CN107607712A - System for predicting bladder cancer patients chemosensitivity - Google Patents
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Abstract
The invention discloses a kind of system for predicting bladder cancer patients chemosensitivity, the system includes gene assaying device, SABC detection means and data processing equipment, wherein, the gene assaying device, the testing result of the immuno-chromatography detection device are analyzed and processed by the data processing equipment；Wherein, the gene assaying device includes DNA extraction kit, primer sets, PCR amplification instrument, PCR purification kits, sequenator and electrophoresis apparatus.The present invention is used for the system for predicting bladder cancer patients chemosensitivity, include FGFR3, HRAS, TERT promoter, TP53, TSC1, PIK3CA, HER2 by using carcinoma of urinary bladder mutator, unconventionality expression albumen includes EGFR, ERCC1, CK20, BRCA1, CD44, RRM1, PD L1, TUBB3, Ki67 and CD71, design prepares corresponding specific primer and antibody and detected, to meet the detection demand of above-mentioned molecular pathology index, it is and easy, economical, Chemotherapy for Bladder Cancer sensitivity prediction accuracy can be greatly improved, for instructing new adjuvant chemotherapy to implement.
Description
Technical field
The present invention relates to biological gene and technical field of immunoassay, and in particular to one kind is used to predict bladder cancer patients
The system for treating sensitiveness.
Background technology
Carcinoma of urinary bladder (bladder cancer, BC) is one of most common tumour of urinary system, and its incidence of disease and the death rate occupy
China's urological cancer is the first.Carcinoma of urinary bladder can be divided into the non-Myometrial involvement carcinoma of urinary bladder of mamillary and Myometrial involvement carcinoma of urinary bladder, preceding
Person often to bladder Intracavity, has high relapse rate, Postoperative recurrent rate is up to 70%；The latter have stronger propagation, invasion and attack,
Mortality transfer can occur for transfer ability, the patient that ratio is up to 50%.Operation is the primary treatment regimen of carcinoma of urinary bladder, for office
Sex-limited carcinoma of urinary bladder, perform the operation prognosis bona；For not limited carcinoma of urinary bladder, especially Myometrial involvement carcinoma of urinary bladder, surgery alone tool
There are higher recurrence rate and DISTANT METASTASES IN risk, do not reach preferable therapeutic effect.Therefore, for the latter, after average of operation periods often
Carry out system chemotherapy is needed to reduce recurrence and metastatic rate.As the systemic chemotherapy before malignant tumour local operation and radiotherapy, newly
NACT can eliminate potential micro metastasis, provide surgical condition for patient, improve surgical radical treatment rate；Tumour can be reduced
Cytoactive, make to be not easy to spread during its operation into blood, reduce and shifted in art, improve carcinoma of urinary bladder postoperative patient prognosis.
Carcinoma of urinary bladder early stage Neoadjuvant Chemotherapy uses cis-platinum, adriamycin, methotrexate (MTX) and vincristine, draws successively later
Novel cytotoxic medicine is entered, such as gemcitabine, taxol, the clinical practice for substantially increasing carcinoma of urinary bladder new adjuvant chemotherapy is imitated
Fruit.However, although new adjuvant chemotherapy can definitely improve carcinoma of urinary bladder totality prognosis, the bladder cancer patients of chemotherapy resistance are produced effects
It is little, and be likely to the treatment for being therefore delayed such patient, in addition, the intrinsic toxicity of new adjuvant chemotherapy can make the treatment pair
The unfavorable increasing of patient.How effectively to screen, predict that patient is alleviated or resisted to new adjuvant chemotherapy application using new adjuvant chemotherapy
Validity it is most important, and be the problem urgently captured of this area.
At present, tumour produces resistance to chemotherapy by a series of molecular mechanism, gene that these molecular mechanisms include, point
The exception of subsignal etc. can reflect the resistance ability of tumour.Therefore, tumor drug resistance is detected, the focus molecular pathology of prognosis correlation refers to
Mark and panel reasonable in design, can predict the sensitiveness of tumours of chemotherapeutic to a certain extent, instruct the execution of chemotherapy regimen.
Traditional iconography or invasive inspection are only capable of intuitively reflecting the attributes such as tumor size, number, invasion and attack scope, are not related to swollen
The generation of knurl, development, resistance mechanism, a quantizating index, the more application of leisure opinion can not be provided to bladder cancer patients chemosensitivity
Prediction in terms of malignant tumor chemotherapy sensitiveness.Detecting single molecular pathology index often has very large deviation, can not ensure pre-
Survey the accuracy of Chemotherapy in Patients sensitiveness.
In summary, traditional equipment can not provide quantizating index to the sensitiveness of bladder cancer patients chemotherapy, and single
Pathological index can not ensure to predict the accuracy of Chemotherapy in Patients sensitiveness, it would be highly desirable to further to improve.
The content of the invention
It is existing to solve it is an object of the invention to provide a kind of system for predicting bladder cancer patients chemosensitivity
There is device can not provide quantizating index to the sensitiveness of bladder cancer patients chemotherapy, can not ensure to predict the standard of Chemotherapy in Patients sensitiveness
True property.
To achieve the above object, the present invention provides a kind of system for predicting bladder cancer patients chemosensitivity, described
System includes gene assaying device, SABC detection means and data processing equipment, wherein, the gene assaying device,
The testing result of the immuno-chromatography detection device is analyzed and processed by the data processing equipment；
Wherein, the gene assaying device includes DNA extraction kit, primer sets, PCR amplification instrument, PCR purified reagents
Box, sequenator and electrophoresis apparatus；The bladder body DNA of the DNA extraction kit extraction is obtained after PCR amplification instrument expands
Product pass through PCR Purification Kits, the purifying DNA obtained after purification passes through sequencer, obtains target gene
DNA sequence dna；
The SABC detection means includes FFPE device, slicer and microscope, and FFPE device will
After bladder body carries out FFPE, bladder tissue sections, the bladder tissue sections, by corresponding are obtained by slicer
Antigen and antibody are handled the bladder tissue sections, and bladder tissue sections detection knot is obtained by the micro- sem observation
Fruit；
The target gene DNA sequence dna and bladder tissue sections testing result input data processing unit that sequenator is obtained enter
Row processing, the target gene DNA sequence dna that sequenator obtains is compared with normal gene DNA sequence dna, determines that bladder body is mutated base
Cause, meanwhile, compared with the testing result of bladder body histotomy is cut into slices into expressing quantity with normal bladder tissue, it is determined that
The expression quantity of specific protein；
According to associated gene mutation species and the data of expressing quantity, resistance or alleviation after Chemotherapy in Patients are judged.
Preferably, the primer sets include gene FGFR3 primer sets：SEQ ID NO.1、SEQ ID NO.2、SEQ ID
NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6；
Gene HRAS primer sets：SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10；
Gene TERT primer sets：SEQ ID NO.11、SEQ ID NO.12；
Gene TP53 primer sets：SEQ ID NO.13、SEQ ID NO.14；
Gene TSC1 primer sets：SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、
SEQ ID NO.19、SEQ ID NO.20；
Gene PIK3CA primer sets：SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24；
Gene HER2 primer sets：SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.28.
Preferably, the albumen be ERCC1, EGFR, CK20, BRCA1, CD44, RRM1, PD-L1, TUBB3, Ki67 and
CD71；
The antibody is corresponding for ERCC1, EGFR, CK20, BRCA1, CD44, RRM1, PD-L1, TUBB3, Ki67 and CD71
Primary antibody；
When detecting TSC1, TERT promoter, PIK3CA gene mutations；The high table of CD71, CD44, Ki67, BRCA1 albumen
Reach, can be resisted after chemotherapy；
When detecting FGFR3, HRAS, HER2, TP53 gene mutation；RRM1、CK20、TUBB3、ERCC1、EGFR、PD-L1
Albumen low expression, it can alleviate after chemotherapy.
Preferably, the system also includes the spectrophotometer for being used to detect DNA concentration.
Preferably, the gene assaying device also includes being used for 96 orifice plates for purifying DNA.
Preferably, the system also includes being used for the centrifugation centrifuge to DNA.
Preferably, the gene assaying device also includesTerminator v3.1cyclesequencing
Kit is as sequencing reaction kit.
Preferably, the data processing equipment is mobile phone or computer.
The invention has the advantages that：
The present invention is used for the system for predicting bladder cancer patients chemosensitivity, includes by using carcinoma of urinary bladder mutator
FGFR3, HRAS, TERT promoter, TP53, TSC1, PIK3CA, HER2, unconventionality expression albumen include EGFR, ERCC1, CK20,
BRCA1, CD44, RRM1, PD-L1, TUBB3, Ki67 and CD71, design prepare corresponding specific primer and antibody and examined
Survey, to meet the detection demand of above-mentioned molecular pathology index, and it is easy, economical, Chemotherapy for Bladder Cancer sensitiveness can be greatly improved
Forecasting accuracy, for instructing new adjuvant chemotherapy to implement.
Brief description of the drawings
Fig. 1 is the composition schematic diagram for being used to predict the system of bladder cancer patients chemosensitivity of the present invention.
Fig. 2 is FGFR3, HRAS, TERT promoter, TP53, TSC1, PIK3CA and HER2 base in embodiments of the invention 1
Graph of a relation between alleviating because of mutation and GC resistances or GC, GC resistance groups TSC1, TERT promoter, PIK3CA gene mutation ratio
Rate is significantly higher than GC alleviation groups；And GC resistance groups FGFR3, HRAS, HER2, TP53 gene mutation ratio are substantially less than GC alleviations
Group.
Fig. 3 be embodiments of the invention 1 in, EGFR, ERCC1, CK20, BRCA1, CD44, RRM1, PD-L1, TUBB3,
Ki67 and CD71 albumen expression quantity and GC resistance or GC alleviate between graph of a relation, GC resistance groups CD71, CD44, Ki67,
BRCA1 expressing quantities are significantly higher than GC alleviation groups；And GC resistance group RRM1, CK20, TUBB3, ERCC1, EGFR, PD-L1 eggs
White expression quantity is substantially less than GC alleviation groups.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
As shown in figure 1, the system for predicting bladder cancer patients chemosensitivity of the present invention, it includes genetic test dress
Put, SABC detection means and data processing equipment, wherein, the detection of gene assaying device, immuno-chromatography detection device
As a result analyzed and processed by data processing equipment；Wherein, gene assaying device include DNA extraction kit, primer sets,
PCR amplification instrument, PCR purification kits, sequenator and electrophoresis apparatus；The bladder body DNA of DNA extraction kit extraction passes through
The product obtained after PCR amplification instrument amplification passes through PCR Purification Kits, and the purifying DNA obtained after purification passes through sequenator
Sequencing, obtain the DNA sequence dna of target gene；
SABC detection means includes FFPE device, slicer and microscope, and FFPE device is by bladder
After tissue carries out FFPE, bladder tissue sections are obtained by slicer, bladder tissue sections, by corresponding antigen and resisted
Body is handled bladder tissue sections, and bladder tissue sections testing result is obtained by micro- sem observation；
At the gene DNA sequence and bladder tissue sections testing result input data processing unit that sequenator is obtained
Reason, the gene DNA sequence that sequenator obtains is compared with normal gene DNA sequence dna, determines bladder body mutator, meanwhile,
Compared with the testing result of bladder body histotomy is cut into slices into expressing quantity with normal bladder tissue, specific protein is determined
Expression quantity.
TSC1, TERT promoter, PIK3CA gene mutations；The high expression of CD71, CD44, Ki67, BRCA1 albumen, promptingization
It can be resisted after treatment, sensitiveness is poor；FGFR3, HRAS, HER2, TP53 gene mutation；RRM1、CK20、TUBB3、ERCC1、EGFR、
PD-L1 albumen low expressions, it can alleviate after prompting chemotherapy, sensitiveness is high.
Specifically, gene assaying device extracts bladder body specimen dna, with Sanger sequencing technologies, carcinoma of urinary bladder group is detected
The gene mutation closely related with carcinoma of urinary bladder in knitting, including FGFR3, HRAS, TERT promoter, TP53, TSC1, PIK3CA and
The catastrophe of HER2 genes, wherein, primer sets include gene FGFR3 primer sets：SEQ ID NO.1、SEQ ID NO.2、SEQ
ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6；Gene HRAS primer sets：SEQ ID NO.7、SEQ ID
NO.8、SEQ ID NO.9、SEQ ID NO.10；Gene TERT primer sets：SEQ ID NO.11、SEQ ID NO.12；Gene
TP53 primer sets：SEQ ID NO.13、SEQ ID NO.14；Gene TSC1 primer sets：SEQ ID NO.15、SEQ ID
NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20；Gene PIK3CA primer sets：
SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24；Gene HER2 primer sets：SEQ ID
NO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.28。
Pcr amplification primer thing group by can respectively specific amplification go out FGFR3, HRAS, TERT promoter, TP53, TSC1,
The primer composition of PIK3CA and HER2 gene orders, specific objective gene sequence and corresponding primer such as table 1.
The objective gene sequence of table 1 and primer composition
The catastrophe step of the gene assaying device detection related gene of the present invention is as follows：
Step 1：Utilize DNA extraction kit extraction bladder body DNA；By the tissue of break process through protease K digesting
Afterwards, tissue DNA is extracted using Qiagen companies DNA extraction kit (QIAamp DNA Mini Kit).
Step 2：Target gene is expanded using the primer and PCR amplification instrument of primer sets, the product of amplification is led to
Cross the electrophoretic band that electrophoresis apparatus electrophoresis obtains target gene PCR primer；
Step 3：The target gene DNA that purifying obtains purifying is carried out to PCR primer by PCR purification kits；Wherein PCR
Purification kit is purified using Qiagen companies PCR purification kits (QIAquick PCR Purication Kit).
Step 4：Sequencing reaction is carried out to target dna by PCR instrument, obtains sequencing reaction product；Wherein, Sanger is sequenced
Reaction usesTerminator v3.1cycle sequencing Kit sequencing kits (Sanger
Sequencing Kits＆Reagents) carry out.
Step 5：The sequencing reaction product of acquisition is purified, obtains sequencing reaction product after purification；
Step 6：Sequencing reaction product after purification is subjected to DNA sequencing by sequenator；Wherein, sequenator uses ABI
3730 sequencers.
Step 7：DNA sequencing result is analyzed and processed using data processing equipment mobile phone or computer.At data
Reason device is analyzed sequencing result using Variant Report v1.1 softwares.
The present invention detects the protein of unconventionality expression in Bladder Cancer, immunohistochemistry dye by SABC device
Color detection carcinoma of urinary bladder sample in unconventionality expression protein include EGFR, ERCC1, CK20, BRCA1, CD44, RRM1, PD-L1,
TUBB3, Ki67 and CD71.And the antibody corresponding to above-mentioned albumen.
The present invention is as follows by the specific detecting step of SABC device：
Step 1：By Bladder Cancer FFPE and cut into slices by FFPE device, obtain paraffin section；
Step 2：Roasting piece, dewaxing aquation are carried out to Bladder Cancer paraffin section by slicer；
Step 3：Tissue antigen recovery；
Step 4：3%H2O2Close endogenous catalase；
Step 5：Antibody hybridization, with EGFR, ERCC1, CK20, BRCA1, CD44, RRM1, PD-L1, TUBB3, Ki67 and
The specific antibody of the protein such as CD71 carries out immunohistochemical staining to section.
Step 6：Colour developing；
Step 7：Redye；
Step 8：Dehydration, transparent and mounting.
SABC device of the present invention is based on Sanger PCR sequencing PCRs and SABC (IHC) technology, is filled by genetic test
Put and the system for predicting bladder cancer patients chemosensitivity of SABC device composition, detection tissue carcinoma of urinary bladder are related
Gene mutation and unconventionality expression albumen, so formed carcinoma of urinary bladder molecular pathology index p anel to patient's bladder tumor recurrence, prognosis,
Drug susceptibility is analyzed, for instructing the implementation of carcinoma of urinary bladder new adjuvant chemotherapy.
Embodiment 1
The present embodiment 1 is to 39 bladder cancer patients tissue specimens from Shanghai Ren Ji hospitals, containing " gemcitabine/cis-platinum
Resistance group is (referred to as：GC is resisted) 23 ", " gemcitabine/cis-platinum alleviation group is (referred to as：GC is alleviated) 16 ", carry out carcinoma of urinary bladder phase
Correlation gene is sequenced and unconventionality expression protein I HC dyeing.Specimen information such as table 3.
The bladder cancer patients tissue sample info of table 2.
1st, materials, Sample storage Bladder Cancer
- 80 DEG C of the bladder body sample or Liquid nitrogen storage that the present invention is obtained.The sample of each patient is divided into two parts, and one
The formaldehyde of part 4% is fixed, and normal temperature preserves, and subsequently gives FFPE and cuts into slices；Another part shreds rear -80 or liquid with tissue shear
Nitrogen preserves, subsequently extracting DNA.
2nd, Sanger is sequenced
Tissue DNA is extracted, the tissue of break process is after protease K digesting, using Qiagen companies DNA extraction kit
(QIAamp DNA Mini Kit) extract tissue DNA, concrete operations by product description carry out, tissue DNA extract after with
The spectrophotometers of Thermo companies NanoDrop 2000 detect sample DNA concentration.
Wherein, the extraction process of tissue DNA is as follows：
A. the tissue (about 30mg or so) after will be broken is placed in centrifuge tube, adds 180 μ l ATL buffer solutions and 20ul eggs
White enzyme K (PK), shakes 20s, and 56 DEG C of water-baths are digested to histolysis；
B. 200ul AL buffer solutions are added, swing 15,10min is acted in 70 DEG C of water-baths；
C. the ethanol of the purity of 200ul more than 95% is added, shakes 15s；
D. prepare centrifugal column and collecting pipe, mixture obtained by previous step is added in centrifugal column, 8000rpm centrifuges
1min, abandon and collect liquid in pipe；
E. 500ul AW1 buffer solutions are carefully added into centrifugal column, 14000rpm centrifuge 3min, abandon collecting pipe
Interior liquid；
F. 500ul AW2 buffer solutions are carefully added into centrifugal column, 14000rpm centrifugation 3min, abandons and collects intraluminal fluid
Body；
G. centrifugal column idle running 1min removes residual AW2 buffer solutions；
H. centrifugal column is placed in the clean EP of 1.5ml, adds 200ul AE buffer solutions or ddH2O, room temperature are protected after placing several minutes
It is stored in -20 refrigerators.
3rd, expanded using PCR amplification instrument, by PCR electrophoresis apparatuses to PCR primer electrophoresis：Such as the upstream and downstream primer pair of table 1
FGFR3, HRAS, TERT promoter, TP53, TSC1, PIK3CA and HER2 gene order carry out specific amplification.Wherein, PCR expands
It is as follows to increase system configurations：10 × PCR buffer, 2.5ul；10 × PCR buffer, 3ul；2.5Mm dNTPs, 3ul；Draw upstream
Thing, 1ul；Anti-sense primer, 1ul；LA Taq enzymes, 0.3ul；Sample DNA templates, 2ul；H2O, 15.2ul.
PCR response procedures：96 DEG C of 2min pre-degenerations, 96 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 2min, 35 circulations, 72 DEG C of extensions
5min, 4 DEG C of ∞.Different genes PCR procedure conditions difference.
4th, PCR primer purifies：Using Qiagen companies PCR purification kits (QIAquick PCRPurication Kit)
PCR primer is purified, purification step is as follows：
A.150ul PB buffer solutions are added to PCR primer, are sufficiently mixed；
B. mixture obtained by upper step is moved into adsorption column, 13000rpm centrifugations 1min；
C. 750ul PE buffer solutions are added into adsorption column, react 5min, 13000 centrifugation 1min, abandons after waste liquid and dallies one
It is secondary, remove remaining waste；
D. adsorption column is placed in clean EP pipes, toward adsorption column center plus 50ulEB or ddH2O, stand several minutes of dissolving absorption
DNA；
E.13000rpm 1min is centrifuged, collects the DNA of dissolving, -20 preserve.
5th, sequencing reaction, useTerminator v3.1cycle sequencing Kit are anti-as sequencing
Answer kit.Reagent：BigDye, 0.4ul；Sequencing Buffer, 0.8ul；Primer (3.2pmol/ μ l), 1ul；(2) step
Gained PCR primer, 1ul；H2O, 1.8ul.
Sequencing reaction configuration scheme is as follows, PCR sequencing reaction programs：96 DEG C of 2min pre-degenerations, 96 DEG C of 10s, 55 DEG C of 5s, 60
DEG C 90s, 25 circulations, 4 DEG C of ∞, different genes PCR procedure conditions difference.
6th, sequencing reaction product purification, using ethanol/EDTA-Na2Method purifies sequencing reaction product, comprises the following steps that：
A. 96 orifice plates are centrifuged, 2.5 μ l EDTA-Na2 (0.125mol/L), the fully ethanol of 40 μ l 85%, shake are added per hole
Swing several minutes, 4 DEG C, 30min is centrifuged under the conditions of 2000-3000g；
B.96 stop immediately when the orifice plate heterogeneous example showing an absence of inverse disconnection between the middle term and the major term heart to centrifugal force reaches 900rpm, 50 μ l70% ethanol are added per hole, fully
1min is shaken, 4 DEG C, 15min is centrifuged under the conditions of 3000g, repeats the step 1 time；
C.96 orifice plate room temperature, 10ul deionized formamide HIDI are added after 15~30min of avoid light place, per hole, centrifugation, 96
DEG C PCR instrument reaction 2min, 4 DEG C of taking-ups.
7th, by the operation of the sequenator specifications of ABI 3730 PCR purified products are sequenced.
8th, using data processing equipment to sequencing peak figure analyze, judge associated gene mutation whether and mutation classification.
Related antigen is detected by the SABC device of the present invention, its step is as follows：
(1) Bladder Cancer FFPE device is embedded, slicer is cut into slices, 60 DEG C of roasting piece 1-2h, dimethylbenzene
Dewax three times, each 10min.
(2) immerse successively in 100%, 95%, 85%, 75% and pure water, each 5min.
(3) distilled water cleans 2 times, each 5min.
(4) 3% hydrogen peroxide close 20min.
(5) EDTA is repaired：20min is maintained after boiling, pays attention to adding EDTA, natural cooling in time.
(6) PBST (PBS+0.025%Triton X-100) buffer solution for cleaning 3 times, each 5min.
(7) serum block closing 1-2h (confining liquids：10% serum+1%BSA+PBST).
(8) after blotting, appropriate primary antibody (volume 50-100ul or so, primary antibody dilution is added dropwise：1%BSA+PBST.In group
Knit on slide, be put into water wet box, 4 DEG C of refrigerator overnights.
(9) PBST buffer solution for cleaning 3 times, each 5min.
(10) appropriate secondary antibody is added dropwise, reacts at room temperature 1 hour.
(11) PBST buffer solution for cleaning 3 times, each 5min.
(12) DAB colour developings 5min or so, Microscopic observation, when tissue color becomes yellowish-brown, immerses in distilled water eventually at any time
Only react.
(13) bush uniformly dyeing core 2-3 minutes, distill water washing, hydrochloride alcohol differentiation 30s, distill water washing, running water or
1% ammoniacal liquor returns indigo plant.
(14) it is dehydrated, is transparent：Invade successively each in 75%, 95%, 100% and dimethylbenzene 1, dimethylbenzene 2, dimethylbenzene 3
1min。
(15) slide is taken out from dimethylbenzene, is dried, neutral gum is added dropwise, is capped slide mounting.
As shown in Fig. 2 gene assaying device is to gene sequencing result, by analyzing gene sequencing data, identification
FGFR3, HRAS, TERT promoter, TP53, TSC1, PIK3CA, HER27 are individual in 39 Bladder Cancer samples of Liao Renji hospitals
Focus gene mutation situation.GC resistance groups TSC1, TERT promoter, PIK3CA gene mutation ratio are significantly higher than GC alleviations
Group；And GC resistance groups FGFR3, HRAS, HER2, TP53 gene mutation ratio are substantially less than GC alleviation groups.
As shown in figure 3, the result of the immunohistochemical staining of the present invention, to Shanghai Ren Ji hospitals 39 to carcinoma of urinary bladder and normal group
Knit sample and carry out IHC coloration results, analyze Bladder Cancer pathological index panel and the state of an illness, chemotherapy necessity, the quick property of sense are commented
Estimate.GC resistance groups TSC1, TERT promoter, PIK3CA gene mutation ratio are significantly higher than GC alleviation groups；And GC resistance groups
FGFR3, HRAS, HER2, TP53 gene mutation ratio are substantially less than GC alleviation groups.GC resistance groups CD71, CD44, Ki67,
BRCA1 expressing quantities are significantly higher than GC alleviation groups；And GC resistance group RRM1, CK20, TUBB3, ERCC1, EGFR, PD-L1 eggs
White expression quantity is substantially less than GC alleviation groups.
In summary, (1) TSC1, TERT promoter, PIK3CA gene mutations；CD71, CD44, Ki67, BRCA1 albumen are high
Expression, it can be resisted after prompting chemotherapy.(2) FGFR3, HRAS, HER2, TP53 gene mutation；RRM1、CK20、TUBB3、ERCC1、
EGFR, PD-L1 albumen low expression, it can alleviate after prompting chemotherapy.With reference to the Bladder Cancer mutator and related egg detected
White unconventionality expression situation, converges into bladder cancer patients molecular pathology index p anel, to conditions of patients, chemotherapy necessity and sensitiveness
Assessed, it is significant to instructing doctor to implement new adjuvant chemotherapy to patient.
The present invention utilizes Sanger gene sequencing goldstandards, and it is direct by PCR for gene mutation site design primer
Amplification sequencing, has high accuracy, simple, fast feature.IHC passes through chemistry according to antigen and antibody specific reaction principle
React the research that relative quantification is carried out to destination protein.The present invention is by can give multiple molecular pathology index p anel simultaneously
The higher prediction effect addition of the present invention, has a good application prospect.
Although above with general explanation and specific embodiment, the present invention is described in detail, at this
On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
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Claims (8)
1. a kind of system for predicting bladder cancer patients chemosensitivity, it is characterised in that the system includes genetic test
Device, SABC detection means and data processing equipment, wherein, the gene assaying device, immunochromatography detection
The testing result of device is analyzed and processed by the data processing equipment；
Wherein, the gene assaying device includes DNA extraction kit, primer sets, PCR amplification instrument, PCR purification kits, survey
Sequence instrument and electrophoresis apparatus；The production that the bladder body DNA of the DNA extraction kit extraction is obtained after PCR amplification instrument expands
Thing passes through PCR Purification Kits, and the purifying DNA obtained after purification passes through sequencer, obtains the DNA sequences of target gene
Row；
The SABC detection means includes FFPE device, slicer and microscope, and FFPE device is by bladder
After tissue carries out FFPE, bladder tissue sections are obtained by slicer, the bladder tissue sections, pass through corresponding antigen
The bladder tissue sections are handled with antibody, bladder tissue sections testing result is obtained by the micro- sem observation；
At the target gene DNA sequence dna and bladder tissue sections testing result input data processing unit that sequenator is obtained
Reason, the target gene DNA sequence dna that sequenator obtains is compared with normal gene DNA sequence dna, determines bladder body mutator, together
When, compared with the testing result of bladder body histotomy is cut into slices into expressing quantity with normal bladder tissue, determine specific
The expression quantity of albumen；According to associated gene mutation species and the data of expressing quantity, judge after Chemotherapy in Patients resistance or
Alleviate.
2. the system according to claim 1 for predicting bladder cancer patients chemosensitivity, it is characterised in that
The primer sets include gene FGFR3 primer sets：SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID
NO.4、SEQ ID NO.5、SEQ ID NO.6；
Gene HRAS primer sets：SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10；
Gene TERT primer sets：SEQ ID NO.11、SEQ ID NO.12；
Gene TP53 primer sets：SEQ ID NO.13、SEQ ID NO.14；
Gene TSC1 primer sets：SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID
NO.19、SEQ ID NO.20；
Gene PIK3CA primer sets：SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24；
Gene HER2 primer sets：SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.28.
3. the system according to claim 2 for predicting bladder cancer patients chemosensitivity, it is characterised in that
The albumen is ERCC1, EGFR, CK20, BRCA1, CD44, RRM1, PD-L1, TUBB3, Ki67 and CD71；
The antibody is one corresponding to ERCC1, EGFR, CK20, BRCA1, CD44, RRM1, PD-L1, TUBB3, Ki67 and CD71
It is anti-；
When detecting TSC1, TERT promoter, PIK3CA gene mutations；The high expression of CD71, CD44, Ki67, BRCA1 albumen, changes
It can be resisted after treatment；
When detecting FGFR3, HRAS, HER2, TP53 gene mutation；RRM1, CK20, TUBB3, ERCC1, EGFR, PD-L1 albumen
Low expression, it can alleviate after chemotherapy.
4. the system according to claim 1 for predicting bladder cancer patients chemosensitivity, it is characterised in that
The system also includes the spectrophotometer for being used to detect DNA concentration.
5. the system according to claim 1 for predicting bladder cancer patients chemosensitivity, it is characterised in that
The gene assaying device also includes being used for 96 orifice plates for purifying DNA.
6. the system according to claim 1 for predicting bladder cancer patients chemosensitivity, it is characterised in that
The system also includes being used for the centrifugation centrifuge to DNA.
7. the system according to claim 1 for predicting bladder cancer patients chemosensitivity, it is characterised in that
The gene assaying device also includesTerminator v3.1 cycle sequencing Kit are as survey
Sequence reaction kit.
8. the system according to claim 1 for predicting bladder cancer patients chemosensitivity, it is characterised in that
The data processing equipment is mobile phone or computer.
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