CN102435734A - Kit used for evaluating ovarian cancer primary chemotherapeutic sensitivity, and application thereof - Google Patents
Kit used for evaluating ovarian cancer primary chemotherapeutic sensitivity, and application thereof Download PDFInfo
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Abstract
The invention discloses a kit used for evaluating ovarian cancer primary chemotherapeutic sensitivity, and an application thereof. The kit comprises a blocking reagent, an antigen-antibody reaction system, an avidin-oxide enzyme reaction system and a counterstaining drug. The invention is provided based on the relevance between the expression level of ERCC2 in serous ovarian cancer cells/tissues and sensitivity of cells, tissues or a tumor patient as a host to a primary chemotherapeutic drug. Through ERCC2 detection, purposes such as chemotherapeutic sensitivity/drug resistance evaluation, tumor patient discrimination, and primary chemotherapeutic drug tolerability evaluation can be achieved. The method provided by the invention is simple and accurate. The requirement on a sample is not high. Therefore, the kit and the method can be used in large-scale screening or trace researches. When the kit and the method is used in clinical diagnosis and medication guidance, ex vivo cancer tissues of a patient can be acquired for detection after operation, and the detection is characterized in no wound, no pain, and noninvasiveness. The detection can easily be accepted by patients. With the kit and the application, patient lives can be prolonged, chemotherapy pains of the patients can be alleviated, and life qualities of the patients after operations can be improved.
Description
Technical field
The present invention relates to a kind of kit of the chemotherapy in ovarian cancer susceptibility that is used for testing and assessing and screen at former chemotherapy sensitivity/resistance test and appraisal, tumor patient, and the application of former chemotherapeutics tolerance assessment.
Background technology
(Epithelial Ovarian Cancer is one of common tumour of female sex organ EOC) to the epithelial ovarian cancer, is the primary killer in the female tumor.Wherein the serosity cancer is the most common, accounts for 80～85% of all oophoromas in western countries, is divided into late period with early stage, and its middle and advanced stage accounts for the overwhelming majority, very rarely account in early days all ovaries＜5%.Think now early, late period the serosity cancer be that two kinds of oophoroma are dissimilar, in fact its morphologic difference has reflected oncobiology difference, is the difference of gene level after all.This disease is difficult for diagnosis in early days, and patient had missed best occasion for the treatment when went to a doctor late period.Because the oophoroma end-stage patients have diffusion in various degree, so patient's postoperative must carry out chemotherapy at once.In the world ovarian cancer post operation patient's chemotherapy regimen mainly is divided at present: downpayment complementary (adjuvant; With platinum is the cis-platinum or the carboplatin/taxol on basis) and later stage rescue property (salvage; Multiple chemotherapeutics compatibility) treatment.Because patient's Gene regulation is different; Its developed by molecule level is also different, thus different to the susceptibility of former chemotherapeutics, thereby be that basic downpayment adjuvant chemotherapy does not have any alleviation concerning 40～50% patients' symptom with platinum; Also claim former resistance; Because treatment does not have specific aim, stopped over best chemotherapy period, be to cause one of dead fast reason of patient.Therefore, predict the susceptibility of patient before the chemotherapy to former chemotherapeutics, most important to the life that prolongs ovarian cancer patients.
On the other hand, the method according to tissue morphology classification is divided into four different histological type with the epithelial ovarian cancer for a long time, that is: serosity, and endometrial-like, mucus and hyaline cell property, wherein common with serosity again.But clinical practice is found; Only organize the typoiogical classification standard that oophoroma is classified according to tradition; Its pathological characters of further research and guiding clinical diagnosis and treatment there are deficiency more, therefore also need a kind of method the tumour under the traditional taxonomy item is further screened and to classify urgently.
Moreover as previously mentioned, the clinical medicine that is used to treat oophoroma does not have any alleviation to quite a few patient's symptom, but this need just can come to light through one quite long clinical period, tends to cause the delay treatment chance, increases less patient suffering.Therefore, also need a kind of fast and effectively high-throughput screening method in the drug development research, come the possible tolerance risk of former chemotherapeutics in the clinical treatment oophoroma of assessment objective pointedly.
Summary of the invention
Find in present inventor's research; The expressing quantity of ERCC2 is higher more than 2 times than mdr cell in sensitive cells; In conjunction with this discovery; The inventor utilizes China's oophoroma patient specimen, adopts simple immunohistochemical method, has confirmed the biomarker effect of ERCC2 the chemotherapy in ovarian cancer patient qualitatively.
Based on above-mentioned result of study; One of the object of the invention is to provide a kind of chemotherapy in ovarian cancer susceptibility test and appraisal kit; Described kit comprises: the confining liquid of being made up of the phosphate buffer of 5% lowlenthal serum, 1%BSA, 0.01% Triton X-100 and pH7.4; The antigen-antibody reaction system that forms by ERCC2 antibody (1: 1000), biotinylation two anti-working fluids and confining liquid; Avidin-oxide enzymatic reaction system of forming by horseradish enzyme labeling strepto-avidin working fluid and DAB reagent damping fluid, and as the haematine of counterstain.
In the embodiment preferred, described kit also comprises the antigen retrieval system that is made up of 0.01M citrate buffer and phosphate buffer, and the elimination endogenous peroxydase system that is made up of 3% hydrogen peroxide and phosphate buffer.Said kit can be applicable to paraffin-embedded ovarian cancer tissue sample pointedly.
Two of the object of the invention is to provide a kind of method of ovarian cancer tissue's sample to former chemotherapy drug susceptibility of testing and assessing; It is characterized in that using the described kit of claim 1; After comprising the steps: to use confining liquid to handle testing sample; Use ERCC2 antibody and biotinylation two anti-working fluids to carry out anti-and two anti-reactions respectively, use horseradish enzyme labeling strepto-avidin working fluid to handle sample then, carry out DAB colour developing and haematoxylin redyeing at last; Dyeing situation is per sample judged the susceptibility to former chemotherapeutics: the sample that is the brown positive reaction is judged to be the ovarian cancer tissue to former chemotherapy medicament sensitive; The sample that is blue look negative reaction is judged to be the ovarian cancer tissue to former chemotherapeutics tolerance.
Above-mentioned test and appraisal ovarian cancer tissue sample more specifically comprises the steps: the method for former chemotherapy drug susceptibility
1. sealing: the tissue at pending sample adds 200 μ l confining liquids, incubated at room 60 minutes;
2. antigen-antibody reaction:
(1) one anti-hatching: add the ERCC2 antibody of 150 μ l organizationally, cover tissue,, clean 3 times each 10 minutes then with PBS in 4 ℃ of incubated overnight to seal film according to 1: 100 dilution proportion;
If negative control group is used with the volume confining liquid to replace ERCC2 antibody.
(2) two anti-hatching: add 150 μ l biotinylations, two anti-working fluids organizationally, incubated at room 60 minutes, PBS cleans 3 times, each 10 minutes;
3. drip horseradish enzyme labeling strepto-avidin working fluid (S-A/HRP) organizationally, incubated at room 60 minutes, PBS cleans 3 times, each 10 minutes;
4. DAB colour developing:
(1) preparation substrate mixed liquor: in the distilled water of 1ml pH value=7.0, add a reagent A, after mixing, reagent B and reagent C respectively one be added dropwise to wherein, mix the substrate mixed liquor;
(2) colour developing: 150 μ l substrate mixed liquors are dripped organizationally color development at room temperature 3～10 minutes, fully colour developing back tap water flushing cessation reaction;
5. dye, dehydration, transparent, mounting: histotomy is immersed the haematine dye liquor 2 times, each 2 seconds; Handle 3 times each 5 minutes then with distilled water; Respectively handle 1 time each 1 minute more successively with 50% ethanol, 70% ethanol, 90% ethanol; Handle 2 times with absolute ethyl alcohol again, each 2 minutes, 1: 1 mixture process of xylene and absolute ethyl alcohol 1 time, 2 minutes, l00% xylene processing 2 times, each 2 minutes; Use gummy mounting at last, preserve;
6. dyeing situation is per sample judged the susceptibility to former chemotherapeutics: the sample that is the brown positive reaction is judged to be the ovarian cancer tissue to former chemotherapy medicament sensitive; The sample that is blue look negative reaction is judged to be the ovarian cancer tissue to former chemotherapeutics tolerance.
Be applicable to the tumor tissue in vitro or the cell sample of any kind on the theoretical method of test and appraisal ovarian cancer tissue sample to former chemotherapy drug susceptibility of the invention described above; Comprise ovarian cancer tissue, the formalin fixed FFPE ovarian cancer tissue of fresh collection, the freezing ovarian cancer tissue of OCT embedding, the fresh nothing that liquid nitrogen is preserved is ovarian cancer tissue etc. fixedly.No matter be applied to the biological specimen of which kind of type, those skilled in the art can confirm the pre-treating method of sample according to prior art information, made sample after the processing be applicable to according to the invention and SABC detect.Several kinds of typical samples and disposal route thereof are respectively:
When (1) said test and appraisal ovarian cancer tissue sample is applied to the test and appraisal of oophoroma formalin fixed paraffin-embedded tissue to the method for former chemotherapy drug susceptibility, The pretreatment is comprised the steps:
A. dewaxing: the conventional section of oophoroma formalin fixed paraffin-embedded tissue, the paraffin organization section of thickness 4～5 μ m is successively through 100% xylene dewaxing 3 times, each 5 minutes; Absolute ethyl alcohol dewaxing 3 times, each 2 minutes; 95% ethanol, 90% ethanol, 70% ethanol, 50% ethanol respectively dewax 1 time, 2 minutes;
B. antigen retrieval: carry out antigen retrieval with the 0.01M citrate buffer, clean 3 times each 5 minutes then with PBS;
C. eliminate endogenous peroxydase: histotomy is placed 3% hydrogen peroxide, and incubated at room 30 minutes is cleaned 3 times each 5 minutes with PBS.
Handling the gained sample directly is used for detecting.
When (2) said test and appraisal ovarian cancer tissue sample is applied to the test and appraisal of ovarian cancer tissue of freezing preservation of OCT embedding to the method for former chemotherapy drug susceptibility, The pretreatment is comprised the steps:
A. fixation of tissue: the about 2x2cm of fresh tumor tissues of excision when the oophoroma patient is performed the operation
3Insert immediately in 4% paraformaldehyde, 4 ℃, fixing 24 hours;
B.PBS cleans 3 times, each 10 minutes;
C. will organize and immerse 30% sucrose, 4 ℃ are spent the night;
D.OCT potpourri (opti-mum cutting temperature compound) embedding is placed on the dry ice;
E. after treating that the OCT potpourri solidifies, cryostat frozen section 7～10um;
Said test and appraisal ovarian cancer tissue sample is applied to fresh nothing that liquid nitrogen preserves fixedly during ovarian cancer tissue to the method for former chemotherapy drug susceptibility, and The pretreatment is comprised the steps:
A. the fresh tumor tissues of oophoroma patient excision is inserted rapidly in the liquid nitrogen, for use;
B. the time spent at first will be organized and immersed in 4% paraformaldehyde, and 4 ℃, fixing 24 hours;
According to the laboratory equipment that the user had, any in feasible above-mentioned two kinds of methods: promptly after FFPE or the OCT embedding process, handle the gained sample and directly be used for detecting.
The purpose of another aspect of the present invention provides the method for a kind of screening to the ovarian cancer patients of former chemotherapy medicament sensitive; This method also need be used the described chemotherapy in ovarian cancer susceptibility test and appraisal of the invention described above kit; Comprise the steps: that with the ovarian cancer patients tumor tissue in vitro be test sample; After using confining liquid to handle testing sample; Use ERCC2 antibody and biotinylation two anti-working fluids to carry out anti-and two anti-reactions respectively, use horseradish enzyme labeling strepto-avidin working fluid to handle sample then, carry out DAB colour developing and haematoxylin redyeing at last; The ovarian cancer patients that dyeing situation is per sample judged sample source is to former chemotherapy drug susceptibility: the sample that is the brown positive reaction is judged to be the ovarian cancer tissue to former chemotherapy medicament sensitive, and the ovarian cancer patients of sample source is to former chemotherapy medicament sensitive; The sample that is blue look negative reaction is judged to be the ovarian cancer tissue to former chemotherapeutics tolerance, and the ovarian cancer patients of sample source tolerates former chemotherapeutics.
The purpose of further aspect of the present invention provides a kind of method that is used to instruct the downpayment chemotherapy medication of ovarian cancer post operation patient; Comprise clinical collection patient ovarian cancer tissue, carry out SABC and detect and judge, confirm step such as therapeutic regimen, be in particular according to judged result:
I. sample collecting
II. the sample SABC detects and result's judgement
1. sealing: the tissue at pending sample adds 200 μ l confining liquids, incubated at room 60 minutes;
2. antigen-antibody reaction:
(1) one anti-hatching: add the ERCC2 antibody of 150 μ l organizationally, cover tissue,, clean 3 times each 10 minutes then with PBS in 4 ℃ of incubated overnight to seal film according to 1: 100 dilution proportion;
If negative control group is used with the volume confining liquid to replace ERCC2 antibody.
(2) two anti-hatching: add 150 μ l biotinylations, two anti-working fluids organizationally, incubated at room 60 minutes, PBS cleans 3 times, each 10 minutes;
3. drip horseradish enzyme labeling strepto-avidin working fluid (S-A/HRP) organizationally, incubated at room 60 minutes, PBS cleans 3 times, each 10 minutes;
4. DAB colour developing:
(1) preparation substrate mixed liquor: in the distilled water of 1ml pH value=7.0, add a reagent A, after mixing, reagent B and reagent C respectively one be added dropwise to wherein, mix the substrate mixed liquor;
(2) colour developing: 150 μ l substrate mixed liquors are dripped organizationally color development at room temperature 3～10 minutes, fully colour developing back tap water flushing cessation reaction;
5. dye, dehydration, transparent, mounting: histotomy is immersed the haematine dye liquor 2 times, each 2 seconds; Handle 3 times each 5 minutes then with distilled water; Respectively handle 1 time each 1 minute more successively with 50% ethanol, 70% ethanol, 90% ethanol; Handle 2 times with absolute ethyl alcohol again, each 2 minutes, 1: 1 mixture process of xylene and absolute ethyl alcohol 1 time, 2 minutes, 100% xylene processing 2 times, each 2 minutes; Use gummy mounting at last, preserve;
6. dyeing situation is per sample judged the susceptibility to former chemotherapeutics: the sample that is the brown positive reaction is judged to be the ovarian cancer tissue to former chemotherapy medicament sensitive; The sample that is blue look negative reaction is judged to be the ovarian cancer tissue to former chemotherapeutics tolerance.
III. interpretation of result and medication guide scheme determination
According to the testing result of Step II, it is high that the source patient of ovarian cancer tissue that can the preliminary judgement positive reaction treats responsive probability to former chemotherapeutics of postoperative; The source patient of the ovarian cancer tissue of negative reaction is high to former chemotherapeutics treatment of postoperative tolerance probability.For the latter, need show to confirm suitable postoperative chemotherapy scheme according to patient physiological pathology characteristic and clinical symptoms thereof.
Former the chemotherapeutics that reaches described in above-mentioned all methods of the present invention is meant and is used for the ovarian cancer post operation patient at the downpayment chemotherapeutics of implementing chemotherapy that this type medicine comprises platinum class, taxol or the combination of the two in the clinical practice.
Described in the technique scheme of the present invention and oophoroma or tumour be meant serous ovarian cancer late period.
The present inventor found expression and cell, the tissue of ERCC2 in tumour cell, especially the serous ovarian cancer cell/tissue or as host's tumor patient to the relevance between former the chemotherapy drug susceptibility.Although existing record about the ERCC2 method for immunohistochemical detection in the prior art, the definite relation between above-mentioned the two are never disclosed, the also unprecedented any teachings of institute in the prior art.Find that based on this application provides a series of methods that are used to judge former chemotherapeutics tolerance, and relevant agent combination.These methods can be applied to multiple sample, and frozen tissue, paraffin-embedded tissue, fresh in vitro tissue etc. can provide multi-level detection information, and the research that can be oophoroma provides support, and also for clinical diagnosis and direction of medication usage dependable detection index is provided.Method of the present invention is simply accurate, and less demanding to sample can be used for extensive examination or retrospective study.When being used for clinical diagnosis and medication guide, the stripped cancer tissue of gathering patients after surgery detects, do not have wound, painless, do not have invasive detection, patient very easily accepts.And visual result is prone to see, rapid sensitive, and is economical and practical.The selection that reasonably is applied to clinical application also helps to prolong patient's life, and the chemotherapy of alleviating patient is painful, improves patient's postoperative life quality.
Description of drawings
Accompanying drawing 4 width of cloth of the present invention:
Fig. 1 embodiment of the invention 1 tissue staining to be detected is example as a result, a wherein, and e=100 *; B, f=200 *; C, g=400 *; D, h=1000 *;
Be ERCC2 serous ovarian cancer postoperative resistance and responsive downpayment chemotherapy patients's expression figure late: the responsive patient's of chemotherapy cancerous tissue epithelium presents tawny positive reaction (shown in the arrow); Chemotherapy resistance patient's cancerous tissue epithelium presents blue look negative reaction (shown in the arrow).The responsive patient of CR=chemotherapy; IR=chemotherapy resistance patient; Enlargement factor: a, e=100X; B, f=200X; C, g=400X; D, h=1000X;
Fig. 2 and Fig. 3 are the gel electrophoresis figure and the statistic analysis result of the expressing quantity of ERCC2 among oophoroma sensitive cells OV2008 and the ovarian cancer drug-resistant cell C13 among the embodiment 2.
Fig. 4 be the immunofluorescence of oophoroma sensitive cells OV2008 and ovarian cancer drug-resistant cell C13 among the embodiment 2 detect (b, e) and immunohistochemical reaction (c, f) testing result figure.
Embodiment
As do not have specified otherwise, the solvent compound method described in the present invention is:
0.01M citrate buffer: the 0.885g citrate is dissolved in the 300ml distilled water, add 94.5 μ l glacial acetic acid adjust pHs to 6.0; Wherein, citrate is available from Tianjin Kermel Chemical Reagent Co., Ltd., batch number: 20070621;
Phosphate buffer (PBS): with 8g sodium chloride, 0.2g potassium chloride, 3.62g sodium hydrogen phosphate (12H
2O) and the 0.24g potassium dihydrogen phosphate dissolve in the 800ml distilled water watery hydrochloric acid adjust pH to 7.4, constant volume 1L;
Sealing damping fluid: 5% lowlenthal serum, 1% bovine serum albumin, pH 7.4 phosphate buffers, 0.01% Triton X-100 (tirtonX-100);
5% lowlenthal serum is available from U.S. ZYMED company, 1091950;
1%BSA is available from U.S. ROCHE, 10735078001;
0.01% Triton X-100 (tirtonX-100) is available from U.S. ROCHE, 0694;
ERCC2 one is anti-available from U.S. Protein Tech Group, Inc., 10818-AP;
Biotinylation two anti-working fluids, horseradish enzyme labeling strepto-avidin working fluid, reagent A (concentrating damping fluid 20x), reagent B (concentrating DAB damping fluid 20x) and reagent C (concentrating hydrogen peroxide damping fluid 20x) all derive from the SABC kit; Available from U.S. ZYMED company, SP-9000;
Triton X-100 (Triton-X 100) is available from Amersco, Cat#0694;
Bovine serum albumin (BSA) is available from Roche, Cat#10735078001;
Other medicine or reagent are analyzes pure level.
Following non-limiting example can make those of ordinary skill in the art more fully understand the present invention, but does not limit the present invention in any way.
I. collection of specimens
Gather 71 examples and organize the paraffin sample by the serous ovarian cancer that pathology department of Dalian Maternity Hospital preserves; Comfortable this hospital of these sample sources carries out radical excision operation serous ovarian cancer patient; Before sample is preserved through routine clinical tissue treatment: i.e. 10% formalin fixed; FFPE, room temperature preservation.
II. sample pre-service comprises the steps:
A. dewaxing: the conventional section of oophoroma formalin fixed paraffin-embedded tissue, the paraffin organization section of thickness 4～5 μ m is successively through 100% xylene dewaxing 3 times, each 5 minutes; Absolute ethyl alcohol dewaxing 3 times, each 2 minutes; 95% ethanol, 90% ethanol, 70% ethanol, 50% ethanol respectively dewax 1 time, 2 minutes;
B. antigen retrieval: carry out antigen retrieval with the 0.01M citrate buffer, clean 3 times each 5 minutes then with PBS;
C. eliminate endogenous peroxydase: histotomy is placed 3% hydrogen peroxide, and incubated at room 30 minutes is cleaned 3 times each 5 minutes with PBS.
III. SABC detects
1. sealing: the tissue at pending sample adds 200 μ l confining liquids, incubated at room 60 minutes;
2. antigen-antibody reaction:
(1) one anti-hatching: add the ERCC2 antibody of 150 μ l organizationally, cover tissue,, clean 3 times each 10 minutes then with PBS in 4 ℃ of incubated overnight to seal film according to 1: 100 dilution proportion;
If negative control group is used with the volume confining liquid to replace ERCC2 antibody.
(2) two anti-hatching: add 150 μ l biotinylations, two anti-working fluids organizationally, incubated at room 60 minutes, PBS cleans 3 times, each 10 minutes;
3. drip horseradish enzyme labeling strepto-avidin working fluid (S-A/HRP) organizationally, incubated at room 60 minutes, PBS cleans 3 times, each 10 minutes;
4. DAB colour developing:
(1) preparation substrate mixed liquor: in the distilled water of 1ml pH value=7.0, add a reagent A, after mixing, reagent B and reagent C respectively one be added dropwise to wherein, mix the substrate mixed liquor;
(2) colour developing: 150 μ l substrate mixed liquors are dripped organizationally color development at room temperature 3～10 minutes, fully colour developing back tap water flushing cessation reaction;
5. dye, dehydration, transparent, mounting: histotomy is immersed the haematine dye liquor 2 times, each 2 seconds; Handle 3 times each 5 minutes then with distilled water; Respectively handle 1 time each 1 minute more successively with 50% ethanol, 70% ethanol, 90% ethanol; Handle 2 times with absolute ethyl alcohol again, each 2 minutes, 1: 1 mixture process of xylene and absolute ethyl alcohol 1 time, 2 minutes, 100% xylene processing 2 times, each 2 minutes; Use gummy mounting at last, preserve;
IV. dyeing situation is per sample judged the susceptibility to former chemotherapeutics: the sample that is brown positive reaction (being the IHC stained positive) is judged to be the ovarian cancer tissue to former chemotherapy medicament sensitive, and the sample that is blue look negative reaction (being that IHC dyeing is negative) is judged to be the ovarian cancer tissue to former chemotherapeutics tolerance.Accompanying drawing 1a～d is the tissue staining photo as a result with typical positive reaction, and the visible tissue epithelium presents obvious tawny; Accompanying drawing 1e～h is the tissue staining photo as a result with typical negative reaction, and the visible tissue epithelium presents obvious blue.
Will be according to routine clinical diagnosis and treatment index gained judged result and the contrast of IHC coloration result, the validity of statistical study the inventive method is like V.
V. testing result and analysis:
According to the routine clinical diagnosis and treatment index of oophoroma chemotherapy of patients prognosis, in the 71 routine patient samples, 44 routine samples are judged to be former chemotherapy medicament sensitive type, and 27 examples are judged to be former chemotherapeutics tolerance type.Judge that concrete grammar is following:
Tumor patient to above-mentioned 71 routine sample sources carries out Information Statistics; Detection level according to patient's postoperative change of serum C A125 in the time of 6 months; Whether the situation of following up a case by regular visits in conjunction with chemotherapy also has new swollen thing to occur such as in 6 months, swollen thing diameter; Whether have continuation abdominal distension, stomachache, lose the appetite, become thin, weak etc. shows that the course of disease still in the clinical symptoms of progress, is divided into two types with patient:
1. chemotherapy responsive type: all measurable disease symptomses with assessment disappear, perhaps in the chemotherapy of patients 6 months the CA125 detection level be lower than＜30U/ml), new swollen thing is less than 3cm
3
2. chemotherapy resistance type: after 6 months chemotherapy, CA125 detects and still is higher than 30U/ml, and with showing the course of disease still in the clinical symptoms of progress, new swollen thing is greater than 3cm
3
Above-mentioned routine clinical index is judged as in the 44 routine samples of chemotherapy responsive type, and 34 examples (account for detect sample 73.3%) sample is judged to be IHC stained positive (to former chemotherapy medicament sensitive), and 11 examples (account for detect sample 26.7%) show that IHC dyeing is negative.
Above-mentioned routine clinical index is judged as in the 27 routine samples of chemotherapy tolerance type, and 19 examples (account for detect sample 70.4%) sample is judged to be IHC dyeing negative (to former chemotherapeutics tolerance), and 8 examples (account for detect sample 29.6%) show the IHC stained positive.
Through statistical results show; Result and contrast method judged result there was no significant difference that IHC dyeing is judged; Explanation is with method of the present invention, and promptly above-mentioned IHC colouring method can carry out the somatotype of former accurately chemotherapeutics resistance/sensitivity to the oophoroma tumor patient.
Embodiment 2
Utilize generally acknowledged former chemotherapy sensitive cells of oophoroma patient (OV2008) of U.S. NCI (National Cancer Institute) and mdr cell (C13) (Yamamoto K; Deng: Heat shock protein 27wasup-regulated in cisplatin resistant human ovarian tumor cell line and associated with the cisplatin resistance.Cancer Lett.2001,168:173-181; Xu G; Deng: Nodal Induces Apoptosis and Inhibits Proliferation in Human Epithelial Ovarian Cancer Cells via Activin Receptor-Like Kinase 7.The Journal of Clinical Endocrinology & Metabolism 2004.89 (11): 5523-5534); Adopt immunofluorescence to detect (immunofluorescence IF); Immunohistochemical reaction (IHC) and Western Blotting improved method (.Perlecan domain V is neuroprotective and proangiogenic following ischemic stroke in rodents.J Clin Invest.2011Jul 11.pii:46358.doi:10.1172/JCI46358 such as Lee B); Verified that ERCC2 is higher 2.49 times than mdr cell in sensitive cells with different expression and ERCC2 protein content in the mdr cell (C13) at former chemotherapy sensitive cells of oophoroma patient (OV2008).Obtained ERCC2 on all four result in chemotherapy sensitivity and resistance patient slices.
I. collection of specimens
Gather oophoroma former the chemotherapy sensitive cells OV2008 and former chemotherapy resistance cell of the oophoroma C13 of international gynecological tumor circle approval.The OV2008 cell comes from epithelium serous ovarian cancer patient (Andrews et al, Cancer Res 1985,45 (12 Pt 1): 6250-6253; Andrews et al:Cancer Chemother Pharmacol 1987; 19 (2): 149-154), be derived from external a plurality of months cultivation and screening (Andrews et al, the Cancer Res 1985 of OV2008 cell inferior 1uM cis-platinums up to a hundred with mdr cell and the OV2008/C13 chemotherapy is responsive; 45 (12Pt 1): 6250-6253); These cells are received in Canadian Chun doctor Peng (York University, Toronto Canada), and conventional cell in vitro was handled before cell was preserved: i.e. cellular incubation; Albumen is purified ,-20 ℃ of preservations.
II. sample pre-service comprises the steps:
1. cellular incubation:
(1) get the one bottle of cell that changes liquid the previous day, the trypsinization with 0.25 after observation of cell becomes circle under the mirror, adds the effect that the 1640 nutrient culture media 1ml that contain 10% hyclone stop pancreatin, and with capillary burette piping and druming, cell is processed suspension;
(2) take a morsel cell with the blue counting of 0.4% placenta, then cell suspension is diluted to 2 * 10
4Individual cell/ml;
(3) will dilute good cell suspension inoculation in 6 orifice plates, every hole 2ml;
(4) insert in 37 ℃ of 5%CO2 incubators and to cultivate 48 hours.
2. albumen is purified:
(1) cell quantity: 6 porocyte culture plates, two every group parallel hole cell extraction albumen;
(2) lysis: with 50mM Tris-HCL PH6.8,2%SDS, 10% glycerine, 1mM PMSF (existing) lysate with add at present, every hole adds the abundant cracking 10～30min of 40 μ l lysates on ice;
(3) the centrifugal 10min of 14000rpm sucts clearly;
(4) BCA kit measurement protein concentration, and be sub-packed in the Ep pipe-70 ℃ of preservations with 30ug.
III.Western Blot detects
1. SDS-PAGE electrophoresis: 5% concentrates glue 100V, 10% separation gel 120V electrophoresis;
2. adopt the NC film, BIO-RAD changes the wet 200～250mA of commentaries on classics of film appearance 45min;
3. the sealing of 5% milk powder room temperature is 1 hour;
4. antibody incubation: anti-(ERCC21: 500, β-actin 1: 1000) 4 ℃ spends the night; Precooling PBST (containing 0.5 ‰ tween-20) washes 10min * 3; Two anti-1: 5000 room temperature 1h; Precooling PBST (containing 0.5 ‰ tween-20) washes 10min * 3;
5. ECL chemiluminescence: luminescence reagent A, B liquid equivalent (0.125ml/cm
2Film) mixes, be added in incubated at room 5min on the film, inhale behind the 5min and remove unnecessary liquid; Wrap with preservative film, put into X-ray magazine exposure (time shutter, the back band was strong and weak according to developing decides), show obvious band in the developer solution after; Tap water cleans; Stop bath photographic fixing 2～3min, the tap water flushing, room temperature is dried film.
6. gel images analysis: film being carried out digital photographing, carry out analyzing and processing through Lab works 4.6 softwares, is the relative density value that benchmark is analyzed each band with internal control gene β-actin.Its result is shown in accompanying drawing 2.A and Fig. 2 .B:
Can find out from Fig. 2 .A: the imaging gray scale of contrast marker protein β-actin two groups of cells is similar; But oophoroma sensitive cells OV2008 is stronger than the imaging gray scale of ovarian cancer drug-resistant cell C13; Thickness is wide, shows that the protein content of ERCC2 is higher than mdr cell in the oophoroma sensitive cells.
Use Excel and SPSS 10.0 softwares and map and statistical study, Fig. 2 .B display analysis is the result prove: the protein content of ERCC2 is higher than 2.49 times in ovarian cancer drug-resistant cell (C13) in oophoroma sensitive cells (OV2008).
IV. immunofluorescence detects:
1. solvent and compound method
I is anti-: ERCC2 (ProteinTech, Catalog No:101818-1-AP)
Fluorescence II is anti-: FITC mark goat anti-rabbit igg (middle China fir Golden Bridge, Catalog No:ZF-0311)
OLYMPUS is inverted and differs fluorescent microscope (Japan, OLYMPUS IX71)
2. Immunofluorescence Reactions
(1) OV2008 and C13 cell are used 0.25% trypsinization, be diluted to 2 * 104/ml and be inoculated in six orifice plates, every hole 2ml cell suspension;
(2) treat that cell 24 hours is adherent after, with fixing 10 minutes of 4%PFA (paraformaldehyde);
(3) PBS washes 5min * 3;
(4) confining liquid (containing 10% sheep blood serum, 30% seralbumin) sealing 1h;
(5) anti-(ERCC21: 150 of the I of confining liquid preparation; ProteinTech, Catalog No:101818-1-AP) 4 degree incubated overnight;
(6) PBS washes 5min * 3;
(7) FITC mark II is anti-[1: 50; FITC mark goat anti-rabbit igg (middle China fir Golden Bridge, Catalog No:ZF-0311)] lucifuge hatches 1h;
(8) PBS washes 5min * 3;
(9) the OLYMPUS inversion differs fluorescent microscope (Japan, OLYMPUS IX71) and observes, takes the photograph sheet.
V. immunohistochemistry detects:
1. solvent and compound method
Described solvent compound method of this method and embodiment 1 said solvent and compound method thereof are identical.
2. immunohistochemical reaction
(1) OV2008 and C13 cell are used 0.25% trypsinization, be diluted to 2 * 104/ml and be inoculated in six orifice plates, every hole 2ml cell suspension;
(2) treat that cell 24 hours is adherent after, with fixing 10 minutes of 4%PFA (paraformaldehyde);
(3) PBS washes 5min * 3;
(4) confining liquid (containing 10% sheep blood serum, 30% seralbumin) sealing 1h;
(5) antigen-antibody reaction:
I. anti-hatching: on cultured cells, add the ERCC2 antibody of 150 μ l,, clean 3 times each 10 minutes then with PBS in 4 ℃ of incubated overnight according to 1: 100 dilution proportion;
Ii two anti-hatching: on cultured cells, add 150 μ l biotinylations, two anti-working fluids, incubated at room 60 minutes, PBS cleans 3 times, each 10 minutes;
(6) on cell, drip horseradish enzyme labeling strepto-avidin working fluid (S-A/HRP), incubated at room 60 minutes, PBS cleans 3 times, each 10 minutes;
(7) DAB colour developing:
I. prepare the substrate mixed liquor: in the distilled water of 1ml pH value=7.0, add a reagent A, after mixing, each one is added dropwise to wherein with reagent B and reagent C, mix the substrate mixed liquor;
Ii colour developing: 150 μ l substrate mixed liquors are dripped on cell color development at room temperature 3～10 minutes, fully colour developing back tap water flushing cessation reaction;
(8) dyeing, dehydration, transparent, mounting: histotomy is immersed the haematine dye liquor 2 times, each 2 seconds; Handle 3 times each 5 minutes then with distilled water; Respectively handle 1 time each 1 minute more successively with 50% ethanol, 70% ethanol, 90% ethanol; Handle 2 times with absolute ethyl alcohol again, each 2 minutes, 1: 1 mixture process of xylene and absolute ethyl alcohol 1 time, 2 minutes, 100% xylene processing 2 times, each 2 minutes; Use gummy mounting at last, preserve;
(9) OLYMPUS is inverted and differs fluorescence microscope, takes the photograph sheet;
(10) immunofluorescence and immunohistochemistry testing result (shown in accompanying drawing 4)
Fig. 4-a, d are the oophoroma cultured cells without any dyeing; Fig. 4-b, e are the oophoroma cultured cells through the immunohistochemistry fluorescent dye, and show: ERCC2 obviously strengthens in the expression of former chemotherapy sensitive cells (OV2008), is strong green, and a little less than the expression very of former chemotherapy resistance cell (C13), is dirty-green.Fig. 4-c, f are the oophoroma cultured cells through immunohistochemical staining, and show: ERCC2 is dark brown in the expression of former chemotherapy sensitive cells (OV2008), obviously is better than the expression at former chemotherapy resistance cell (C13), is light brown.It is in full accord that this result and ERCC2 institute in chemotherapy sensitivity and resistance patient slices obtains the result.
Claims (8)
1. former chemosensitivity of oophoroma test and appraisal kit; It is characterized in that this kit comprises: the confining liquid of forming by the phosphate buffer of 5% lowlenthal serum, 1%BSA, 0.01% Triton X-100 and pH7.4; The antigen-antibody reaction system that forms by ERCC2 antibody, biotinylation two anti-working fluids and confining liquid; Avidin-oxide enzymatic reaction system of forming by horseradish enzyme labeling strepto-avidin working fluid and DAB reagent damping fluid, and as the haematine of counterstain.
2. the described kit of claim 1; It is characterized in that said kit also comprises the antigen retrieval system that is made up of 0.01M citrate buffer and phosphate buffer, and the elimination endogenous peroxydase system that forms by 3% hydrogen peroxide and phosphate buffer.
3. test and appraisal ovarian cancer tissue sample is to the method for former chemotherapy drug susceptibility; It is characterized in that using the described kit of claim 1; After comprising the steps: to use confining liquid to handle testing sample; Use ERCC2 antibody and biotinylation two anti-working fluids to carry out anti-and two anti-reactions respectively, use horseradish enzyme labeling strepto-avidin working fluid to handle sample then, carry out DAB colour developing and haematoxylin redyeing at last;
Dyeing situation is per sample judged the susceptibility to chemotherapeutics: the sample that is the brown positive reaction is judged to be the ovarian cancer tissue to former chemotherapy medicament sensitive; The sample that is blue look negative reaction is judged to be the ovarian cancer tissue to former chemotherapeutics tolerance.
4. the described method of claim 3 comprises the steps:
1. sealing: the tissue at pending sample adds 200 μ l confining liquids, incubated at room 60 minutes;
2. antigen-antibody reaction:
(1) one anti-hatching: add the ERCC2 antibody of 150 μ l organizationally, cover tissue,, clean 3 times each 10 minutes then with PBS in 4 ℃ of incubated overnight to seal film according to 1: 100 dilution proportion;
If negative control group is used with the volume confining liquid to replace ERCC2 antibody;
(2) two anti-hatching: add 150 μ l biotinylations, two anti-working fluids organizationally, incubated at room 60 minutes, PBS cleans 3 times, each 10 minutes;
3. drip horseradish enzyme labeling strepto-avidin working fluid (S-A/HRP) organizationally, incubated at room 60 minutes, PBS cleans 3 times, each 10 minutes;
4. DAB colour developing:
(1) preparation substrate mixed liquor: in the distilled water of 1ml pH value=7.0, add a reagent A, after mixing, reagent B and reagent C respectively one be added dropwise to wherein, mix the substrate mixed liquor;
(2) colour developing: 150 μ l substrate mixed liquors are dripped organizationally color development at room temperature 3～10 minutes, fully colour developing back tap water flushing cessation reaction;
5. dye, dehydration, transparent, mounting: histotomy is immersed the haematine dye liquor 2 times, each 2 seconds; Handle 3 times each 5 minutes then with distilled water; Respectively handle 1 time each 1 minute more successively with 50% ethanol, 70% ethanol, 90% ethanol; Handle 2 times with absolute ethyl alcohol again, each 2 minutes, 1: 1 mixture process of xylene and absolute ethyl alcohol 1 time, 2 minutes, 100% xylene processing 2 times, each 2 minutes; Use gummy mounting at last, preserve;
6. dyeing situation is per sample judged the susceptibility to chemotherapeutics: the sample that is the brown positive reaction is judged to be the ovarian cancer tissue to former chemotherapy medicament sensitive; The sample that is blue look negative reaction is judged to be the ovarian cancer tissue to former chemotherapeutics tolerance.
5. the described method of claim 3 is characterized in that: when said method is applied to the test and appraisal of oophoroma formalin fixed paraffin-embedded tissue, also comprise the The pretreatment step:
A. dewaxing: the conventional section of oophoroma formalin fixed paraffin-embedded tissue, the paraffin organization section of thickness 4～5 μ m is successively through 100% xylene dewaxing 3 times, each 5 minutes; Absolute ethyl alcohol dewaxing 3 times, each 2 minutes; 95% ethanol, 90% ethanol, 70% ethanol, 50% ethanol respectively dewax 1 time, 2 minutes;
B. antigen retrieval: carry out antigen retrieval with the 0.01M citrate buffer, clean 3 times each 5 minutes then with PBS;
C. eliminate endogenous peroxydase: histotomy is placed 3% hydrogen peroxide, and incubated at room 30 minutes is cleaned 3 times each 5 minutes with PBS.
6. screening is to the method for the ovarian cancer patients of former chemotherapy medicament sensitive; It is characterized in that using the described kit of claim 1; Comprise the steps: that with the ovarian cancer patients tumor tissue in vitro be test sample, after the use confining liquid is handled testing sample, use ERCC2 antibody and biotinylation two anti-working fluids to carry out anti-and two anti-reactions respectively; Use horseradish enzyme labeling strepto-avidin working fluid to handle sample then, carry out DAB colour developing and haematoxylin redyeing at last;
The ovarian cancer patients that dyeing situation is per sample judged sample source is to former chemotherapy drug susceptibility: the sample that is the brown positive reaction is judged to be the ovarian cancer tissue to former chemotherapy medicament sensitive, and the ovarian cancer patients of sample source is to former chemotherapy medicament sensitive; The sample that is blue look negative reaction is judged to be the ovarian cancer tissue to former chemotherapeutics tolerance, and the ovarian cancer patients of sample source tolerates former chemotherapeutics.
7. the described method of arbitrary claim in the claim 3～6 is characterized in that described former chemotherapeutics is cis-platinum, taxol or the combination of the two.
8. the described method of arbitrary claim in the claim 3～6 is characterized in that described oophoroma is a serous ovarian cancer.
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