CN106520949B - Biomarker is used to prepare the purposes of cystitis glandularis diagnostic reagent - Google Patents
Biomarker is used to prepare the purposes of cystitis glandularis diagnostic reagent Download PDFInfo
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Abstract
The invention discloses the purposes that biomarker is used to prepare cystitis glandularis diagnostic reagent, cystitis glandularis is divided into chronic inflammatory type, folliculus edema type, papilloma type and enteric adenoma template, and biomarker includes miR-372-3p shown in SEQ ID NO.1, miR-377-3p shown in miR-383-3p and SEQ ID NO.3 shown in SEQ ID NO.2.When cystitis glandularis patient and bladder normal person are distinguished in use in conjunction miR-372-3p, miR-383-3p and miR-377-3p diagnosis, area (AUC) is 0.964 under ROC curve, and sensitivity and specificity are respectively 96.2% and 95.6%.Not passing through intervention means using diagnostic reagent of the present invention can be to cystitis glandularis diagnosis typing, it is only necessary to which a small amount of blood plasma of patient, when inspection do not avoid, and overcome cystoscopic deficiency.
Description
Technical field
The invention belongs to diagnostic reagent fields, and in particular to biomarker is used to prepare cystitis glandularis diagnosis typing reagent
Purposes.
Background technique
Cystitis glandularis (cystitis glandularis, CG) is a kind of more rare Non-cancerous inflammatory lesion,
It is a kind of epithelial proliferation and the simultaneous lesion of metaplasia, process is that epithelial proliferation is recessed into Brunn nest, is inside split
Gap forms branch-like, cyclic annular lumen, and center gland metaplasia occurs and forms gland structure, with exist simultaneously at this time lymphocyte and
The infiltration of thick liquid cell, therefore referred to as cystitis glandularis.In simple terms, cystitis glandularis is exactly under a kind of bladder mucosa and mucous membrane
Layer mucus body of gland abnormality proliferation, metaplasia venereal disease become, and disease incidence increasingly increases, and with bladder cancer closely (Yang Anqing etc.,
Expression and its clinical meaning of the p27 albumen in different parting cystitis glandularis, 2016).Whether cystitis glandularis is precancerosis
Become, presently, there are disputes.Although its canceration rate is lower, clinically still there is potential canceration to be inclined to (Yang Anqing etc., p27 albumen
It is associated in the expression of cystitis glandularis and its with bladder cancer, 2016).
Cystitis glandularis has the characteristics that special pathological development process and clinical onset, and the cause of disease is still unclear at present, may
It is related with the diseases such as bladder chronic inflammation, calculus, obstruction, neurogenic bladder, ectopia vesicae.In trigone of urinary bladder, neck of urinary bladder
Equal positions are easier to occur around portion and orificium ureteris.
There are mainly three types of the diagnostic methods of cystitis glandularis: cystoscope, ultrasound and CT.The gland bladder of different stages of development
Scorching clinic and ultrasonographic manifestation is different, and ultrasonic examination, which is easy to happen, fails to pinpoint a disease in diagnosis or mistaken diagnosis is bladder cancer.The ultrasound of cystitis glandularis
Parting includes nodular type, tubercle incrassation type, sheet incrassation type and diffuses incrassation type, and it is bladder cancer that wherein nodular type, which is easy mistaken diagnosis,
And the bladder wall without obviously thicken or nodositas protrusion person's ultrasound inspection Check be easy to happen and fail to pinpoint a disease in diagnosis (Ke Liming etc., cystitis glandularis it is super
Sound parting and Misdiagnosis are analyzed, and 2014).CT examination is easy, quick, noninvasive, can observe lesion from different perspectives, can be used as morning
Phase finds the effective means of follow-up after cystitis glandularis or treatment, and the characteristic performance of CT can clarify a diagnosis to a certain extent,
But cystoscope biopsy (yellow lead river etc., the CT diagnosis imaging of cystitis glandularis can only be relied on for some atypical cases of performance
It learns and shows, 2011).Therefore, cystitis glandularis most authoritative diagnostic method or cystoscope.Clinic is mainly according to cystoscopy
And histopathologic type is respectively grouped cystitis glandularis.Chronic inflammatory type, folliculus oedema are grouped into seen in cystoscope
Type, papilloma type, four kinds of enteric adenoma template；Histopathology is grouped into no metaplasia type, intestinal metaplasia type, three kinds of prostate type
(Fang Haiwei etc., expression and clinical meaning of p53, Ki67 tumor markers in different parting cystitis glandularis, 2010).
As previously mentioned, the prior art needs by means of cystoscope cystitis glandularis parting, it is the recognized standard.But
Cystoscopy has many taboos, and if urethra, bladder are in acute inflammation stage and should not be checked, bladder capacity is too small should not be into
Row checks that woman month menstrual period or gestation 3 months or more should not be checked.Moreover, Preoperative Method and post surgery treatment are cumbersome.These
Increase the body and mental anguish of examiner.
Biomarker can assess human body to the exception of the neurological susceptibility of disease or detection body, be usually used in examining for pathological state
Disconnected and detection, the progress of predictive disease, or assessment curative effect.It is different (such as year from some risk factors to play a role in disease
The factor related with disease such as age, obesity, smoking), biomarker needs not participate in the progression of disease.Biomarker
Screening technique it is varied, such as by metabolism group method, genomics method, Proteomic methods find small molecule metabolic indicator
Object, gene marker and protein markers, circulation microRNA and tissue microRNA etc..
MiRNA can by pairing with the area target gene 3'-UTR (pairing or portion paired completely) in conjunction with come degrade mRNA or
Inhibit mRNA translation, to regulate and control expression of target gene level.As regulatory molecule, most miRNA have and other gene expressions
Height between the common feature of regulatory molecule, i.e. cell and tissue specificity, expression timing, structural variability and different plant species
Homology.There is also similar with Circulating DNA, circulating cells in the body fluid such as blood, urine, gastric juice, and it is originated from primary lesion
Recycle miRNA molecule.These circulations miRNA is with good stability, be placed at room temperature for for 24 hours, thawing, RNA enzyme and DNA
Without obvious degradation under the conditions of enzyme effect and storage temperature change etc., consider to be present in apoptotic body, microvesicle, outer dye with miRNA
It is closely related in the lipids such as colour solid or lipoprotein complexes.Accordingly, with respect to other diagnostic methods, recycles miRNA and detect wound
It is small, and have certain disease correlation specificity and stability, have as new diagnostic markers potential value (Li Yue etc.,
Progress of the circulation microRNA in the screening of diagnosing human disease's marker, 2014).
The concept of metabolism group is that Nicholson etc. took the lead in proposing in 1999.Relative to genomics, transcription group
And proteomics, metabolism group are more concerned about the phenotype of organism, therefore be also in the screening of disease potential source biomolecule marker most
Promising developing direction.It combines a variety of separation means to biosystem in physiology by means of nuclear magnetic resonance spectroscopy, mass spectrum
Or the dynamic change of all endogenous small molecule metabolites is analyzed under pathological state, to describe bioagent metabolism wheel
Exterior feature explores the relationship between endogenous small-molecule substance and disease development.Small molecule of the molecular weight less than 1000 dalton
Close the main target that object is metabolism group concern.It is compared to gene, transcription and protein research, metabolism group reflection is thin
The final product of intracellular activity, so also most stable.Since when maintaining function, the moment is all being metabolized cell, metabolism group
Credit analysis can reflect the function and state of organism within a certain period of time, gene and protein expression under certain morbid state
Minor change can be amplified on metabolin, and the variation that many cannot be embodied from both substances can be in metabolism group
It is detected.The generation for having proven to disease is studied at present and progress is closely related with metabolic disorder, and metabolism group can analyze disease
All metabolins generated in sick occurrence and development identify valuable potential marker, so that the diagnosis and parting for disease are opened
Ward off new way (Wu Jie etc., application progress of the metabonomic analysis in tumor markers screening, 2016).
Applicant is retrieved with " cystitis glandularis " and " marker " in Chinese Periodicals Database, retrieves 3 correlations
Document: (1) 40 patients are grouped by Fang Haiwei etc. by cystoscopy and histopathologic type, using immunohistochemistry
SABC method, the expression of inspection p53, Ki67 in each parting cystitis glandularis, and be compared with 10 normal bladder mucous membrane groups,
As a result, it has been found that papilloma type, enteric adenoma type cystitis glandularis have higher p53, Ki67 expression rate, and with normal bladder mucous membrane
Comparing has significant difference；Then p53, Ki67 expression rate are lower for chronic inflammatory type, folliculus edema type, with normal bladder mucous membrane indifference
It is different；Enteric epithelium type, prostate type cystitis glandularis p53 expression rate are higher, and have conspicuousness compared with normal bladder mucous membrane group
Difference；Enteric epithelium type, prostate type cystitis glandularis Ki67 expression rate are higher, compared with bladder normal mucosa group, prostate type
Group has significant difference, and there was no significant difference for enteric epithelium type, and (Fang Haiwei etc., p53, Ki67 tumor markers are in different parting glands
Expression and clinical meaning in cystitis, 2010)；(2) Cheng Yue etc. takes 96 determinations to have abnormal pathologic to change parallel operative treatment
Patient's sample, wherein cystitis glandularis (GC) 40, bladder mucosa squamous metaplasia (SMBM) 32 merge two kinds of pathology
Change (GC-SMBM) 24, separately taking 21 normal bladder mucous membranes is control group, detects biopsy specimen using immunohistochemical method
The expression of middle P53, RasP21, as a result GC group and SMBM group P53 and RasP21 positive rate are expressed and the equal nothing of control group difference
Statistical significance, GC-SMBM group P53, RasP21 and the two, which express positive expression rate and control group difference simultaneously, statistics
Meaning (grind by the expression of Cheng Yue etc., monoclonal antibody P53 and RasP21 in cystitis glandularis and bladder mucosa squamous metaplasia
Study carefully, 2012)；(3) Song Yahui etc. supports that cystitis glandularis is normal bladder tissue into bladder cancer development from pathologic angle
Between the stage, Survivin can be considered as diagnosis early stage adenocarcinoma of bladder sensitive indicator (Song Yahui etc., Survivin are in gland wing
Expression and clinical meaning in Guang inflammation and adenocarcinoma of bladder, 2014).
Applicant is carried out with " cystitis glandularis " and " biomarker " in Web of Science database
Retrieval, retrieves 2 pertinent literatures: (1) Wei ZF etc. has found in mamillary cystitis glandularis patient mucous membrane of urinary bladder tissue
HTERT, p53 and PCNA are more significantly higher than the level of bladder normal human bladder's mucous membrane, and mamillary cystitis glandularis is prompted to compare appearance
Easily occur canceration (Expression of hTERT, p53and PCNA in cystitis glandularis, 2007)；(2)
Velickovic, LJ etc. have found mucin CD10 in the mucosal tissue of intestinal metaplasia type cystitis glandularis using the method for immunohistochemistry
In do not express, can be used for distinguishing intestinal metaplasia type and without metaplasia type cystitis glandularis (Differences in the
expression of mucins in various forms of cystitis glandularis,2007)。
Applicant's retrieval does not find that the prior art examines cystitis glandularis using metabolism group and circulation miRNA molecule
It is disconnected, and different type cystitis glandularis is distinguished, the more application without open related associated biomarkers in this respect.
Since cystitis glandularis clinical manifestation lacks characteristic, there is respective defect and deficiency in existing detection methods, it is necessary to open again
It sends out diagnostic reagent relevant and diagnosis typing is carried out to cystitis glandularis.
Summary of the invention
The purpose of the present invention is to provide the purposes that biomarker is used to prepare cystitis glandularis diagnosis typing reagent, specifically
Ground: an object of the present disclosure is to provide blood circulation miRNA molecule for diagnosing cystitis glandularis, and to different type (with
Parting seen in cystoscope, i.e. chronic inflammatory type, folliculus edema type, papilloma type, four kinds of enteric adenoma template) cystitis glandularis
It distinguishes；The present invention second is designed to provide plasma metabolism small molecule for diagnosing cystitis glandularis, and to different type
The gland wing of (with parting seen in cystoscope, i.e. chronic inflammatory type, folliculus edema type, papilloma type, four kinds of enteric adenoma template)
Guang inflammation distinguishes；The present invention can provide the diagnostic reagent of cystitis glandularis diagnosis typing from different perspectives.
Above-mentioned purpose of the invention is achieved by following technical solution:
It is a kind of for diagnosing the biomarker group of cystitis glandularis, the cystitis glandularis is divided into chronic inflammatory type, filter
Steeping edema type, papilloma type and enteric adenoma template, biomarker group includes miR-372- shown in SEQ ID NO.1
MiR-377-3p shown in miR-383-3p and SEQ ID NO.3 shown in 3p, SEQ ID NO.2.Use in conjunction miR-372-
When cystitis glandularis patient and bladder normal person are distinguished in 3p, miR-383-3p and miR-377-3p diagnosis, area under ROC curve
It (AUC) is 0.964, under best cutoff value, sensitivity and specificity are respectively 96.2% and 95.6%.
Further, the cystitis glandularis is chronic inflammatory type, and biomarker group further includes shown in SEQ ID NO.4
MiR-485-3p and SEQ ID NO.5 shown in miR-501-3p.In miR-372-3p, miR-383-3p and miR-377-3p
On the basis of, chronic inflammatory type patient and bladder normal person are distinguished in further use in conjunction miR-485-3p and miR-501-3p diagnosis
When, area is 0.943 under ROC curve, and sensitivity and specificity are respectively 95.3% and 94.2%.
Further, the cystitis glandularis is folliculus edema type, and biomarker group further includes shown in SEQ ID NO.6
MiR-96-3p and SEQ ID NO.7 shown in miR-93-5p.In miR-372-3p, miR-383-3p and miR-377-3p base
On plinth, when folliculus edema type patient and bladder normal person are distinguished in further use in conjunction miR-96-3p and miR-93-5p diagnosis,
Area is 0.969 under ROC curve, and sensitivity and specificity are respectively 96.5% and 95.8%.
Further, the cystitis glandularis is papilloma type, and biomarker group further includes shown in SEQ ID NO.8
MiR-223-5p and SEQ ID NO.9 shown in miR-183-3p.In miR-372-3p, miR-383-3p and miR-377-3p
On the basis of, papilloma type patient and bladder normal person are distinguished in further use in conjunction miR-223-5p and miR-183-3p diagnosis
When, area is 0.984 under ROC curve, and sensitivity and specificity are respectively 96.2% and 96.6%.
Further, the cystitis glandularis is enteric adenoma template, and biomarker group further includes SEQ ID NO.10 institute
MiR-128-3p shown in miR-584-3p the and SEQ ID NO.11 shown.In miR-372-3p, miR-383-3p and miR-
On the basis of 377-3p, enteric adenoma template patient and bladder are distinguished in further use in conjunction miR-584-3p and miR-128-3p diagnosis
When normal person, area is 0.973 under ROC curve, and sensitivity and specificity are respectively 96.0% and 95.8%.
Further, the biomarker is derived from blood plasma.
Purposes of the above-mentioned biomarker group in terms of preparing cystitis glandularis diagnostic reagent.
It is a kind of for diagnosing the biomarker group of cystitis glandularis, the cystitis glandularis is divided into chronic inflammatory type, filter
Steeping edema type, papilloma type and enteric adenoma template, biomarker group includes methyl crotonic acylcarnitine, hemolytic phosphatidyl second
Hydramine and arachidonic acid.Use in conjunction methyl crotonic acylcarnitine, lysophosphatidyl ethanolamine and arachidonic acid diagnosis are distinguished
When cystitis glandularis patient is with bladder normal person, area (AUC) is 0.958 under ROC curve, and sensitivity and specificity are respectively
96.6% and 96.1%, individually area is respectively less than 0.7 under ROC curve when application.
Further, the cystitis glandularis is chronic inflammatory type, and biomarker group further includes indoxyl sulfate and coffee
Coffee because.On the basis of methyl crotonic acylcarnitine, lysophosphatidyl ethanolamine and arachidonic acid, further use in conjunction sulfuric acid Yin
When chronic inflammatory type patient and bladder normal person are distinguished in diindyl phenol and caffeine diagnosis, area is 0.952 under ROC curve, sensitivity
It is respectively 96.3% and 95.4% with specificity.
Further, the cystitis glandularis is folliculus edema type, and biomarker group further includes lysophosphatidyl choline
(18:1), chenodeoxycholic acid and gangliosides.In methyl crotonic acylcarnitine, lysophosphatidyl ethanolamine and arachidonic acidic group
On plinth, folliculus water is distinguished in further use in conjunction lysophosphatidyl choline (18:1), chenodeoxycholic acid and gangliosides diagnosis
When swollen type patient is with bladder normal person, area is 0.962 under ROC curve, and sensitivity and specificity are respectively 95.8% He
95.2%.
Further, the cystitis glandularis is papilloma type, and biomarker group further includes lysophosphatidyl choline
(18:1), phosphatidylserine and biphosphate acetone.In methyl crotonic acylcarnitine, lysophosphatidyl ethanolamine and arachidonic
On the basis of acid, further use in conjunction lysophosphatidyl choline (18:1), phosphatidylserine and biphosphate acetone diagnostic region
When dividing papilloma type patient and bladder normal person, area is 0.979 under ROC curve, and sensitivity and specificity are respectively
95.5% and 96.0%.
Further, the cystitis glandularis is enteric adenoma template, and biomarker group further includes lysophosphatidyl choline
(16:0) and hyaluronic acid.On the basis of methyl crotonic acylcarnitine, lysophosphatidyl ethanolamine and arachidonic acid, further join
When closing using lysophosphatidyl choline (16:0) and hyaluronic acid diagnosis differentiation enteric adenoma template patient and bladder normal person, ROC
Area under the curve is 0.981, and sensitivity and specificity are respectively 96.7% and 96.1%.
Further, the biomarker is derived from blood plasma.
Purposes of the above-mentioned biomarker group in terms of preparing cystitis glandularis diagnostic reagent.
Present contribution to the art:
(1) present invention demonstrates that, use in conjunction miR-372-3p, miR-383-3p and miR-377-3p can be with Accurate Diagnosis areas
Divide cystitis glandularis patient and bladder normal person；Further use in conjunction miR-485-3p and miR-501-3p can be with Accurate Diagnosis
Distinguish chronic inflammatory type patient and bladder normal person；Further use in conjunction miR-96-3p and miR-93-5p can be with Accurate Diagnosis
Distinguish folliculus edema type patient and bladder normal person；Further use in conjunction miR-223-5p and miR-183-3p can be examined accurately
It is disconnected to distinguish papilloma type patient and bladder normal person；Further use in conjunction miR-584-3p and miR-128-3p can be accurate
Enteric adenoma template patient and bladder normal person are distinguished in diagnosis.Cystitis glandularis can be diagnosed simultaneously by this 11 miRNAs
Parting can be prepared into the diagnostic reagent that cystitis glandularis is distinguished in diagnosis.
(2) present invention demonstrates that, use in conjunction methyl crotonic acylcarnitine, lysophosphatidyl ethanolamine and arachidonic acid can be with
Accurate Diagnosis distinguishes cystitis glandularis patient and bladder normal person；Further use in conjunction indoxyl sulfate and caffeine can be quasi-
Make a definite diagnosis disconnected differentiation chronic inflammatory type patient and bladder normal person；Further use in conjunction lysophosphatidyl choline (18:1), goose go
Oxycholic acid and gangliosides can distinguish folliculus edema type patient and bladder normal person with Accurate Diagnosis；Further use in conjunction is molten
Serium inorganic phosphorus phosphatidylcholine (18:1), phosphatidylserine and biphosphate acetone can with Accurate Diagnosis distinguish papilloma type patient with
Bladder normal person；Further use in conjunction lysophosphatidyl choline (16:0) and hyaluronic acid can distinguish mamillary with Accurate Diagnosis
Tumor type patient and bladder normal person.Cystitis glandularis can diagnose and parting by this 13 blood plasma metabolins, it can be with
It is prepared into the diagnostic reagent that cystitis glandularis is distinguished in diagnosis.
(3) using diagnostic reagent provided by the invention do not pass through intervention means can to cystitis glandularis diagnosis typing,
Only need a small amount of blood plasma of patient, when inspection does not avoid, and overcomes cystoscopic deficiency.
Specific embodiment
Essentiality content of the invention is specifically introduced below with reference to embodiment, but protection model of the invention is not limited with this
It encloses.The routine test operation that the test operation not being described in detail in experiment is well known to the skilled person.
Diagnostic reagent of the embodiment 1 based on blood circulation miRNA molecule
First part: screening stage
One, experimental material and experimental method
1, plasma sample: research object 23, the age 45~55 years old, 51.3 years old average；Male 12, female 11.Through bladder
Mirror makes a definite diagnosis grouping: chronic inflammation group (chronic inflammatory type 6,3M/3F), folliculus oedema group (folliculus edema type 5,3M/2F), cream
Head tumor group (5, papilloma type, 2M/3F), enteric adenoma sample group (enteric adenoma template 7,4M/3F).Each case is first visit,
Without history of medications.Separately there is 5 bladders normally artificial control group.The age of each group research object, there was no significant difference.All trouble
Person and bladder normally have normal cardiopulmonary liver kidney and hematopoiesis function per capita.Bladder normal person refers to that bladder mucosa and submucosa are glutinous
Liquid body of gland proliferation without exception, the healthy volunteer without the change of metaplasia venereal disease.Collect the peripheral vein of above-mentioned patient and bladder normal person
Blood blood plasma, blood sampling time are early morning fasting state.
2, blood plasma Total RNAs extraction: taking 400 μ L blood plasma, and using mirVanaTM RNAIsolation kit, (Ambion is public
Department) extracted total RNA, it is operated according to kit specification, using the quality of the extracted RNA of spectrophotometric determination.
3, separation marking: by 1 μ g RNA, 7 μ L DEPC water, 2 μ L RNA Spike Control Oligospoly, 5 μ
Rapid centrifugation after the solution mixing that LATP concentration is 1mmol/L Tris.1.5 μ L 25mmol/L are added to above-mentioned solution
MnCl2, 1.5 μ 10 × reaction of L buffer, 1.0 μ L acid phosphatases incubate for 37 DEG C after 1.0 μ L dilutedATP Mix
Educate 15min；Fluorescent marker connection, takes on 15 μ L rapid centrifugation postposition ice bath of said mixture, 2 μ L T4DNA ligases of addition, and 4
μ L 5 × FlashTag connection Mix biotin, rapid centrifugation after mixing, 25 DEG C of incubation 0.5h.
4, chip hybridization: Affymetrix company miRNA chip agent box is used, kit specification is deferred to and is grasped
Make.The preparation of hybridization solution: 50 μ L 2 × hybridization Mix, 5 μ L 20 × Hybridization Controls objects, 5 μ L deionized formamides, 10 μ L DEPC
Water, 1.7 μ L reference material oligonucleotide B2And 10 μ L dimethyl sulfoxide.99 DEG C after above-mentioned hybridization solution is mixed with biotin sample
5min, 45 DEG C of incubation 5min are incubated for, take 100 μ L for microarray hybridization, 48 DEG C, 60r/min is incubated for 16h.
5, chip scanning and image real time transfer: pass through GenePix 4000B microarray scanner (Axon
Instruments, Foster City, CA) scanner acquires fluorescence intensity to chip scanning.Then pass through Genepix again
Pro 6.0software (Axon) image analysis software carries out digital assay to the hybridization image of acquisition, with each probe
The background value that original signal value subtracts the point obtains the correction value of each probe；It is repaired on every chip in the experiment of same batch
The non-control probe of positive value >=50 standardizes, and marks using this part probe intermediate value as point of the normalization factor to whole chip
Quasi-ization processing.After standard post-processes, respectively with chronic inflammation group/control group, folliculus oedema group/control group, papilloma
Group/control group, enteric adenoma sample group/control group calculate the fold differences of corresponding miRNAs molecule, select difference greater than 1.5 or are less than
0.5 miRNA is as the miRNAs for having differential expression, i.e. acquisition primary dcreening operation as a result, for further verifying.
6, expression verifying: having the miRNAs of differential expression to verify primary dcreening operation, and primer is referring to GenBank database base
Because sequence is designed.According to qPCR Reverse Transcriptase kit specification, 20 μ L reaction systems, 37 DEG C of incubation 60min, 95 DEG C of incubations
5min terminates reaction, and reverse transcription is stored for future use at -20 DEG C after cDNA.In accordance with qPCR kit specification, 20 μ L reaction systems, 95
DEG C initial denaturation 2min, 40 circulations, reaction condition are 95 DEG C, 15s and 60 DEG C, 60s.It is obtained by 7500 quantitative PCR apparatus of ABI
Ct value, single reaction are repeated 2 times.
Two, experimental result
1, the miRNA of patient's variant expression compared with bladder normal person
The analysis of chip results is as the result is shown: compared with the control group, chronic inflammation group, folliculus oedema group, papilloma group
With miR-222-3p, miR-146a-5p, miR-485-3p, miR-501-3p, miR-96-3p, miR- in enteric adenoma sample group blood plasma
93-5p、miR-223-5p、miR-183-3p、miR-584-3p、miR-128-3p、miR-425-3p、miR-372-3p、miR-
This 18 kinds of miRNAs expression of 342-3p, miR-124-5p, miR-155-5p, miR-183-5p, miR-383-3p, miR-377-3p
Up-regulation is lowered, and differential expression is significant (P < 0.05).
2, qPCR expresses verification result
QPCR verifying is carried out to the above-mentioned otherness miRNA in disease group and control group blood plasma, as the result is shown:
With control group ratio, miR-372- in chronic inflammation group, folliculus oedema group, papilloma group and enteric adenoma sample group blood plasma
3p lowers 0.3~0.5 times, and miR-383-3p raises 1.8~2.2 times, and miR-377-3p lowers 0.2~0.4 times；With control group
Than miR-485-3p raises 1.9~2.3 times in chronic inflammation group blood plasma, and miR-501-3p raises 1.7~2.0 times；With control group
Than miR-96-3p raises 2.1~2.4 times in folliculus oedema group blood plasma, and miR-93-5p raises 2.5~2.8 times；With control group
Than miR-223-5p raises 2.6~2.9 times in papilloma group blood plasma, and miR-183-3p raises 1.8~2.1 times；With control group
Than miR-584-3p raises 3.2~3.4 times in enteric adenoma sample group blood plasma, and miR-128-3p raises 2.4~2.6 times.Verification result
As shown in table 1.
The qPCR of 1 otherness miRNA of table expresses verification result
Second part: Qualify Phase
Plasma sample: research object 81, the age 45~55 years old, 50.4 years old average；Male 42, female 39.Through cystoscope
Make a definite diagnosis grouping: chronic inflammation group (chronic inflammatory type 20,11M/9F), folliculus oedema group (folliculus edema type 22,11M/
11F), papilloma group (21, papilloma type, 11M/10F), enteric adenoma sample group (enteric adenoma template 18,8M/10F).Respectively
Case is first visit, no history of medications.Separately there is 15 bladders normally artificial control group.The age of each group research object is without significant
Sex differernce.All patients and bladder normally have normal cardiopulmonary liver kidney and hematopoiesis function per capita.Bladder normal person refers to that bladder is glutinous
Film and submucosa mucus body of gland proliferation without exception, the healthy volunteer without the change of metaplasia venereal disease.Collect above-mentioned patient and bladder just
The peripheric venous blood blood plasma of ordinary person, blood sampling time are early morning fasting state.
Other test materials and the same screening stage of method.
Receiver operating curve (ROC) analysis: building ROC curve expresses the miRNAs verified to verify above-mentioned qPCR
To the ability of cystitis glandularis diagnosis typing.
As the result is shown: cystitis glandularis is distinguished in use in conjunction miR-372-3p, miR-383-3p and miR-377-3p diagnosis
When patient is with bladder normal person, area (AUC) is 0.964 under ROC curve, and sensitivity and specificity are respectively 96.2% He
95.6% (under best cutoff value, similarly hereinafter), individually area is respectively less than 0.7 under ROC curve when application；Further use in conjunction
When chronic inflammatory type patient and bladder normal person are distinguished in miR-485-3p and miR-501-3p diagnosis, area is under ROC curve
0.943, sensitivity and specificity are respectively 95.3% and 94.2%；Further use in conjunction miR-96-3p and miR-93-5p are examined
When disconnected differentiation folliculus edema type patient is with bladder normal person, area is 0.969 under ROC curve, and sensitivity and specificity are respectively
96.5% and 95.8%；Papilloma type patient and wing are distinguished in further use in conjunction miR-223-5p and miR-183-3p diagnosis
When Guang normal person, area is 0.984 under ROC curve, and sensitivity and specificity are respectively 96.2% and 96.6%；Further joint
When distinguishing enteric adenoma template patient and bladder normal person using miR-584-3p and miR-128-3p diagnosis, area under ROC curve
It is 0.973, sensitivity and specificity are respectively 96.0% and 95.8%.Due to miR-372-3p, miR-383-3p and miR-
Up-regulation or downward situation basic one of the 377-3p in chronic inflammation group, folliculus oedema group, papilloma group, enteric adenoma sample group
It causes, so chronic inflammation group, folliculus only cannot be distinguished by miR-372-3p, miR-383-3p and miR-377-3p use in conjunction
Oedema group, papilloma group and enteric adenoma sample group.
Analysis of conclusion: in ROC curve evaluation method, the area value AUC under ROC curve more connects in the case where being greater than 0.5
It is bordering on 1, illustrates that diagnosis effect is better.AUC has lower accuracy at 0.5~0.7, and AUC has certain accurate at 0.7~0.9
Property, AUC has high accuracy at 0.9 or more.The above results show use in conjunction miR-372-3p, miR-383-3p and
MiR-377-3p can distinguish cystitis glandularis patient and bladder normal person with Accurate Diagnosis；Further use in conjunction miR-485-3p
With miR-501-3p chronic inflammatory type patient and bladder normal person, high sensitivity, high specificity can be distinguished with Accurate Diagnosis；Into one
Folliculus edema type patient and bladder normal person can be distinguished with Accurate Diagnosis by walking use in conjunction miR-96-3p and miR-93-5p, sensitive
Degree is high, high specificity；Further use in conjunction miR-223-5p and miR-183-3p can distinguish papilloma type with Accurate Diagnosis
Patient and bladder normal person, high sensitivity, high specificity；Further use in conjunction miR-584-3p and miR-128-3p can be quasi-
Make a definite diagnosis disconnected differentiation papilloma type patient and bladder normal person, high sensitivity, high specificity.It can be right by this 11 miRNAs
Cystitis glandularis diagnose and parting.ROC curve evaluation result is shown in Table 2.
2 ROC curve evaluation result of table (circulation miRNAs)
The contribution of the present embodiment:
This example demonstrates that use in conjunction miR-372-3p, miR-383-3p and miR-377-3p can be with Accurate Diagnosis areas
Divide cystitis glandularis patient and bladder normal person；Further use in conjunction miR-485-3p and miR-501-3p can be with Accurate Diagnosis
Distinguish chronic inflammatory type patient and bladder normal person；Further use in conjunction miR-96-3p and miR-93-5p can be with Accurate Diagnosis
Distinguish folliculus edema type patient and bladder normal person；Further use in conjunction miR-223-5p and miR-183-3p can be examined accurately
It is disconnected to distinguish papilloma type patient and bladder normal person；Further use in conjunction miR-584-3p and miR-128-3p can be accurate
Enteric adenoma template patient and bladder normal person are distinguished in diagnosis.Cystitis glandularis can be diagnosed simultaneously by this 11 miRNAs
Parting can be prepared into the diagnostic reagent that cystitis glandularis is distinguished in diagnosis.
Diagnostic reagent of the embodiment 2 based on blood plasma small molecule metabolites
First part: screening stage
One, experimental material and experimental method
1, plasma sample: research object 23, the age 45~55 years old, 51.3 years old average；Male 12, female 11.Through bladder
Mirror makes a definite diagnosis grouping: chronic inflammation group (chronic inflammatory type 6,3M/3F), folliculus oedema group (folliculus edema type 5,3M/2F), cream
Head tumor group (5, papilloma type, 2M/3F), enteric adenoma sample group (enteric adenoma template 7,4M/3F).Each case is first visit,
Without history of medications.Separately there is 5 bladders normally artificial control group.The age of each group research object, there was no significant difference.All trouble
Person and bladder normally have normal cardiopulmonary liver kidney and hematopoiesis function per capita.Bladder normal person refers to that bladder mucosa and submucosa are glutinous
Liquid body of gland proliferation without exception, the healthy volunteer without the change of metaplasia venereal disease.Collect the peripheral vein of above-mentioned patient and bladder normal person
Blood blood plasma, blood sampling time are early morning fasting state.
2, the preparation of LC-MS sample for analysis
50 μ l of blood plasma is taken, 200 μ l acetonitriles, ultrasonic mixing 20min after hand mix 30s is added, then with 10,000 × g is centrifuged
30min collects supernatant in separate type glass tube, and precipitating continues the methanol extraction with 200 μ l volumetric concentrations 50%, by methanol
The supernatant of extraction merges with the supernatant of acetonitrile extraction, is dried with nitrogen, and residue is saved in -80 DEG C.Before LC-MS analysis, use
The acetonitrile solution of 100 μ l volumetric concentrations 5% dissolves residue, and 14,000 × g is centrifuged 5min, supernatant sample introduction is analyzed (8h
It is interior effective).
3, LC-MS analyzes parameter
Chromatographic system: WatersAcquity HPLC/Q-TOF Micro MS
Chromatographic column: Acquity UPLCTM BEH C18(2.1m×100mm×1.7μm)
Mobile phase: A Xiang Weishui (contains 0.1% formic acid)；B phase is acetonitrile (containing 0.1% formic acid)
Gradient condition: 0-2min, 2-50%B；2-3min, 50-98%B；3-4.5min, 98%B；4.5-6min, 2%B.
Flow velocity: 0.5mL/min；Column temperature: 45 DEG C.
MS condition: electron spray (UPLC/Q-TOF/MS ESI) ion source, under positive and negative ion mode, scanning range m/
z100-1000；Capillary voltage 3.0kV, orifice potential 35kV, 100 DEG C of ion source temperature, dry temperature degree is 450 DEG C, dry
Gas velocity 900L/h, taper hole throughput 50L/h.
4, Data Management Analysis and difference are metabolized Structural Identification
The data that LC-MS is obtained import SIMCA software (version 13.0.2, Umetrics) and carry out multivariate statistics point
Analysis.By establishing OPLS-DA model, find chronic inflammation group and control group, folliculus oedema group and control group, papilloma group and
Metabolic profile contributes the metabolin of larger (VIP > 1.0 and p < 0.01) between control group, enteric adenoma sample group and control group, then
By these metabolins, cutting protonatomic mass data are committed to online database KEGG (http://wwwkeg.com), METLIN really
(http://www.metlin.scipps.edu), HMDB (http://www.hmdb.ca) are searched, and parse compound mass spectrum, right
Compound carries out Structural Identification, eventually by the structure of these compound standard product confirmation difference metabolin.
5, quantitative verification: it is quantitative to difference metabolin using mass spectrum, measure concentration of the difference metabolin in each group sample
It is horizontal.
Two, experimental result
Mass spectrum quantitative result is shown: with control group ratio, chronic inflammation group, folliculus oedema group, papilloma group and enteric adenoma
Methyl crotonic acylcarnitine raises 2.4~2.6 times in sample group blood plasma, and lysophosphatidyl ethanolamine lowers 0.5~0.6 times, peanut four
Olefin(e) acid raises 3.6~3.9 times；With control group ratio, indoxyl sulfate lowers 0.4~0.5 times in chronic inflammation group blood plasma, caffeine
1.8~2.0 times of up-regulation；With control group ratio, lysophosphatidyl choline (18:1) lowers 0.5~0.6 times in folliculus oedema group blood plasma,
Chenodeoxycholic acid raises 2.7~2.8 times, and gangliosides raise 2.3~2.5 times；With control group ratio, in papilloma group blood plasma
Lysophosphatidyl choline (18:1) lowers 0.5~0.6 times, and phosphatidylserine lowers 0.5~0.6 times, on biphosphate acetone
Adjust 1.7~1.9 times；With control group ratio, lysophosphatidyl choline (16:0) lowers 0.4~0.5 times in enteric adenoma sample group blood plasma, thoroughly
Bright matter acid raises 3.1~3.3 times.The results are shown in Table 3 for mass spectrum quantitative verification.
The mass spectrum quantitative verification result of 3 otherness blood plasma metabolin of table
Second part: Qualify Phase
Plasma sample: research object 81, the age 45~55 years old, 50.4 years old average；Male 42, female 39.Through cystoscope
Make a definite diagnosis grouping: chronic inflammation group (chronic inflammatory type 20,11M/9F), folliculus oedema group (folliculus edema type 22,11M/
11F), papilloma group (21, papilloma type, 11M/10F), enteric adenoma sample group (enteric adenoma template 18,8M/10F).Respectively
Case is first visit, no history of medications.Separately there is 15 bladders normally artificial control group.The age of each group research object is without significant
Sex differernce.All patients and bladder normally have normal cardiopulmonary liver kidney and hematopoiesis function per capita.Bladder normal person refers to that bladder is glutinous
Film and submucosa mucus body of gland proliferation without exception, the healthy volunteer without the change of metaplasia venereal disease.Collect above-mentioned patient and bladder just
The peripheric venous blood blood plasma of ordinary person, blood sampling time are early morning fasting state.
Other test materials and the same screening stage of method.
Receiver operating curve (ROC) analysis: ROC curve is constructed to verify the difference generation of above-mentioned mass spectrum quantitative verification
Thank to object to the ability of cystitis glandularis diagnosis typing.
As the result is shown: use in conjunction methyl crotonic acylcarnitine, lysophosphatidyl ethanolamine and arachidonic acid diagnosis are distinguished
When cystitis glandularis patient is with bladder normal person, area (AUC) is 0.958 under ROC curve, and sensitivity and specificity are respectively
96.6% and 96.1%, individually area is respectively less than 0.7 under ROC curve when application；Further use in conjunction indoxyl sulfate and coffee
When coffee distinguishes chronic inflammatory type patient and bladder normal person because of diagnosis, area is 0.952 under ROC curve, sensitivity and specificity
Respectively 96.3% and 95.4%；Further use in conjunction lysophosphatidyl choline (18:1), chenodeoxycholic acid and ganglioside
When folliculus edema type patient and bladder normal person are distinguished in rouge diagnosis, area is 0.962 under ROC curve, sensitivity and specificity point
It Wei 95.8% and 95.2%；Further use in conjunction lysophosphatidyl choline (18:1), phosphatidylserine and biphosphate
When papilloma type patient and bladder normal person are distinguished in acetone diagnosis, area is 0.979 under ROC curve, sensitivity and specificity
Respectively 95.5% and 96.0%；Enteraden is distinguished in further use in conjunction lysophosphatidyl choline (16:0) and hyaluronic acid diagnosis
When tumor template patient is with bladder normal person, area is 0.981 under ROC curve, and sensitivity and specificity are respectively 96.7% He
96.1%.Since methyl crotonic acylcarnitine, lysophosphatidyl ethanolamine and arachidonic acid are in chronic inflammation group, folliculus oedema
Group, papilloma group, the up-regulation in enteric adenoma sample group or lower situation it is almost the same, so only by methyl crotonic acylcarnitine,
Chronic inflammation group, folliculus oedema group, papilloma group cannot be distinguished in lysophosphatidyl ethanolamine and arachidonic acid use in conjunction
With enteric adenoma sample group.
The above results show that use in conjunction methyl crotonic acylcarnitine, lysophosphatidyl ethanolamine and arachidonic acid can be with
Accurate Diagnosis distinguishes cystitis glandularis patient and bladder normal person；Further use in conjunction indoxyl sulfate and caffeine can be quasi-
Make a definite diagnosis disconnected differentiation chronic inflammatory type patient and bladder normal person；Further use in conjunction lysophosphatidyl choline (18:1), goose go
Oxycholic acid and gangliosides can distinguish folliculus edema type patient and bladder normal person with Accurate Diagnosis；Further use in conjunction is molten
Serium inorganic phosphorus phosphatidylcholine (18:1), phosphatidylserine and biphosphate acetone can with Accurate Diagnosis distinguish papilloma type patient with
Bladder normal person；Further use in conjunction lysophosphatidyl choline (16:0) and hyaluronic acid can distinguish enteric adenoma with Accurate Diagnosis
Template patient and bladder normal person.To cystitis glandularis can diagnose simultaneously parting by this 13 blood plasma metabolins.ROC is bent
Line assessment the results are shown in Table 4.
4 ROC curve evaluation result of table (blood plasma metabolin)
The contribution of the present embodiment:
This example demonstrates that use in conjunction methyl crotonic acylcarnitine, lysophosphatidyl ethanolamine and arachidonic acid can be with
Accurate Diagnosis distinguishes cystitis glandularis patient and bladder normal person；Further use in conjunction indoxyl sulfate and caffeine can be quasi-
Make a definite diagnosis disconnected differentiation chronic inflammatory type patient and bladder normal person；Further use in conjunction lysophosphatidyl choline (18:1), goose go
Oxycholic acid and gangliosides can distinguish folliculus edema type patient and bladder normal person with Accurate Diagnosis；Further use in conjunction is molten
Serium inorganic phosphorus phosphatidylcholine (18:1), phosphatidylserine and biphosphate acetone can with Accurate Diagnosis distinguish papilloma type patient with
Bladder normal person；Further use in conjunction lysophosphatidyl choline (16:0) and hyaluronic acid can distinguish mamillary with Accurate Diagnosis
Tumor type patient and bladder normal person.Cystitis glandularis can diagnose and parting by this 13 blood plasma metabolins, it can be with
It is prepared into the diagnostic reagent that cystitis glandularis is distinguished in diagnosis.
The effect of above-described embodiment is specifically to introduce essentiality content of the invention, but those skilled in the art should know
Protection scope of the present invention should not be confined to the specific embodiment by road.
SEQUENCE LISTING
<110>Nanjing Tuo Ruipu Gene Tech. Company Limited
<120>biomarker is used to prepare the purposes of cystitis glandularis diagnostic reagent
<130> 1
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213>homo sapiens
<400> 1
aaagugcugc gacauuugag cgu 23
<210> 2
<211> 19
<212> DNA
<213>homo sapiens
<400> 2
acagcacugc cuggucaga 19
<210> 3
<211> 22
<212> DNA
<213>homo sapiens
<400> 3
aucacacaaa ggcaacuuuu gu 22
<210> 4
<211> 22
<212> DNA
<213>homo sapiens
<400> 4
gucauacacg gcucuccucu cu 22
<210> 5
<211> 22
<212> DNA
<213>homo sapiens
<400> 5
aaugcacccg ggcaaggauu cu 22
<210> 6
<211> 22
<212> DNA
<213>homo sapiens
<400> 6
aaucaugugc agugccaaua ug 22
<210> 7
<211> 23
<212> DNA
<213>homo sapiens
<400> 7
caaagugcug uucgugcagg uag 23
<210> 8
<211> 22
<212> DNA
<213>homo sapiens
<400> 8
cguguauuug acaagcugag uu 22
<210> 9
<211> 22
<212> DNA
<213>homo sapiens
<400> 9
gugaauuacc gaagggccau aa 22
<210> 10
<211> 22
<212> DNA
<213>homo sapiens
<400> 10
ucaguuccag gccaaccagg cu 22
<210> 11
<211> 21
<212> DNA
<213>homo sapiens
<400> 11
ucacagugaa ccggucucuu u 21
Claims (6)
1. purposes of the biomarker group in terms of preparing cystitis glandularis diagnostic reagent, the cystitis glandularis includes chronic inflammation
Disease type, folliculus edema type, papilloma type and enteric adenoma template, it is characterised in that: the biomarker group includes SEQ ID
MiR-377- shown in miR-383-3p and SEQ ID NO.3 shown in miR-372-3p, SEQ ID NO.2 shown in NO.1
3p。
2. purposes according to claim 1, it is characterised in that: the cystitis glandularis is chronic inflammatory type, biological marker
Object group further includes miR-501-3p shown in miR-485-3p and SEQ ID NO.5 shown in SEQ ID NO.4.
3. purposes according to claim 1, it is characterised in that: the cystitis glandularis is folliculus edema type, biological marker
Object group further includes miR-93-5p shown in miR-96-3p and SEQ ID NO.7 shown in SEQ ID NO.6.
4. purposes according to claim 1, it is characterised in that: the cystitis glandularis is papilloma type, biological marker
Object group further includes miR-183-3p shown in miR-223-5p and SEQ ID NO.9 shown in SEQ ID NO.8.
5. purposes according to claim 1, it is characterised in that: the cystitis glandularis is enteric adenoma template, biological marker
Object group further includes miR-128-3p shown in miR-584-3p and SEQ ID NO.11 shown in SEQ ID NO.10.
6. -5 any purposes according to claim 1, it is characterised in that: the biomarker group is derived from blood plasma.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103314298A (en) * | 2010-11-12 | 2013-09-18 | 环太平洋生物技术有限公司 | Novel marker for detection of bladder cancer and/or inflammatory conditions of the bladder |
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| CN103314298A (en) * | 2010-11-12 | 2013-09-18 | 环太平洋生物技术有限公司 | Novel marker for detection of bladder cancer and/or inflammatory conditions of the bladder |
Non-Patent Citations (4)
| Title |
|---|
| Cyclooxygenase-2 and B-cell lymphoma-2 expression in cystitis glandularis and primary vesicle adenocarcinoma;Li et al.;《BMC Urology》;20140103;第14卷(第2期);第1-5页 |
| Differences in the expression of mucins in various forms of cystitis glandularis;L. Jankovic Velickovic et al.;《Pathology-Research and Practice》;20071231;第203卷;第653-658页 |
| Expression of hTERT, p53 and PCNA in Cystitis Glandularis;WEI Zhifeng et al.;《Journal of Huazhong University of Science and Technology 》;20071231;第27卷(第4期);第437-439页 |
| 肿瘤标志物p53、Ki67在不同分型腺性膀胱炎组织中阳性表达率的相关性分析;王相平等;《现代泌尿生殖肿瘤杂志》;20160831;第8卷(第4期);第231-232页 |
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