CN109762898A - A kind of application of tumor markers CA9 and UCA1 in the kit for preparing the probability that Noninvasive testing suffers from bladder cancer - Google Patents
A kind of application of tumor markers CA9 and UCA1 in the kit for preparing the probability that Noninvasive testing suffers from bladder cancer Download PDFInfo
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Abstract
The present invention discloses a kind of application of tumor markers CA9 and UCA1 in the kit for preparing the probability that Noninvasive testing suffers from bladder cancer.The present invention can be used for the preliminary screening and Follow-up After of doubtful bladder cancer, reduce cystoscopy frequency, reduce patient suffering and risk, significant to the blank for filling up the external molecular diagnosis product of domestic bladder cancer.
Description
Technical field
The present invention relates to detection kit fields, and in particular to a kind of tumor markers CA9 and UCA1 is preparing non-intruding
Application in the kit for the probability that bladder cancer is suffered from formula detection.
Background technique
Bladder cancer refers to that the most common malignant tumour of urinary system occurred in bladder mucosa, average age of onset are about
65 years old, wherein about 90% is transitional cell carcinoma, other 10% be mainly squamous carcinoma and gland cancer, the national tumour registration area in 2012 years
The disease incidence of bladder cancer be 6.61/10 ten thousand, the 9th of column Cancer Mortality.Since there are apparent poor prognosis, easy to recur
Feature, and 5 annual survival rate of bladder cancer patients of early discovery, early treatment is greater than 90%, therefore early diagnosis has weight to the treatment of the disease
The meaning wanted.Cystoscopy is the most reliable method of current diagnosis bladder cancer, by cystoscopy it can be found that bladder is
It is no to have tumour, but because its is invasive and expensive, patient dependence is poor, and easily stimulation the bladder wall tumour, may cause swollen
The undesirable extention and transfer of tumor, are not suitable for extensive screening；Urine sediment inspection is high to bladder transitional cell carcinoma specificity,
But to being classified, low bladder cancer susceptibility is low, easily formation false negative result, and cell be not true to type or retrogression, urinary system
The factors such as observational bias is larger between infection, calculus, bladder instillation to treat and different examiner also can have shadow to urinary cytology inspection
It rings.Therefore, the tumor markers method that noninvasive easy, sample easily obtains is in the early diagnosis of bladder cancer and answering for postoperative context of detection
With the concern for causing more researchers.
Bladder cancer Related product has bladder cancer cell chromosome and gene unconventionality detection kit (fluorescence original currently on the market
Position hybrid method) detection cast-off cells in 3,7, No. 17 chromosome aneuploids and P16(9p21) missing and related neoplasms
Marker product such as tumor of bladder related antigen BTA quantitative determination reagent kit (microwell plate chemoluminescence method), urine nuclear matrix protein
22(NMP-22) detection kit (colloidal gold method) etc..Wherein urine Exfoliative cells FISH technology is relatively suitable for bladder transitional cell carcinoma
The detection of postoperative recurrence, but to low level bladder transitional cell carcinoma, lacking genetic alteration relevant to bladder transitional cell carcinoma will lead to leakage
It examines, easily formation false negative result.NMP-22 is a kind of tridimensional network albumen for participating in maintaining cell kernel function, can be by thin
Born of the same parents' apoptosis and be discharged into urine, thus be considered closely related with urothelial tumor, some researches show that it in bladder cancer
Content decades of times higher than normal urothelium in chrotoplast.Document different to NMP-22 susceptibility and specific reporter (47%
~90%), there are larger disputes.Tumor of bladder related antigen (BTA) is also known as complement factor H GAP-associated protein GAP, is swollen by urothelium
Oncocyte and macrophage generate, and are the compounds that bladder is discharged into tumor of bladder growth course, when urine BTA level compared with
Gao Shi prompts the generation of urothelial tumor, therefore its early detection that can be used for urothelial tumor and recurrence monitoring.Bladder
Cancer diagnoses and treatment guide is pointed out: BTA is the relatively early tumor marker for being used to detect bladder cancer, mostly uses BTA Stat and BTA
The detection of Trak method, wherein BTA Stat is a kind of qualitative rapid techniques, reports susceptibility and specificity point according to pertinent literature
It Wei 29%~74% and 56%~86%；BTA Trak is enzyme linked immunological quantitative approach, susceptibility and specificity be respectively 60%~
83% and 60%~79%, susceptibility is with tumor grade and rises by stages and improves.NMP-22 and BTA detection is noninvasive, conveniently, fastly
Victory, but work as and the disease in the urological systems such as blood urine, urinary tract infections, stone in urinary system, hyperplasia of prostate, especially row irrigation of bladder occur
After chemotherapy, NMP-22 and BTA detection false positive rate are higher, therefore the bladder cancer tumor markers of high specific and susceptibility are still
Urgently develop.
Excretion body is that a kind of of cell active secretion shuttles in iuntercellular, diameter is 30 ~ 150 nm, density be 1.10 ~
The bilayer structure vesica sample corpusculum of 1.18 g/ml, can be fallen off release by different type cell (including tumour cell),
Most of body fluid such as peripheral blood, urine, saliva, ascites, amniotic fluid, milk, cerebrospinal fluid, joint fluid, BAL fluid etc.
In can be detected.Since excretion body includes the ingredients such as protein, DNA, RNA, the selective package of energy/discharge in its cell
Hereditary information, and there is now in more document report excretion body inclusion in fields such as cell-cell communication, tumour immunity and treatments
Research achievement, thus extract extracellular excretion body disease, in terms of diagnosis, monitoring have huge application potential.
The country is concentrated mainly on the separation and detection level of excretion body about the patent of excretion body at present, but extracts excretion body in urine and answer
Open report for bladder cancer auxiliary diagnosis is less, and a Zhejiang University discloses a kind of urine excretion body separation, enrichment and inspection
The integrated testing method and detection chip of survey, by the cancer information in detection urine excretion body to for the noninvasive of bladder cancer
Diagnosis, monitoring, it is quick that this method using excretion body transmembrane protein CD63 this distinctive marker establishes a kind of chip ELISA
Diagnostic method, but do not carry out the screening of bladder cancer specific tumour marker.
If the tumor markers of high specific and susceptibility can be found and be filtered out in the urine specimen excretion body of extraction
And be used for quickly detecting by Molecular tools, it is great for the early diagnosis and postoperative Clinical significance of detecting that assist bladder cancer.
Summary of the invention
To solve the above problems, the present invention provides a kind of tumor markers CA9 and UCA1 to prepare Noninvasive testing
Suffer from the application in the kit of the probability of bladder cancer.
The forward primer of CA9 is SEQ.4:CATCCTAGCCCTGGTTTTTGG,
The reverse primer of CA9 is SEQ.5:CCTTCTGTGCTGCCTTCTCAT,
The TaqMan probe of CA9 is SEQ.6:CTGTCACCAGCGTCGCGTTCCTT,
The forward primer of UCA1 is SEQ.7:GCCCTCATTCCGTGAAGAGA,
The reverse primer of UCA1 is SEQ.8:ATTTGAAATTGGTGAGATGTTCCTT,
The TaqMan probe of UCA1 is SEQ.9:CCACCTGCGACCTCGGGTCCT.
A and d respectively represent the fluorescent quantitation of CA9 and UCA14 gene as a result,
Logit (P)=1.168*a+0.855*d-1.177, or
Logit (P)=1.048*a+0.679*d-1.344,
The value of Logit (P) is used to judge the probability for suffering from bladder cancer.
It further include tumor markers CK20.
The forward primer of CK20 is SEQ.1:AAAAGGAGCATCAGGAGGAAGTC,
The reverse primer of CK20 is SEQ.2:GCATCAACCTCCACATTGACA,
The TaqMan probe of CK20 is SEQ.3:ATGGCCTACACAAGCATCTGGGCAA.
The fluorescent quantitation result of the gene of the CK20 is b,
Logit (P)=1.146*a-0.068*b+0.692*d-0.924, or
Logit (P)=1.274*a-0.077*b+0.84*d-0.714,
The value of Logit (P) is used to judge the probability for suffering from bladder cancer.
It further include the primer and needle sequence of endogenous control gene GAPDH and β-actin.
The forward primer of GAPDH is SEQ.13:CACATGGCCTCCAAGGAGTAA,
The reverse primer of GAPDH is SEQ.14:TGAGGGTCTCTCTCTTCCTCTTGT,
The TaqMan probe of GAPDH is SEQ.15:CTGGACCACCAGCCCCAGCAAG,
The forward primer of β-actin is SEQ.16:TGCCGACAGGATGCAGAAG,
The reverse primer of β-actin is SEQ.17:CTCAGGAGGAGCAATGATCTTGA,
The TaqMan probe of β-actin is SEQ.18:ATCACTGCCCTGGCACCCAGCA.
The kit uses when being preliminary screening or Follow-up After.
The present invention can be used for the noninvasive auxiliary diagnosis of bladder cancer by detecting the information of urine excretion body, while urine falls off
Some tumor markers (such as CA9, CK20 and UCA1) are the good sign objects for diagnosing bladder cancer in cell, can be passed through
The expression quantity of tumor markers in urine excretion body is detected to realize the non-invasive diagnosing of bladder cancer.
Compared with prior art, beneficial effects of the present invention are as follows:
(1) urine excretion body is used for the diagnosis of bladder cancer by the present invention, is a kind of diagnostic method of non-intrusion type, and materials are convenient can
It repeats, patient is easy to receive and easy to operate, though cystoscopy cannot be substituted completely, is able to cooperate cystoscopy, reduces wing
Guang spectroscopy frequency reduces patient suffering and risk.
(2) research object of the invention is the excretion body in urine, since the excretion body of tumor cell secretion is relatively normal thin
The secretory volume of born of the same parents is more, itself also acts as the role of tumor markers, and excretion vesicle structure is stablized, and what is carried is swollen
Specifically the stabilities of molecule such as relevant nucleic acid, protein, can be really anti-better than the complex environment and blood free state of cell for tumor
The physiology and pathologic function state of secretory cell are reflected, specificity is more preferable.
(3) present invention detects this 3 tumor markers of CA9, CK20 and UCA1 in urine excretion body simultaneously for the first time, and passes through
Diagnosis of Bladder model to be established, specificity is increased to 95% or more, susceptibility reaches 71% or more, and positive prediction rate is greater than 92%,
With the urine sediment inspection (sensibility 13%-75%, specific 85%-95%) and other bladder cancer immunodiagnosises production in clinic
Product such as NMP22(sensibility 47%-90%, specific 55%-98%) compared to showing more preferably diagnosis performance.
(4) present invention is the supplement strong to existing diagnosis of bladder cancer technology, to filling up the external molecule of domestic bladder cancer
The blank of diagnostic products is significant.
Detailed description of the invention
Fig. 1 is the concentration scatter plot and endogenous control base that bladder cancer group and control group excretion body RNA in case is embodied
The scatter plot of the expression CT value of cause；
Fig. 4 is tetra- gene combination of two analysis bladders of excretion body CA9, CK20, IGF2 and UCA1 in specific implementation case
Differential expression between cancer group and control group；
Fig. 5 is three combinatory analysis bladder cancer groups of excretion body CA9, CK20, IGF2 and UCA1 gene and right in specific implementation case
According to the differential expression between group；
Fig. 6 is the ROC curve analysis (Fig. 6 A) that tetra- gene combination of two of CA9, CK20, IGF2 and UCA1 in case are embodied
(Fig. 6 B) is analyzed with the ROC curve of 3 genes, 4 assortments of genes.
Specific embodiment
Below in conjunction with the drawings and specific embodiments to the present invention is based on the Noninvasive testing sides of urine excretion body bladder cancer
Explanation is described in detail in method.
Inventor has collected 176 through wing in Zhongshan Univ. Cancer Cure Center to during in March, 2018 in September, 2017
Guang mirror and pathological diagnosis are diagnosed as the urine specimen of bladder cancer patients, are defined as bladder cancer group, in addition also have collected 123 health
The urine specimen of volunteer, is defined as control group.Sample supplier in this research is told object of this investigation, and signs
Informed consent form is affixed one's name to.
The specific detecting step of non-invasive inspection methods based on urine excretion body bladder cancer is as follows:
1, urine specimen is handled
The preoperative urina sanguinis of bladder cancer patients and healthy control group urina sanguinis collect 30ml, obtain 176 bladder cancer group samples and 123 altogether
Example healthy control group sample, wherein satisfactory sample is respectively 170 patient's samples and 111 normal healthy controls samples.Urine
Liquid sample 3000g is centrifuged 15 minutes, takes supernatant spare.
, excretion body enrichment
According to the explanation of supplier, excretion body extracts kit (System Biosciences, ExoQuick-TC, cat. are used
No. EXOTC50A-1) excretion body is extracted from urine supernatant.Specific step is as follows:
(1) resulting urine supernatant 20ml after sampling present treatment, is added 4ml excretion body and extracts reagent, mix well, 4 DEG C of placements
16 hours.
(2) mixed liquor 5000g, is centrifuged 30 minutes by 4 DEG C；Supernatant is carefully completely removed, collection is precipitated as excretion body.
, excretion body RNA extract
It is heavy from excretion body using RNA extracts kit (Qiagen, miRNeasy Micro Kit, cat. no. 217084)
Excretion body total serum IgE is extracted in shallow lake:
(1) the excretion body precipitating collected is added 700uL QIAzol Lysis Reagent and sufficiently blows and beats mixing, mixed liquor transfer
Into new RNase-free 1.5mL centrifuge tube；
(2) it is placed at room temperature for 5min, is sufficiently cracked；
(3) 140uL chloroform is added in mixed liquor, acutely mixes 15s；
(4) it is stored at room temperature 2-3min；
(5) 4 DEG C of centrifuge are used, 12000xg is centrifuged 15min；
(6) upper strata aqueous phase solution is carefully drawn into new 1.5mL centrifuge tube；
(7) dehydrated alcohol of 1.5 times of volumes is added, mixes well；
(8) mixed liquor being transferred in adsorption column (RNeasy MinElute spin column), adsorption column is placed in collecting pipe,
Using 4 DEG C of centrifuge, 12000xg is centrifuged 15s, abandons waste liquid, adsorption column is put back in collecting pipe；
(9) it is added whether 700uL Buffer RWT(preoperation inspection is added dehydrated alcohol as required into adsorption column), 4 DEG C,
12000xg is centrifuged 15s, abandons waste liquid, adsorption column is put back in collecting pipe；
(10) it is added whether 500uL Buffer RPE(preoperation inspection is added dehydrated alcohol as required into adsorption column), 4 DEG C,
12000xg is centrifuged 15s, abandons waste liquid, adsorption column is put back in collecting pipe;
(11) 80% ethyl alcohol of 500uL is added into adsorption column, 4 DEG C, 12000xg is centrifuged 2min, abandons waste liquid；
(12) adsorption column is transferred in new collecting pipe, 4 DEG C, 12000xg is centrifuged 5min；
(13) adsorption column is transferred in new 1.5mL RNase-free centrifuge tube, 14uL RNase-free H is added2O, 4
DEG C, 12000xg is centrifuged 1min, is gained RNA solution in 1.5mL centrifuge tube.
RNA concentration obtained is measured using spectrophotometer (NanoDrop).In view of next step quantitative PCR experiment
Sample of the RNA measurement concentration lower than 8 ng/uL is excluded from research, obtains 168 bladder cancer patients and 100 by feasibility
The sample of normal healthy controls, and the research of next step is carried out to it.
, primer and probe design
According to primer and probe design principle, to CK20, CA9, UCA1, IGF2 and 2 endogenous control gene β-of 4 target genes
Actin, GAPDH design primer and probe, specific gene information are shown in Table 1, and primer probe sequence is shown in Table 2.
Table 1: the list of 4 target genes and 2 endogenous controls that the present patent application is analyzed
| Gene symbol | Gene ID | Gene Name |
| CK20 | 54474 | Keratin 20 |
| CA9 | 768 | Carbonic anhydrase 9 |
| UCA1 | 652995 | Urothelium cancer associated antigen 1 |
| IGF2 | 3481 | Insulin-like growth factor 2 |
| β-actin | 60 | Actin |
| GAPDH | 2597 | Glyceraldehyde-3-phosphate dehydrogenase |
Table 2: the primer and probe sequence of 4 target genes and 2 endogenous controls analyzed in the present patent application
5, real-time quantitative PCR
According to availability, the total serum IgE that is obtained to 500 ng from urine sample using 100 ng by RNA reverse transcriptase (Invitrogen,
Article No.: 4304134) TaqMan Reverse Transcription Reagents obtains complementary DNA (cDNA).It uses
PCR Taq enzyme (Applied Biosystems, TaqMan Universal PCR Master Mix, article No.: 4304437) to 4
The cDNA of a target gene and 2 endogenous control genes carries out quantitative fluorescent PCR reaction amplification, amplification instrument Applied
7500 type real-time fluorescence quantitative PCR instrument of Biosystems ABI.
Table 3: quantitative fluorescent PCR reaction system
Quantitative fluorescent PCR condition is as follows:
95 DEG C of 10min,
6, data are analyzed
With amplification instrument configure 7500 Software v2.3 softwares handle quantitative PCR data, establish each gene threshold value and
Baseline obtains the CT value that each sample reacts corresponding gene.With 2 endogenous control genes (β-actin and GAPDH) geometric average
It is worth standardized data.In order to ensure the reliability of result data, it is believed that endogenous control gene CT value is greater than 30 sample RNA mass
It is lower, and it is excluded from analysis, finally obtain the significant figure of 158 bladder cancer patients samples and 88 normal healthy controls samples
According to.The relative expression quantity of target gene uses the algorithm of 2- Δ Δ ct in sample, and final result is indicated with RQ.
Think that CT value is poor greater than 36 gene expression, Δ CT is calculated by the minimum delta CT of the gene.Utilize SPSS
Software carries out statistical analysis to the RQ value of all data, and it is aobvious whether the expression of t survey bladder cancer group and control group has
Sex differernce is write, and constructs the scatter plot of corresponding gene；The susceptibility and specificity of ROC curve calculating gene；Logistic is returned
Analysis is to construct the Diagnosis of Bladder model based on 4 genes, in conjunction with the susceptibility of ROC curve analysis and evaluation model and special
Property.
, result
To 176 bladder cancer groups and 123 control group sample analyses, the concentration of excretion body RNA and the expression of endogenous control gene
There is significant difference (as shown in Figure 1) between the two groups.To the expression analysis hair of this 4 genes of CA9, CK20, IGF2 and UCA1
Existing 4 genes all have significant difference (as shown in Figure 2) in two groups of expression, the AUC areas of ROC 4 genes of analysis between
Between 0.7304-0.8076 (as shown in Figure 3), wherein the specificity of CK20 reaches 97.73%, but susceptibility is lower, and CA9's is quick
Sensitivity is relatively high, and the AUC area of IGF2 is maximum, UCA1 low expression in bladder cancer, with other three genes and bladder cancer
Relationship is opposite.In view of the different manifestations of every kind of gene data, 4 genes are combined point using logistic regression equation
Analysis, establishes 11 kinds of computation models based on 2 kinds (C1-C6) or 3 kinds (C7-C10) or 4 kinds of (C11) assortment of genes, and every kind of model exists
It is all had between bladder cancer group and control group significant difference (Fig. 4-5), the result that ROC calculates every kind of model equation is divided
Analysis obtains AUC area between 0.6988-0.9064, and wherein C11C8 combined diagnosis performance highest, AUC area are
0.9064, susceptibility 71.52%, and specificity is 95.45%(table 4, Fig. 6), total coincidence rate is 81.7%.According to C11 model
Logistic regression equation Logit (the P)=1.146*a-0.068*b+0.692*d-0.924 obtained can be predicted unknown sample and suffer from
The probability of bladder cancer.
Table 4: the diagnosis performance of 4 genes of the present patent application analysis and combinations thereof
SN: susceptibility；SP: specificity；PPV: positive prediction rate；NPV: negative predictive rate；Cutoff: cutoff value；A, b, c, d points
The fluorescent quantitation result of each gene in CA9, CK20, IGF2 and UCA14 genes is not represented
The preferred embodiment of the present invention has been described in detail above, it should be understood that the ordinary skill of this field is without creativeness
Labour, which according to the present invention can conceive, makes many modifications and variations.Therefore, all technician in the art are according to this
Inventive concept is in prior art basis by logic analysis, reasoning or according to the limited available technical side of experiment
Case, should be among the protection scope determined by the claims.
Claims (9)
1. a kind of tumor markers CA9 and UCA1 answering in the kit for preparing the probability that Noninvasive testing suffers from bladder cancer
With.
2. tumor markers CA9 and UCA1 according to claim 1 is preparing probability that Noninvasive testing suffers from bladder cancer
Application in kit, which is characterized in that
The forward primer of CA9 is SEQ.4:CATCCTAGCCCTGGTTTTTGG,
The reverse primer of CA9 is SEQ.5:CCTTCTGTGCTGCCTTCTCAT,
The TaqMan probe of CA9 is SEQ.6:CTGTCACCAGCGTCGCGTTCCTT,
The forward primer of UCA1 is SEQ.7:GCCCTCATTCCGTGAAGAGA,
The reverse primer of UCA1 is SEQ.8:ATTTGAAATTGGTGAGATGTTCCTT,
The TaqMan probe of UCA1 is SEQ.9:CCACCTGCGACCTCGGGTCCT.
3. tumor markers CA9 and UCA1 according to claim 1 or 2 suffers from the general of bladder cancer preparing Noninvasive testing
Application in the kit of rate, which is characterized in that a and d respectively represent the fluorescent quantitation of CA9 and UCA14 gene as a result,
Logit (P)=1.168*a+0.855*d-1.177, or
Logit (P)=1.048*a+0.679*d-1.344,
The value of Logit (P) is used to judge the probability for suffering from bladder cancer.
4. tumor markers CA9 and UCA1 according to claim 1 or 2 suffers from the general of bladder cancer preparing Noninvasive testing
Application in the kit of rate, which is characterized in that further include tumor markers CK20.
5. tumor markers CA9 and UCA1 according to claim 4 is in the probability for preparing Noninvasive testing and suffering from bladder cancer
Kit in application, which is characterized in that
The forward primer of CK20 is SEQ.1:AAAAGGAGCATCAGGAGGAAGTC,
The reverse primer of CK20 is SEQ.2:GCATCAACCTCCACATTGACA,
The TaqMan probe of CK20 is SEQ.3:ATGGCCTACACAAGCATCTGGGCAA.
6. tumor markers CA9 and UCA1 according to claim 4 is in the probability for preparing Noninvasive testing and suffering from bladder cancer
Kit in application, which is characterized in that the fluorescent quantitation result of the gene of the CK20 be b,
Logit (P)=1.146*a-0.068*b+0.692*d-0.924, or
Logit (P)=1.274*a-0.077*b+0.84*d-0.714,
The value of Logit (P) is used to judge the probability for suffering from bladder cancer.
7. tumor markers CA9 and UCA1 according to claim 1 is in the probability for preparing Noninvasive testing and suffering from bladder cancer
Kit in application, which is characterized in that further include the primer and needle sequence of endogenous control gene GAPDH and β-actin.
8. tumor markers CA9 and UCA1 according to claim 7 is in the probability for preparing Noninvasive testing and suffering from bladder cancer
Kit in application, which is characterized in that
The forward primer of GAPDH is SEQ.13:CACATGGCCTCCAAGGAGTAA,
The reverse primer of GAPDH is SEQ.14:TGAGGGTCTCTCTCTTCCTCTTGT,
The TaqMan probe of GAPDH is SEQ.15:CTGGACCACCAGCCCCAGCAAG,
The forward primer of β-actin is SEQ.16:TGCCGACAGGATGCAGAAG,
The reverse primer of β-actin is SEQ.17:CTCAGGAGGAGCAATGATCTTGA,
The TaqMan probe of β-actin is SEQ.18:ATCACTGCCCTGGCACCCAGCA.
9. tumor markers CA9 and UCA1 according to claim 1 is in the probability for preparing Noninvasive testing and suffering from bladder cancer
Kit in application, which is characterized in that the kit be preliminary screening or Follow-up After when use.
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| CN110541030A (en) * | 2019-08-22 | 2019-12-06 | 广州医科大学附属肿瘤医院 | bladder cancer detection kit and application thereof |
| CN111154879A (en) * | 2020-03-06 | 2020-05-15 | 北京恩泽康泰生物科技有限公司 | Biomarker and kit for bladder cancer diagnosis or recurrence monitoring |
| CN113122640A (en) * | 2021-05-31 | 2021-07-16 | 中国医学科学院肿瘤医院 | Use of DNA copy number variation of CEP63 and FOSL2 in diagnosis of urothelial carcinoma of bladder |
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