WO2013033933A1

WO2013033933A1 - Kit, process and use for measuring and evaluating sensitivity of ovarian cancer to primary chemotherapy

Info

Publication number: WO2013033933A1
Application number: PCT/CN2011/079885
Authority: WO
Inventors: 崔世英; 丁艳芳; 赵瑾瑶; 杨亮; 毕丽华; 姜继勇
Original assignee: 大连医科大学
Priority date: 2011-09-08
Filing date: 2011-09-20
Publication date: 2013-03-14
Also published as: CN102435734A

Abstract

A kit, method and use for measuring and evaluating sensitivity of ovarian cancer to primary chemotherapy, wherein the kit comprises a sealing fluid, an antigen-antibody reaction system, an avidin-oxidase reaction system and an afterstain. The present invention is based on the expression level of ERCC2 in serous ovarian cancer cells/tissues correlated with the sensitivity of cells, tissues or tumor patients as the host to primary chemotherapy medicament. Detection of ERCC2 may be used for multiple purposes of measurement and evaluation of chemotherapy sensitivity/resistance, identification of tumor patients, and evaluation of resistance to primary chemotherapy medicament.

Description

Kit, method and application for measuring primary chemotherapy sensitivity of ovarian cancer

The present invention relates to a kit for measuring chemosensitivity of ovarian cancer and its use in assessment of primary chemotherapy sensitivity/resistance, screening of tumor patients, and assessment of tolerance of primary chemotherapy drugs. Background technique

Epithelial Ovarian Cancer (ECC) is one of the common tumors in female reproductive organs and the leading killer in female tumors. Among them, serous carcinoma is the most common, accounting for 80-85% of all ovarian cancers in Western countries, divided into late and early stages, of which the late stage accounts for the vast majority, and early rare is about 5% of all ovaries. It is now believed that early and late serous carcinomas are two different types of ovarian cancer, and their morphological differences actually reflect differences in tumor biology, which is ultimately the difference in gene levels. The disease is difficult to diagnose early, and the patient has missed the best treatment opportunity at the time of the late visit. Because patients with advanced ovarian cancer have varying degrees of spread, patients must be treated immediately after surgery. At present, the international chemotherapy regimens for postoperative patients with ovarian cancer are mainly divided into: first-stage adjuvant (adjuvant; platinum-based cisplatin or carboplatin/paclitaxel) and late salvage (salvage; multiple chemotherapy drugs) treatment . Because the patient's gene regulation is different, and their molecular expression levels are different, the sensitivity to primary chemotherapy drugs varies. Therefore, platinum-based first-stage adjuvant chemotherapy does not have any symptoms for 40 to 50% of patients. Relief, also known as primary resistance, is one of the reasons for the rapid death of patients because the treatment is not targeted and delays the optimal chemotherapy period. Therefore, predicting the sensitivity of patients to primary chemotherapy before chemotherapy is critical to prolonging the lives of patients with ovarian cancer.

On the other hand, epithelial ovarian cancer has long been classified into four different pathological types according to histomorphological classification, namely: serous, endometrial, mucinous and transparent cell, of which serousity is most common. . However, clinical practice has found that ovarian cancer is classified only according to the traditional tissue morphological classification criteria. It is insufficient for further study of its pathological features and guiding clinical diagnosis and treatment. Therefore, there is an urgent need for a method for tumors under traditional taxonomy. Carry out further screening and classification.

Furthermore, as mentioned above, the clinical use of drugs for the treatment of ovarian cancer does not relieve the symptoms of a considerable number of patients, but this takes a long period of clinical time to be discovered, often leading to delays in treatment, increasing patients pain. Therefore, there is a need for a rapid and efficient high-throughput screening method for drug research and development to specifically assess the potential tolerance risk of a target primary chemotherapy drug in clinical treatment of ovarian cancer. Summary of the invention

The inventors of the present application found that the protein expression level of E CC2 is higher in sensitive cells than in resistant cells 2 More than this time, in combination with this finding, the inventors used the simple anatomical immunohistochemical method in our country to qualitatively confirm the biomarker effect of ERCC2 in ovarian cancer chemotherapy patients.

Based on the above findings, one of the objects of the present invention is to provide a kit for measuring the sensitivity of primary chemotherapy for ovarian cancer, the kit comprising: 5% goat serum, 1% BSA, 0.01% polyethylene Blocking solution consisting of diol octyl phenyl ether and phosphate buffer pH 7.4, antigen-antibody reaction system consisting of ERCC2 antibody (1: 1000), biotinylated secondary antibody working solution and blocking solution, by horseradish enzyme An avidin-oxidase reaction system consisting of a streptavidin working solution and a DAB reagent buffer, and a hematoxylin as a counterstain.

In a preferred embodiment, the kit further comprises an antigen retrieval system consisting of 0.01 M citrate buffer and phosphate buffer, and an endogenous peroxidation consisting of 3% hydrogen peroxide and phosphate buffer. Enzyme system. The kit can be specifically adapted for use in paraffin-embedded ovarian cancer tissue samples.

A second object of the present invention is to provide a method for measuring sensitivity of a ovarian cancer tissue sample to a primary chemotherapeutic drug, characterized in that the kit of claim 1 is used, comprising the steps of: treating a sample to be tested with a blocking solution; , using ERCC2 antibody and biotinylated secondary antibody working solution for primary and secondary antibody reaction respectively, and then treating the sample with horseradish-labeled streptavidin working solution, and finally performing DAB color development and hematoxylin counterstaining; Sensitivity of the staining condition to the primary chemotherapeutic drug: The sample with a brown positive reaction was judged to be an ovarian cancer tissue sensitive to the primary chemotherapeutic drug; the sample with a blue negative reaction was determined to be ovarian cancer resistant to the primary chemotherapeutic drug. organization.

The above method for measuring the sensitivity of an ovarian cancer tissue sample to a primary chemotherapeutic drug more specifically includes the following steps:

1 blocking: Add 200 μl blocking solution to the tissue of the sample to be treated, and incubate for 60 minutes at room temperature;

2 antigen antibody response:

(1) Primary antibody incubation: 150 μl of ERCC2 antibody diluted 1:100 in tissue, covered with a parafilm, incubated at 4 ° C overnight, and then washed 3 times with PBS for 10 minutes each time;

A negative control group was used, and the same volume of blocking solution was used instead of ERCC2 antibody.

(2) Incubation of secondary antibody: Add 150 μl of biotinylated secondary antibody working solution to the tissue, incubate for 60 minutes at room temperature, and wash 3 times with PBS for 10 minutes each time;

3 Add the horseradish enzyme-labeled streptavidin working solution (S-A/HRP) to the tissue, incubate for 60 minutes at room temperature, and wash 3 times with PBS for 10 minutes each time;

4 DAB color development:

(1) preparing a substrate mixture: in 1 ml of pH=7.0 in double distilled water, adding a drop of reagent A, mixing uniformly, adding one drop of reagent B and reagent C, and uniformly mixing the substrate mixture; (2) Color development: 150μ1 substrate mixed droplets were added to the tissue, and the color was developed at room temperature for 3 to 10 minutes. After sufficient color development, the tap water rinse was used to terminate the reaction;

5 staining, dehydration, transparency, sealing: The tissue sections were immersed in hematoxylin staining solution 2 times for 2 seconds each time; then treated with distilled water 3 times for 5 minutes each time; then 50% ethanol, 70% ethanol, 90% ethanol was treated once for 1 minute each time; then treated twice with absolute ethanol for 2 minutes, 1 :1 mixture of xylene and absolute ethanol, 2 minutes, 100% xylene treatment 2 Times, 2 minutes each time; finally seal with a gum, save;

6 Depending on the staining of the sample, the sensitivity to the primary chemotherapeutic drug was determined: the sample with a brown positive reaction was determined to be an ovarian cancer tissue sensitive to the primary chemotherapeutic drug; the sample with a blue negative reaction was judged to be resistant to the primary chemotherapeutic drug. Ovarian cancer tissue.

The above method for measuring the sensitivity of an ovarian cancer tissue sample to a primary chemotherapeutic drug is theoretically applicable to any type of ex vivo tumor tissue or cell sample, including freshly collected ovarian cancer tissue, formalin-fixed paraffin-embedded ovary. Cancer tissue, OCT-embedded frozen ovarian cancer tissue, fresh, non-fixed ovarian cancer tissue preserved by liquid nitrogen. Regardless of the type of biological sample applied, those skilled in the art can determine the pretreatment method of the sample based on prior art information, and the treated sample has been adapted for use in the immunohistochemical assays described herein. Several typical samples and their treatment methods are:

(1) When the method for measuring sensitivity of a ovarian cancer tissue sample to a primary chemotherapeutic drug is applied to the evaluation of fixed paraffin-embedded tissue of ovarian cancer formalin, the pretreatment of the sample includes the following steps:

a. Dewaxing: routine section of formalin-fixed paraffin-embedded tissue of ovarian cancer, paraffin section of thickness 4~5μιη was dewaxed by 100% xylene three times for 5 minutes each time; dehydrated ethanol for 3 times , 2 minutes each time; 95% ethanol, 90% ethanol, 70% ethanol, 50% ethanol, each dewaxed once, 2 minutes;

b. Antigen retrieval: antigen retrieval with 0.01 M citrate buffer, then washed 3 times with PBS for 5 minutes each time;

c Elimination of endogenous peroxidase: Tissue sections were placed in 3% hydrogen peroxide, incubated for 30 minutes at room temperature, and washed 3 times with PBS for 5 minutes each time.

The resulting sample was processed directly for testing.

(2) When the method for measuring sensitivity of a ovarian cancer tissue sample to a primary chemotherapeutic drug is applied to an OCT-embedded cryopreserved ovarian cancer tissue, the pretreatment of the sample includes the following steps:

a. Tissue fixation: Fresh tumor tissue excised during ovarian cancer surgery is placed into 4% paraformaldehyde immediately after 2x2 cm ³ , 4 . C, fixed for 24 hours;

b. Wash PBS 3 times, 10 minutes each time; c. Immerse the tissue in 30% sucrose at 4 ° C overnight;

d. OCT, opti-mum cutting temperature compound is embedded, placed on the dry 3⁄4; e. After the OCT mixture is solidified, the cryostat is frozen and sliced 7~10um;

When the method for measuring sensitivity of a ovarian cancer tissue sample to a primary chemotherapeutic drug is applied to fresh non-fixed ovarian cancer tissue preserved by liquid nitrogen, the pretreatment of the sample includes the following steps:

a. Freshly resected fresh tumor tissue from ovarian cancer patients is quickly placed in liquid nitrogen for use;

b. The tissue is first immersed in 4% paraformaldehyde at 4 ° C for 24 hours;

Depending on the laboratory equipment the user has, either of the above two methods is possible: After the paraffin embedding or OCT embedding process, the treated sample is directly used for testing.

Another object of the present invention is to provide a method for screening an ovarian cancer patient susceptible to a primary chemotherapeutic drug, which method also requires the use of the ovarian cancer chemosensitivity assessment kit of the present invention described above, comprising the following steps: Taking the isolated tumor tissue of ovarian cancer patients as a test sample, after treating the sample to be tested with a blocking solution, the primary and secondary antibodies were reacted with E CC2 antibody and biotinylated secondary antibody working solution, respectively, and then labeled with horseradish-labeled streptococcus. Samples were treated with avidin working solution, and finally DAB coloration and hematoxylin counterstaining were performed; susceptibility of primary chemotherapeutic drugs to sample-derived ovarian cancer patients was determined based on sample staining: Samples with brown positive reaction were judged as primary chemotherapy Drug-sensitive ovarian cancer tissue, sample-derived ovarian cancer patients are sensitive to primary chemotherapy drugs; samples that are blue-negative are judged to be ovarian cancer tissues that are resistant to primary chemotherapy drugs, and sample-derived ovarian cancer patients are primary Chemotherapy drugs are tolerated.

Another object of the present invention is to provide a method for guiding a first-stage chemotherapy drug for a patient after ovarian cancer surgery, including clinically collecting ovarian cancer tissue, performing immunohistochemical detection and judgment, and determining a medication plan according to the judgment result. Specifically:

I. Sample collection

II. Sample immunohistochemistry detection and result judgment

2 antigen antibody response:

(2) Incubation of secondary antibody: Add 150 μl of biotinylated secondary antibody working solution to the tissue, incubate for 60 minutes at room temperature, and wash 3 times with PBS for 10 minutes each time; 3 Add horseradish enzyme-labeled streptavidin working solution (SA/HRP) to the tissue, incubate for 60 minutes at room temperature, and wash 3 times with PBS for 10 minutes each time;

4 DAB color development:

(1) Preparation of substrate mixture: In 1 ml of double distilled water with pH value of 7.0, add a drop of reagent A, mix well, add one drop of reagent B and reagent C, and mix to obtain a mixture of substrates;

(2) Color development: 150 μl substrate mixed droplets were added to the tissue, and the color was developed at room temperature for 3 to 10 minutes. After sufficient color development, the tap water rinse was used to terminate the reaction;

5 staining, dehydration, transparency, sealing: The tissue sections were immersed in hematoxylin staining solution 2 times for 2 seconds each time; then treated with distilled water 3 times for 5 minutes each time; then 50% ethanol, 70% ethanol, 90% ethanol was treated once for 1 minute each time; then treated with absolute ethanol for 2 times, 2 minutes each time, 1 :1 mixture of xylene and absolute ethanol was treated once, 2 minutes, 100% xylene treatment 2 Times, 2 minutes each time; finally seal with a gum, save;

III. Analysis of results and determination of medication guidance program

According to the test result of step II, the source of the ovarian cancer tissue which can be positively judged positively is highly sensitive to the postoperative primary chemotherapy drug treatment; the source of the negative reaction ovarian cancer tissue is resistant to the postoperative primary chemotherapy drug treatment The probability is high. For the latter, it is necessary to determine a suitable postoperative chemotherapy regimen based on the patient's physiopathological characteristics and clinical symptoms.

The primary chemotherapeutic agent described in all of the above methods of the present invention refers to a first-stage chemotherapeutic drug for performing chemotherapy in patients after ovarian cancer surgery, and such drugs include platinum, paclitaxel or a combination of the two in clinical practice.

The ovarian cancer or tumor described in the above technical solution of the present invention refers to the advanced stage of serous ovarian cancer.

The inventors of the present application have found a correlation between the expression level of ERCC2 in tumor cells, particularly serous ovarian cancer cells/tissues, and the sensitivity of cells, tissues or tumor patients as hosts to primary chemotherapeutic drugs. Although there has been a description of the ERCC2 immunohistochemical detection method in the prior art, the above-described determination relationship between the two is not disclosed in the prior art, and there has not been any technical suggestion. Based on this finding, the present application provides a series of methods for determining the tolerance of a primary chemotherapeutic drug, as well as related reagent combinations. These methods can be applied to a variety of samples, frozen tissue, paraffin-embedded tissue, fresh ex vivo tissue, etc., can provide multi-level detection information, can support the research of ovarian cancer, and provide dependable for clinical diagnosis and guidance medication. Test indicators. The method of the invention is simple and accurate, has low requirements on samples, and can be used for large-scale screening or retrospective research. For clinical diagnosis and medication guidance, the patient's isolated cancer tissue is collected after surgery, non-invasive, painless, non-invasive, and the patient is highly acceptable. And the results are intuitive, quick and sensitive, economical and practical. The rational use of clinical medications also helps to prolong the patient's life, relieve the patient's chemotherapy pain, and improve the patient's postoperative quality of life. DRAWINGS

Figure 4 of the present invention:

Figure 1 is an example of the results of tissue dyeing to be detected in Example 1 of the present invention, wherein a, e = 100 _; b, f = 200x; c, g = 400x; d, h = lOOOOx;

It is the expression of ERCC2 in patients with drug-resistant and sensitive first-stage chemotherapy after advanced serous ovarian cancer: The cancerous epithelium of chemotherapy-sensitive patients showed a yellow-brown positive reaction (arrow); the cancer tissue epithelium of chemotherapy-resistant patients A blue negative reaction (shown by the arrow) appears. 0 = chemotherapy-sensitive patients; 11 = chemotherapy-resistant patients; magnification multiples: a, e = 100X; b, f = 200X; c, g = 400X; d, h = 1000X;

Fig. 2 and Fig. 3 are gel electrophoresis patterns and statistical analysis results of protein expression levels of ERCC2 in ovarian cancer sensitive cell OV2008 and ovarian cancer resistant cell C13 in Example 2.

Fig. 4 is a graph showing the results of immunofluorescence detection (b, e) and immunohistochemistry (c, f) of ovarian cancer sensitive cell OV2008 and ovarian cancer resistant cell C13 in Example 2. detailed description

Unless otherwise stated, the solvent preparation method described in the present invention is:

0.01 M citrate buffer: 0.885 g of citrate was dissolved in 300 ml of distilled water, and 94.5 μl of glacial acetic acid was added to adjust the value to 6.0; wherein citrate was purchased from Tianjin Komi Chemical Reagent Co., Ltd., product batch number : 20070621;

Phosphate buffer (PBS): 8 g of sodium chloride, 0.2 g of potassium chloride, 3.62 g of disodium hydrogen phosphate (123⁄40) and 0.24 g of potassium dihydrogen phosphate were dissolved in 800 ml of distilled water, and the pH was adjusted to 7.4 with dilute hydrochloric acid. Constant volume 1L;

Blocking buffer: 5% goat serum, 1% bovine serum albumin, pH 7.4 phosphate buffer, 0.01% polyethylene glycol octyl phenyl ether (tirton X-100);

5% goat serum was purchased from ZYMED, USA, 1091950;

1% BSA was purchased from the US ROCHE, 10735078001;

0.01% polyethylene glycol octyl phenyl ether (tirtonX-100) was purchased from the United States ROCHE, 0694;

E CC2 primary antibody was purchased from Protein Tech Group, Inc., 10818-AP; Biotinylated secondary antibody working solution, horseradish-labeled streptavidin working solution, reagent A (concentration buffer 20x), reagent B (concentrated DAB buffer 20x) and reagent C (concentrated hydrogen peroxide buffer 20x) From the immunohistochemistry kit, purchased from ZYMED, USA, SP-9000;

Polyethylene glycol octylphenyl ether (Triton-X 100) was purchased from Amersco, Cat# 0694;

Bovine serum albumin (BSA) was purchased from Roche, Cat# 10735078001;

Other drugs or reagents are of analytical grade.

The following non-limiting examples are provided to enable those of ordinary skill in the art to understand the invention. Example 1

I. Specimen collection

Seventy-one samples of serous ovarian cancer tissues preserved by the Department of Pathology, Dalian Maternity Hospital were collected. These samples were obtained from patients undergoing radical resection of serous ovarian cancer in the hospital. The samples were processed by clinical routine before storage: 10 % formalin fixed, embedded in paraffin, stored at room temperature.

II. Sample pretreatment, including the following steps:

a. Dewaxing: ovarian cancer formalin fixed paraffin-embedded tissue conventional sections, paraffin tissue sections of thickness 4~5μηι were dewaxed by 100% xylene three times, each time for 5 minutes; anhydrous ethanol dewaxing 3 times , 2 minutes each time; 95% ethanol, 90% ethanol, 70% ethanol, 50% ethanol, each dewaxed once, 2 minutes;

III. Immunohistochemistry

2 antigen antibody response:

(1) Primary antibody incubation: 150 μl of ERCC2 antibody diluted 1:100 in tissue, covered with a parafilm, incubated overnight at 4 ,, and then washed 3 times with PBS for 10 minutes each time;

3 Add horseradish enzyme-labeled streptavidin working solution (SA/HRP) to the tissue and incubate for 60 minutes at room temperature. Wash 3 times with PBS for 10 minutes each time;

4 DAB color development:

IV. Sensitivity to primary chemotherapeutic drugs based on sample staining: Samples that were brown-positive (ie, positive for IHC staining) were identified as ovarian cancer tissues sensitive to primary chemotherapeutic drugs, with a blue-negative response (ie, IHC staining) The negative sample was judged to be an ovarian cancer tissue that was resistant to the primary chemotherapy drug. Figures la~d are photographs of tissue staining results with a typical positive reaction, showing that the tissue epithelium is clearly yellowish brown; Figure le~! ! It is a photograph of tissue staining results with a typical negative reaction, showing that the tissue epithelium is clearly blue.

The results of the method according to the conventional clinical diagnosis and treatment index are compared with the IHC staining results, and the effectiveness of the method of the present invention is statistically analyzed, for example.

V. Test results and analysis:

According to the routine clinical diagnosis and treatment index of chemotherapy prognosis in patients with ovarian cancer, of the 71 patients, 44 samples were identified as primary chemotherapy-sensitive, and 27 were judged to be resistant to primary chemotherapy. The specific methods were as follows: Information on the 71 patients with tumors from the above samples was analyzed according to the level of serum CA125 at 6 months after surgery, combined with chemotherapy follow-up, such as whether there were new tumors within 6 months. Appearance, size of the tumor, presence of persistent abdominal distension, abdominal pain, loss of appetite, loss of phlegm, fatigue, and other clinical symptoms that indicate that the disease is still progressing, the patients are divided into two categories:

1 Chemotherapy-sensitive: All predictable and assessed disease symptoms disappeared, or the patient's CA125 detection level was less than < 30 U/ml within 6 months of chemotherapy, and the new tumor was less than 3 cm ³ .

2 Chemotherapy-resistant type: After 6 months of chemotherapy, the CA125 test is still higher than 30 U/ml, and is accompanied by clinical symptoms showing that the disease course is still progressing, and the new tumor is larger than 3 cm ³ .

Of the 44 samples that were judged to be chemotherapy-sensitive, 34 of them (73.3% of the specimens) were positive for IHC staining (sensitive to primary chemotherapy) and 11 (26.7% of the specimens). Show IHC Negative staining.

Of the 27 samples of the above-mentioned conventional clinical indicators that were judged to be resistant to chemotherapy, 19 (70.4% of the specimens) were judged to be negative for IHC staining (tolerant to primary chemotherapy), and 8 (29.6 of the specimens) %) shows positive IHC staining.

Statistical analysis showed that there was no significant difference between the results of IHC staining judgment and the judgment results of the control method, indicating that the method of the present invention, that is, the above IHC staining method can accurately and sensitively administer primary chemotherapy drugs to patients with ovarian cancer. Type of. Example 2

Using NCI (National Cancer Institute) recognized ovarian cancer patients with primary chemotherapy-sensitive cells (OV2008) and drug-resistant cells (C13) (Yamamoto, etc.: Heat shock protein 27 was up-regulated in cisplatin resistant human ovarian tumor cell line Cancer Lett. 2001 , 168: 173-181 ; Xu G, et al: 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(ll): 5523-5534), using immunofluorescence IF, immunohistochemistry (IHC) and Western Blotting, Lee B et al. Perlecan domain V is neuroprotective and proangiogenic following ischemic stroke in Rodents. / Clin Invest. 201 1 Jul 1 1. pii: 46358. doi: 10.1 172/JCI46358 ) , validated the difference in ERCC2 between primary chemotherapy-sensitive cells (OV2008) and resistant cells (C 13) in patients with ovarian cancer Expression, and ERCC2 protein content in sensitive cells The drug-resistant cells were 2.49 times higher. Obtained consistent results for ERCC2 in chemosensitive and resistant patient sections.

I. Specimen collection

The ovarian cancer primary chemotherapy-sensitive cells OV2008 and ovarian cancer primary chemotherapy-resistant cells C13 were collected from the international gynecological oncology community. OV2008 cells are derived from patients with epithelial serous ovarian cancer (Andrews et al, Cancer Res 1985, 45 (12 Pt l): 6250-6253; Andrews et al: Cancer Chemother Pharmacol 1987, 19(2): 149-154), and OV2008/C 13 Chemotherapy-sensitive and drug-resistant cells were cultured and screened in vitro for hundreds of luM cisplatins in OV2008 cells (Andrews et al, Cancer Res 1985, 45 (12 Pt l): 6250-6253) These cells were obtained from Dr. Chun Peng (Toronto, Canada) for routine in vitro cell processing before cell preservation: cell culture, protein purification, storage at -20 ° C.

II. Sample pretreatment, including the following steps:

1 cell culture: (1) Take one bottle of cells changed the day before, digest with 0.25 trypsin, observe the rounding of the cells under microscope, add 1ml of 1640 medium containing 10% fetal bovine serum to stop the action of trypsin, and use capillary dropper Blowing, making the cells into suspension;

(2) taking a small amount of cells and counting with 0.4% placenta blue, and then diluting the cell suspension to 2χ 10 ⁴ cells/ml;

(3) Inoculate the diluted cell suspension in a 6-well plate at 2 ml per well _;

(4) Incubate in a 37 ° C 5% CO 2 incubator for 48 hours.

2 protein purification:

(1) Number of cells: 6-well cell culture plates, and two parallel-well cells in each group extract proteins;

(2) Cell lysis: 50 mM Tris-HCL PH6.8, 2% SDS, 10% glycerol, ImM PMSF (currently used) lysate, 40 μl of lysate per well, fully lysed on ice for 10 to 30 min ;

(3) Centrifuge at 14000 rpm for 10 min;

(4) The protein concentration was determined by BCA kit and stored in Ep tubes at 30 ug, and stored at -70 °C.

III. Western Blot detection

1 SDS-PAGE electrophoresis: 5% concentrated gel 100V, 10% separation gel 120V electrophoresis;

2 using NC film, BIO-RAD film transfer machine wet transfer 200~250mA 45min;

3 5% milk powder was sealed at room temperature for 1 hour;

4 antibody incubation: primary antibody (ERCC2 1 :500, β -actin 1 : 1000 ) overnight at 4 ° C; pre-cooled PBST (containing 0.5% ₀ tween-20) washed 10 min X 3 ; secondary antibody 1: 5000 room temperature lh; Cold PBST (containing 0.5%. tween-20) washed lOmin X 3 ;

5 ECL chemiluminescence: illuminating reagent VIII, B solution equal amount (0.125ml/cm ² membrane), add to the membrane and incubate for 5min at room temperature. After 5min, absorb excess liquid, wrap it with plastic wrap, and put it into X-ray cassette. Exposure (exposure time depends on the strength of the strip after development), after the visible band appears in the developer, the tap water is cleaned, the fixer is fixed for 2 to 3 minutes, the tap water is rinsed, and the film is air-dried at room temperature.

6 Gel image analysis: The film was digitally photographed, analyzed by Lab Works 4.6 software, and the relative density values of each strip were analyzed based on the internal reference gene -actin. The results are shown in Fig. 2. A and Fig. 2.B: It can be seen from Fig. 2.A that the contrast gray level of the control marker protein β-actin is similar in the two groups of cells, but the ovarian cancer sensitive cell OV2008 is more than the ovary. The imaging gray-staining cell C13 has strong gray scale and wide thickness, indicating that the protein content of ERCC2 is higher than that of drug-resistant cells in ovarian cancer sensitive cells.

Using Excel and SPSS 10.0 software for mapping and statistical analysis, Figure 2.B shows that the analysis results show that the protein content of ERCC2 is 2.49 times higher than that of ovarian cancer resistant cells (C13) in ovarian cancer sensitive cells (OV2008). IV. Immunofluorescence detection:

1 solvent and preparation method

I resistance: ERCC2 (ProteinTech, Catalog No: 101818-1-AP)

Fluorescent II antibody: FITC-labeled goat anti-rabbit IgG (Zhongsu Jinqiao, Catalog No: ZF-0311)

OLYMPUS inverted phase contrast fluorescence microscope (Japan, OLYMPUS 1X71)

2 immunofluorescence reaction

(1) OV2008 and C13 cells were digested with 0.25% trypsin, diluted to 2 x 104 cells/ml and inoculated into a six-well plate, 2 ml of cell suspension per well;

(2) After the cells were attached for 24 hours, they were fixed with 4% PFA (paraformaldehyde) for 10 minutes;

(3) PBS wash 5minx3;

(4) blocking solution (containing 10% sheep serum, 30% serum albumin) blocked lh;

(5) The I antibody (ERCC2 1 : 150; ProteinTech, Catalog No: 101818-1-AP) prepared in the blocking solution was incubated at 4 degrees overnight;

(6) PBS wash 5 minx 3;

(7) FITC-labeled II anti-[1:50; FITC-labeled goat anti-rabbit IgG (Zhongsu Jinqiao, Catalog No: ZF-0311)] was incubated in the dark for lh;

(8) Wash with PBS for 5 min 3;

(9) OLYMPUS inverted phase contrast fluorescence microscope (Japan, OLYMPUS 1X71) observation, filming.

V. Immunohistochemical detection:

1 solvent and preparation method

The solvent preparation method described in the method is identical to the solvent described in Example 1 and the preparation method thereof.

2 Immunohistochemistry

(3) PBS wash 5minx3;

(5) Antigen antibody response:

i. Primary antibody incubation: 150 μl of ERCC2 antibody diluted 1:100 was added to the cultured cells, incubated at 4 ° C overnight, and then washed 3 times with PBS for 10 minutes each time; Ii. Incubation of secondary antibody: Add 150 μl of biotinylated secondary antibody working solution to the cultured cells, incubate for 60 minutes at room temperature, and wash 3 times with PBS for 10 minutes each time;

(6) Add horseradish-labeled streptavidin working solution (S-A/HRP) to the cells, incubate for 60 minutes at room temperature, and wash 3 times with PBS for 10 minutes each time;

(7) DAB color development:

i. Prepare the substrate mixture: add 1 drop of reagent A in 1 ml of double distilled water with pH value of 7.0, mix well, add one drop of reagent B and reagent C, and mix to obtain a mixture of substrates;

Ii. Color development: 150μ1 substrate mixed droplets are added to the cells, and the color is developed at room temperature for 3~10 minutes. After sufficient color development, the tap water is rinsed to terminate the reaction;

(8) Dyeing, dehydration, transparency, and mounting: The tissue sections were immersed in hematoxylin staining solution twice for 2 seconds each time; then treated with distilled water for 3 times for 5 minutes each time; then 50% ethanol, 70% ethanol and 90% ethanol were treated once each for 1 minute; then treated with absolute ethanol for 2 times, each time for 2 minutes, and a mixture of xylene and absolute ethanol was treated once for 1 minute, 2 minutes, 100% Xylene treatment 2 times, each time 2 minutes; finally sealed with gum, save;

(9) OLYMPUS inverted phase contrast fluorescence microscope observation, film;

00) Immunofluorescence and immunohistochemistry results (as shown in Figure 4)

Figure 4-a, d is an ovarian cancer cultured cell without any staining; Figure 4-b, e is an immunohistochemical staining of ovarian cancer cultured cells, showing: expression of ERCC2 in primary chemotherapy-sensitive cells (OV2008) Significantly enhanced, strong green, and the expression of primary chemotherapy-resistant cells (C13) was weak and dark green. Figure 4-c, f is an immunohistochemical staining of ovarian cancer cultured cells, showing that ERCC2 expression in primary chemotherapy-sensitive cells (OV2008) is dark brown, significantly stronger than primary chemotherapy-resistant cells (C13) Expressed, light brown. This result is completely consistent with the results obtained by ERCC2 in chemosensitive and resistant patient sections.

Claims

Claim

A kit for measuring the sensitivity of primary chemotherapy for ovarian cancer, characterized in that the kit comprises: 5% goat serum, 1% BSA, 0.01% polyethylene glycol octyl phenyl ether and pH 7.4 A blocking solution consisting of phosphate buffer, an antigen-antibody reaction system consisting of ERCC2 antibody, biotinylated secondary antibody working solution and blocking solution, and avidin composed of horseradish-labeled streptavidin working solution and DAB reagent buffer A protein-oxidase reaction system, and hematoxylin as a counterstain.

2. The kit according to claim 1, characterized in that the kit further comprises an antigen retrieval system consisting of 0.01 M citrate buffer and phosphate buffer, and consisting of 3% hydrogen peroxide and phosphate buffer. Elimination of the endogenous peroxidase system.

3. A method for measuring sensitivity of a ovarian cancer tissue sample to a primary chemotherapeutic drug, characterized in that the kit of claim 1 is used, comprising the steps of: using a blocking solution to treat a sample to be tested, respectively using ERCC2 antibody and biotin The secondary antibody working solution is subjected to primary antibody and secondary antibody reaction, and then the sample is treated with horseradish-labeled streptavidin working solution, and finally DAB coloring and hematoxylin counterstaining are performed;

Sensitivity to chemotherapeutic drugs was determined based on sample staining: Samples with a brown-positive reaction were identified as ovarian cancer tissues sensitive to primary chemotherapeutic drugs; samples with a blue-negative response were identified as ovarian cancer resistant to primary chemotherapeutic drugs organization.

4. The method of claim 3, comprising the steps of:

2 antigen antibody response:

A negative control group was used, and the same volume of blocking solution was used instead of ERCC2 antibody;

4 DAB color development:

(2) Color development: 150μ1 substrate mixed droplets were added to the tissue, and the color was developed at room temperature for 3 to 10 minutes. Rinse with water to stop the reaction;

6 Depending on the staining of the sample, the sensitivity to the chemotherapeutic drug was determined: the sample with a brown positive reaction was determined to be an ovarian cancer tissue sensitive to the primary chemotherapeutic drug; the sample with a blue negative reaction was judged to be an ovary resistant to the primary chemotherapeutic drug. Cancer tissue.

5. The method of claim 3, wherein: the method is applied to the evaluation of ovarian cancer formalin-fixed paraffin-embedded tissue, and further includes a pre-treatment step of the sample:

A method for screening an ovarian cancer patient susceptible to a primary chemotherapeutic drug, comprising the use of the kit of claim 1, comprising the steps of: treating a sample of an ovarian cancer patient with an isolated tumor tissue as a test sample, using a blocking solution After measuring the sample, the primary antibody and the secondary antibody were reacted with the ERCC2 antibody and the biotinylated secondary antibody working solution, respectively, and then the sample was treated with horseradish-labeled streptavidin working solution, and finally DAB color development and hematoxylin counterstaining were performed. Depending on the staining of the sample, the susceptibility of the sample to the ovarian cancer patient is sensitive to the primary chemotherapeutic drug: The sample with a brown positive reaction is determined to be an ovarian cancer tissue sensitive to the primary chemotherapeutic drug, and the sample is derived from the ovarian cancer patient to the primary chemotherapeutic drug. Sensitive; samples with a blue-negative response were identified as ovarian cancer tissues that were resistant to primary chemotherapy drugs, and sample-derived ovarian cancer patients were resistant to primary chemotherapy drugs.

A method for guiding a first-stage chemotherapy administration of a patient after ovarian cancer, characterized in that the kit according to claim 1 is used, comprising the steps of: clinically collecting a patient's ovarian cancer tissue as a test sample, and treating the sample to be tested with a blocking solution; The ERCC2 antibody and the biotinylated secondary antibody working solution were respectively used for primary and secondary antibody reaction, and then the sample was treated with horseradish-labeled streptavidin working solution, and finally DAB color development and hematoxylin counterstaining were performed; First, the sensitivity to the primary chemotherapy drug was determined based on the staining of the sample: The sample with a brown positive reaction was determined to be an ovarian cancer tissue sensitive to the primary chemotherapy drug; the sample with a blue negative reaction was judged to be resistant to the primary chemotherapy drug. Ovarian cancer tissue;

According to the results of the sensitivity of the above samples to the primary chemotherapy drugs, further determination: The source of positive ovarian cancer tissue is highly sensitive to postoperative primary chemotherapy drug treatment; the source of negative ovarian cancer tissue is Post-primary chemotherapy drugs have a high tolerance to treatment.

8. The method of any of claims 3-7, wherein the primary chemotherapeutic agent is cisplatin, paclitaxel or a combination of the two.

9. A method according to any one of claims 3 to 7 wherein the ovarian cancer is serous ovarian cancer.