that cell reduce（Fig. 5）.
Therefore,, can be by non B-sIgG staining conditions in tissue using the kit in the present invention based on above-mentioned discovery
As the index for assessing adenocarcinoma of lung transfer case.Contribute to whether look-ahead patients with lung adenocarcinoma has the tendency of transfer, in advance
Early prevention and treatment work is carried out, improves the survival rate of patient.
The non B-sIgG of embodiment 5 are used to assess adenocarcinoma of lung patient's prognosis situation
Single argument survival technology analysis is carried out by Kaplan-Meier, we are to non B-sIgG staining conditions and lung gland
The correlation of carninomatosis people's prognosis is assessed.As a result show, patients with lung adenocarcinoma positive RP215（Contain a large amount of non in tissue
B-sIgG）, its 5 year life cycle be substantially less than the negative staining patients of non B-sIgG, illustrate that non B-sIgG stained positives are pre-
Show poor prognosis（Fig. 6）.After being controlled to Clinical and pathologic features（Age, sex, smoking history, differentiation degree, TNM
By stages）, Cox regression analyses show non B-sIgG dyeing be adenocarcinoma of lung independent hazard factor, relative risk 5.71（CI
1.26~25.95）, it is shown in Table 4.In addition, without related between age, sex, smoking history, differentiation degree, TNM stage and survival rate
Property.
Table 4
| Pathological characters | Hazard Ratio | 95%CI | P |
| Age (≤60/>60 ) | 0.47 | 0.15-1.45 | 0.19 |
| Sex (male/female) | 2.01 | 0.45-8.99 | 0.36 |
| Smoking history (non-smoking/smoking) | 1.26 | 0.36-4.37 | 0.72 |
| Differentiation (middle and high differentiation/low differentiation) | 0.69 | 0.19-2.51 | 0.57 |
| TNM stage (I, II/III, IV) | 1.34 | 0.49-3.70 | 0.57 |
| RP215 dyeing (negative/positive) | 5.71 | 1.26-25.95 | 0.02 |
Therefore, can be pre- as adenocarcinoma of lung tumour patient is assessed using RP215 staining conditions using the kit in the present invention
The index of situation afterwards, the prognosis of patient can be assessed, carry out early prevention and treatment, improve survival.
Inhibited proliferations of the non B-sIgG of embodiment 6 to T lymphocytes
Method：
1st, albumen is extracted from tumor tissues, purifies wherein IgG with protein G-beads（tIgG).
2nd, mononuclearcell is separated from normal human peripheral blood with lymphocytes separating solution, is 10 by cell modulation concentration6/
ML is cultivated, and after PHA activated T lymphocytes, cell, wherein normal person's venous blood IgG are handled with the tIgG of various concentrations
(IVIG) compare.
3rd, inhibited proliferations of the tIgG to T lymphocytes is observed with CFSE methods.
Experimental result shows, non B-sIgG to the depression effect of T lymphocytes is IVIG 5-10 times.
Growth facilitation actions of the non B-sIgG of embodiment 7 to tumour
Experiment in vitro and experiment in vivo are carried out respectively.Method is as follows：
1st, mouse melanin tumour cell (B16) is cultivated.
2nd, C57BL/6 mouse 20 are bought, B16 is injected by oxter daily（2.5×105/ point）, form it into tumour.
3rd, it is divided into 5 groups：
PBS groups：Negative control group, injects PBS solution, and dosage is isometric with experimental group；
The μ g groups of tIgG 10：Sialylated non B-IgG are injected, dosage is the μ g of per injection 10；
The μ g groups of tIgG 50：Sialylated non B-IgG are injected, dosage is the μ g of per injection 50；
The μ g groups of IVIG 10：IVIG is injected, dosage is the μ g of per injection 10；
The μ g groups of IVIG 50：IVIG is injected, dosage is the μ g of per injection 50.
Sialylated non B-IgG were injected from the 5th day knurl week, injects every other day and measures gross tumor volume, put to death within the 17th day, take
Go out tumour, compare the size of tumour between each group.
Experimental result is shown in Fig. 7 ~ 9, as a result shows, with the extension of time, tIgG groups promote tumour growth effect obvious high
In other groups, show that sialylated non B-IgG can remarkably promote tumour growth, it can be as the mark of tumour growth
Detected.
The explanation of above example is only intended to help the core concept for understanding the present invention.It should be pointed out that for this technology
For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improvement can also be carried out to the present invention
And modification, these are improved and modification is also fallen into the protection domain of the claims in the present invention.
Claims (3)
- Applications of the 1.non B-sIgG as ancestral cells mark, the application are specifically with non B-sIgG and/or are somebody's turn to do IgG sialylated glycosyl site prepares generation and/or the transfer detection reagent of tumour as target spot, prepares the medicine of oncotherapy Thing, or prepare the application in tumor prognosis evaluation reagent；Sialylated glycosyl glycosylates not related with O- on the IgG；Institute It is tumour ancestral cells to state ancestral cells.
- 2. application according to claim 1, it is characterised in that the tumour is epithelial tumour.
- 3. application according to claim 1 or 2, it is characterised in that be with non B-sIgG and/or comprising the IgG glycosyls The immunogen immune animal of epitope prepares antibody；It is using non B-sIgG and/or immune as target spot preparation comprising the IgG glycosyls site Cell, albumen and/or small molecule.
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| CN108610414B (en) * | 2018-02-12 | 2022-09-13 | 北京大学 | IgG epitope and application thereof as target spot |
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| US8974786B2 (en) * | 2012-06-13 | 2015-03-10 | Vancouver Biotech Ltd. | Humanized antibodies to CA215 |
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