CN111751531A - Kit and detection method for detecting expression of peripheral blood circulating tumor cell AR-V7 protein of prostate cancer patient - Google Patents

Abstract

The invention provides a kit and a detection method for detecting the expression of a prostate cancer patient peripheral blood circulating tumor cell AR-V7 protein, wherein the kit comprises a diluent, a destaining solution, a staining solution A, a staining solution B, a mouse anti-human AR-V7 primary antibody, a horse radish peroxidase-labeled goat anti-mouse secondary antibody, 0.1% Triton X-100, 0.3% H₂O₂A reagent A; the detection method comprises the steps of separating and obtaining the peripheral blood circulating tumor cells CTC by using a membrane filtration device, and detecting the AR-V7 expression condition of the peripheral blood CTC by using an immunohistochemical technology. By using the detection method provided by the invention, the AR-V7 expression condition of a patient with late or recurrent hepatocellular carcinoma can be detected without obtaining a tissue sample by puncture biopsy, and real-time dynamic detection can be realized by using a minimally invasive technology; by adopting the separation device and the diluent, the circulating tumor cells are well separated, the interference of blood cells is avoided, and the condition that the circulating tumor cells can be separated in the dyeing process is avoidedThe method can generate false positive results caused by edge effects, has good stability, reduces the loss of cells, and improves the detection accuracy.

Description

Kit and detection method for detecting expression of peripheral blood circulating tumor cell AR-V7 protein of prostate cancer patient

Technical Field

The invention provides a kit and a detection method for detecting the expression of a prostate cancer patient peripheral blood circulating tumor cell AR-V7 protein, and belongs to the technical field of molecular biology.

Background

Worldwide, prostate cancer incidence is on the 2 nd malignant tumor in men and cancer-specific deaths are on the 6 th. 2015 annual report of Chinese tumor registration shows that in 2011, the incidence rate of Chinese prostate cancer is on the 9 th position of malignant tumor, and the incidence rate and the mortality rate of urban male prostate cancer are on the 6 th position and the 9 th position respectively in male malignant tumor. The difficulty of the prostate cancer treatment is the castration resistance phenomenon after metastatic prostate cancer, particularly endocrine treatment, the treatment means after the castration resistance of the metastatic prostate cancer is limited at present, and all conventional treatment means (radiotherapy, chemotherapy, endocrine treatment and targeted treatment) are ineffective.

With the development of molecular biology, immunotherapy of middle and late stage prostate cell carcinoma is gradually emerging, and a primary clinical test also obtains a better curative effect, and a plurality of phase III clinical tests are currently carried out. AR-V7 (Androgen ReceptorVariant-7) is a splice variant of the Androgen Receptor (AR) that initiates nuclear translocation of the AR by forming heterodimers with the AR in the absence of ligands such as testosterone and dihydrotestosterone, which in turn stimulates transcriptional expression of downstream target genes involved in the Androgen axis. The detection of the expression level of the AR-V7 protein in the tissues of the patient can simply and accurately predict the treatment result of the mCRPC patient receiving abiraterone or enzalutamide: patients with high expression level of AR-V7 protein have poor effect of orally taking abiraterone. However, there is little chance of obtaining pathological tissue from metastatic lesions in patients with metastatic castration resistant prostate cancer.

Circulating Tumor Cells (CTC) are tumor cells that have been shed from solid tumors into the peripheral blood circulation, and since their discovery in 1989, various methods have been used to detect Circulating tumor cells in the peripheral blood. Recent studies have shown that their detection is of great clinical significance for assessing the prognosis of patients with tumours, especially patients with advanced tumours, and for selecting appropriate individualized treatments. CTC detection is called liquid biopsy of tumor because of its characteristics of minimal invasion, real-time detection, etc.

Aiming at the current clinical practice, the sample for detecting the AR-V7 of the prostate cancer patient is mainly tumor tissue, which is from operation or puncture biopsy and is difficult to detect for many times or in real time. Therefore, the detection of the expression of the AR-V7 in the Circulating Tumor Cells (CTC) has important value for the prognosis of the prostate cancer and the evaluation of the curative effect of immunotherapy.

At present, units such as Shandong province first medical university, Shandong province drug research institute combined with Shandong Qixin Biotechnology limited company, Shandong well-known Biotechnology limited company, Jinan Xin Biotechnology limited company, Shandong discovery biotechnology limited company and the like, carry out industrialized popularization on the key technology of detection and identification of circulating tumor cells, the project is a Shandong province major scientific and technological innovation project, the project takes the Shandong province drug research institute in Jinan school of Shandong first medical university as the core, realizes a registration system, relies on the core diagnostic technology of detection and identification of circulating tumor cells, and further registers, identifies and diagnoses a kit, and comprises PD1, PD-L1, ER, PR, Her-2, GPC-3, VEGF, P53, Vimentin, EGFR, RAS, CK, ALK-D5F3, CD20, ALK/EML4, Beta-catendin, E-Cacatenin, EP-CAM, HPV, IDH-1, PSA, PSMA, VEGF, GFAP, cytokeratin, AE1/AE3, estrogen receptor, progestin receptor, BCA-225, CA 125, CEA, EMA, ERCC1, HPV, Ki-67, P53, TOP2A and the like are used as tracers for expression of CTCs, and the identification and diagnosis kit is registered to be an ultrasensitive, ultrafast, high-coverage, low-cost, accurate and specific kit, and industrially popularized by cooperating with Shandong Qicheng Xin Biotech Co., Ltd, Shandong well-known Biotech Co., Ltd, Jinan En Biotech Co., Ltd, Shandong discovery Biotech Co., Ltd registered in Jinan.

Disclosure of Invention

Aiming at the defects that in the prior art, a patient with advanced tumor or recurrent prostate cancer cannot puncture to obtain a tissue specimen in real time or repeatedly and further cannot evaluate the real-time dynamic state of AR-V7 of the patient, and the prior detection method is easy to generate false positive and false negative, the invention provides a kit and a detection method for detecting the expression of AR-V7 protein of peripheral blood circulation tumor cells of the patient with prostate cancer: separating and obtaining Circulating Tumor Cells (CTC) in peripheral blood of a patient with advanced prostate cancer by using a membrane filtration device, and further detecting the expression condition of AR-V7 on the CTC by using an immunohistochemical technology.

The invention is realized by the following technical scheme:

a kit for detecting the expression of the peripheral blood circulating tumor cell AR-V7 protein of a prostate cancer patient is characterized by comprising 45mL of diluent, 1mL of destaining solution, 0.5mL of staining solution A, 1mL of staining solution B, 100 muL of primary mouse anti-human AR-V7 antibody, 100 muL of secondary horse radish peroxidase-labeled goat anti-mouse antibody, 0.1% Triton X-100100 muL and 0.3% H₂O₂100. mu.L of reagent A1 mL.

Preferably, the diluent is 1mmol/L EDTA +0.1% BSA +0.3% ferric sulfate +0.5% sucrose.

Preferably, the decoloring liquid is composed of 95% alcohol and 100% xylene in a volume ratio of 1: 1.

Preferably, the staining solution A is a DAB staining solution; the staining solution B is hematoxylin staining solution.

Preferably, the reagent A is ethanol and polyformaldehyde according to a volume ratio of 3: 1.

In the invention, the method for detecting the expression of the AR-V7 protein in the peripheral blood circulation tumor cells of the prostate cancer patients by using the kit for non-diagnosis purposes comprises the following steps:

(1) separating and obtaining peripheral blood of patients with advanced or recurrent prostate cancer who can not obtain tissue specimens by using a membrane filtration device: collecting 5ml of peripheral blood of the median elbow vein of a patient with advanced stage or recurrent prostate cancer who cannot obtain a tissue specimen;

(2) peripheral blood sample pretreatment: diluting the collected peripheral blood sample by 10 times by using a diluent, and adding polyformaldehyde to fix the peripheral blood sample for 10 minutes after dilution, wherein the fixed final concentration is 0.25%;

(3) and (3) filtering the peripheral blood sample by using a membrane filtration tumor cell separation device, and separating to obtain peripheral blood CTC: adding the pretreated peripheral blood sample into a blood sample container of a membrane filtration tumor cell separation device, and naturally filtering the blood sample by means of gravity;

(4) after the filtration is finished, taking the filter out of the membrane filtration tumor cell separation device, adding 0.5ml of circulating tumor cell staining solution A into the filter, staining for 3min, and washing with PBS buffer solution; adding 1ml of staining solution B after completely filtering the filtrate, staining for 2min, washing for 2 times by using 1ml of pure water, taking down the filter membrane, placing on a glass slide, drying, and observing under a microscope to determine whether CTC exists;

(5) detecting the expression condition of AR-V7 protein of CTC in peripheral blood by using immunohistochemical technology.

Preferably, the specific method for detecting AR-V7 protein expression of peripheral blood CTC in step (5) is as follows:

(1) and (3) decoloring: taking down the filter membrane with CTC from the glass slide, soaking in a decolorizing solution for 4-6 hours, and removing the CTC staining solution;

(2) dropping 100 μ l of 0.1% Triton X-100, incubating at room temperature for 15min, and washing with DI water for 2min × 3 times;

(3) 100 μ l of 0.3% H was added dropwise₂O₂Incubating at room temperature for 10min, washing with PBS for 2min × 3 times;

(4) dripping 100 μ l of mouse anti-human AR-V7 primary antibody, incubating at room temperature for 2h or overnight at 4 deg.C, washing with PBS for 2min × 3 times;

(5) dripping 100 mu l of horseradish peroxidase labeled goat anti-mouse secondary antibody, incubating for 20min at the temperature of 18-26 ℃, and washing for 2min multiplied by 3 times by PBS;

(6) dripping 100 mul of DAB color development solution, incubating at 18-26 ℃, and observing the color development condition under a microscope at any time, wherein the observation time is 3-10 min;

(7) after the color development is finished, discarding DAB color development liquid, flushing with running water for 5min, and dyeing with hematoxylin for 5 min;

(8) the hydrochloric acid alcohol is differentiated for 8 seconds, and tap water is rewound for 5 min;

(9) dehydrating the rewound CTC by using 75% ethanol (1 min), 95% ethanol (1 min) and 100% ethanol (1 min) in a gradient manner, then adding 1mL of reagent A, shaking and mixing uniformly, centrifuging and precipitating, and sealing the precipitate by using neutral resin;

(10) and (5) performing microscopic examination by using an optical microscope.

The device for separating and circulating tumor cells by membrane filtration comprises a filter, a blood sample container, a waste liquid tank and an iron stand, wherein the iron stand is provided with a base, a vertical frame and a support, the blood sample container is arranged at the upper part of the iron stand through the support, the filter is arranged below the blood sample container, the filter is communicated to the waste liquid tank through an infusion apparatus, and the waste liquid tank is arranged on the base.

The filter comprises a filter upper opening, a filter membrane carrying platform and a filter lower opening, and the filter membrane is arranged on the filter membrane carrying platform; the upper port of the filter is connected with a blood sample container, and the lower port of the filter is connected with a waste liquid cylinder through an infusion apparatus.

The filter membrane is made of hydrophobic material, and filter holes with the caliber of 8 microns are uniformly distributed on the filter membrane; tumor cells are typically greater than 15 microns in diameter, while blood cells (including red blood cells, white blood cells) are typically less than 8 microns in diameter, so that when peripheral blood containing CTCs is filtered, the blood cells are filtered by being smaller in diameter than the filter pores, and the CTCs are retained on the filter membrane by being larger in diameter than the filter pores.

Advantageous effects

(1) The detection method provided by the invention can detect the AR-V7 protein expression condition of a patient with advanced or recurrent prostate cancer without obtaining a tissue sample by puncture biopsy, and can realize real-time dynamic detection by utilizing a minimally invasive technology;

(2) the method provided by the invention has the advantages of good separation of circulating tumor cells, capability of avoiding the interference of blood cells, capability of avoiding false positive results caused by edge effect possibly generated in the dyeing process, good stability, reduction of cell loss and improvement of detection accuracy.

Drawings

FIG. 1 is a schematic structural view of a membrane filtration apparatus according to the present invention;

FIG. 2 is a schematic sectional view showing the structure of a filter of the membrane filtration apparatus of the present invention;

FIG. 3 is a schematic view showing the structure of a filter membrane of the membrane filtration apparatus of the present invention;

FIG. 4 is a diagram of a circulating tumor cell image obtained by separating peripheral blood of a prostate cancer patient;

in the figure: 1 iron stand, 2 blood sample containers, 3 filters, 4 transfusion devices, 5 waste liquid jars, 6 filter upper ports, 7 filter membranes, 8 filter membrane platforms, 9 filter lower ports, 10 filter holes, 11 bases, 12 vertical frames and 13 supports.

Detailed Description

The invention is elucidated below with reference to the figures and embodiments.

The specific specification of the kit used in the embodiment of the invention is shown in table 1:

TABLE 1

The technical method is applied to the embodiment of separating, obtaining and identifying 8 prostate cancer patients (and detecting 8 normal human samples as negative controls).

Example 1

Firstly, separating and acquiring CTCs in peripheral blood of patients with advanced or recurrent prostate cancer, wherein the patients cannot obtain tissue specimens, and determining whether the CTCs exist:

collecting 5ml of fasting 8-12 hours fasting blood from the median cubital vein, diluting peripheral blood with 45ml of diluent, and then adding 3ml of 4% paraformaldehyde to fix the diluted blood sample for 10 minutes;

at fixed intervals, a membrane filtration device was assembled: as shown in fig. 1, 2 and 3, the filter device comprises a filter 3, a filter membrane 7, a blood sample container 2, a waste liquid tank 5 and an iron stand 1;

wetting the filter 3 with 10ml of PBS, then adding the fixed peripheral blood sample into the blood sample container 2 of the membrane filtration device, allowing it to naturally filter by gravity, and the CTC being trapped on the filter membrane 7;

the tumor cells are typically larger than 15 microns in diameter, while the blood cells (including red blood cells, white blood cells) are typically smaller than 8 microns in diameter, so that when peripheral blood containing CTCs is filtered, the blood cells can be filtered by being smaller than filter pores 10, while the CTCs are retained on filter membrane 7 by being larger than filter pores 10.

After the filtration is finished, taking the filter 3 from the filter device, opening and removing the upper opening 6 of the filter, adding 0.5ml of circulating tumor cell staining solution A into the filter, staining for 3min, and washing with PBS buffer solution; filtering the filtrate completely, adding solution B, 1ml, staining for 2min, and pure water 1ml, washing filter 3 with PBS buffer solution, taking down filter membrane 7 with ophthalmic forceps with cell surface facing upwards, and placing on glass slide;

the filters were dried and observed under a microscope to determine the presence of CTCs, with the results shown in table 2.

By observation, no CTCs were detected in 8 healthy volunteers; CTC was detected in 5 cases (Table 2) except that CTC was not detected in 2 cases of patients with intermediate stage prostate cancer and 1 case of patients with advanced stage prostate cancer, and the positive rate of this detection was 62.5%. It is worth noting that when the diluent contains 0.3% of ferric sulfate and 0.5% of sucrose, the circulating tumor cells in human peripheral blood can be effectively separated, and blood cells (red blood cells, white blood cells and the like) are prevented from being adhered to the tumor cells, so that the color development of CTC is influenced, and the final detection result is influenced. FIG. 4 is a photograph of a circulating tumor cell isolated from peripheral blood of a patient with prostate cancer, which has a large nucleus, irregular shape of nucleus and high nuclear to cytoplasmic ratio.

Table 2 example 1 CTC assay results

Secondly, detecting the expression condition of the AR-V7 protein of the CTC by using an immunohistochemical technology:

taking down the filter membrane 7 carrying CTC on the glass slide from the glass slide, soaking in a destaining solution of 95% alcohol and 100% xylene uniformly mixed according to a volume ratio of 1:1 for 4-6 hours, removing the CTC staining solution, dripping 100 mul of 0.1% Triton X-100, incubating at room temperature for 15min, washing with DI water for 2min × 3 times, dripping 100 mul of 0.3% H₂O₂Incubating for 10min at room temperature, washing for 2min × 3 times by PBS, dripping 100 mul of rat-anti-human AR-V7 primary antibody, incubating for 2h (or overnight at 4 ℃), washing for 2min × 3 times by PBS, dripping 100 mul of horse radish peroxidase-labeled goat-anti-mouse secondary antibody, incubating for 20min at room temperature (18-26 ℃), washing for 2min × 3 times by PBS, dripping 100 mul of DAB developing solution, incubating at room temperature (18-26 ℃) and observing the developing condition under a microscope at any time (generally 3-10 min, the time can not exceed 10 min), discarding the DAB developing solution after developing, and running waterWashing for 5min, and staining with hematoxylin for 5 min; the hydrochloric acid alcohol is differentiated for 8 seconds, and tap water is rewound for 5 min; dehydrating with 75% ethanol (1 min), 95% ethanol (1 min), 100% ethanol (1 min), adding reagent A, shaking, mixing, centrifuging, precipitating, air drying, and sealing with neutral resin; and (4) performing microscopic examination under an optical microscope, reading by a cytopathologist, and judging the expression condition of the AR-V7 protein according to the staining degree of cell membranes and cytoplasm.

The reagent A is a mixed solution of ethanol and polyformaldehyde with a volume ratio of 3:1, and after the mixed solution is sealed by neutral resin, CTC cells are complete in shape, do not wrinkle or swell, and reagents with other proportions or a single reagent cannot achieve the effect, so that the accuracy of the test is influenced.

The detected circulating tumor cells are applied to immunohistochemistry to confirm the expression of AR-V7 protein and are compared with the results of a prostate cancer general sample AR-V7, the difference is observed, the targeted therapy of the prostate cancer is guided mainly aiming at patients with the general sample AR-V7 protein negative expression and the circulating tumor cells positive expression, and a new thought is provided for the targeted therapy of the prostate cancer.

Claims (7)

1. A kit for detecting the expression of the peripheral blood circulating tumor cell AR-V7 protein of a prostate cancer patient is characterized by comprising 45mL of diluent, 1mL of destaining solution, 0.5mL of staining solution A, 1mL of staining solution B, 100 muL of primary mouse anti-human AR-V7 antibody, 100 muL of secondary horse radish peroxidase-labeled goat anti-mouse antibody, 0.1% Triton X-100100 muL and 0.3% H₂O₂100. mu.L of reagent A.

2. The kit of claim 1, wherein the diluent is 1mmol/L EDTA +0.1% BSA +0.3% ferric sulfate +0.5% sucrose.

3. The kit of claim 1, wherein the destaining solution is comprised of 95% alcohol to 100% xylene in a volume ratio of 1: 1.

4. The kit according to claim 1, wherein the staining solution A is DAB staining solution; the staining solution B is hematoxylin staining solution.

5. The kit according to claim 1, wherein the reagent A is ethanol and polyformaldehyde in a volume ratio of 3: 1.

6. A method for detecting the expression of AR-V7 protein in peripheral blood circulating tumor cells of a patient with prostate cancer using the kit of any one of claims 1-5 for non-diagnostic purposes, comprising the steps of:

(1) separating and obtaining peripheral blood of patients with advanced or recurrent prostate cancer who can not obtain tissue specimens by using a membrane filtration device: collecting 5ml of peripheral blood of the median elbow vein of a patient with advanced stage or recurrent prostate cancer who cannot obtain a tissue specimen;

(2) peripheral blood sample pretreatment: diluting the collected peripheral blood sample by 10 times by using a diluent, and adding polyformaldehyde to fix the peripheral blood sample for 10 minutes after dilution, wherein the fixed final concentration is 0.25%;

(3) and (3) filtering the peripheral blood sample by using a membrane filtration tumor cell separation device, and separating to obtain peripheral blood CTC: adding the pretreated peripheral blood sample into a blood sample container of a membrane filtration tumor cell separation device, and naturally filtering the blood sample by means of gravity;

(4) after the filtration is finished, taking the filter out of the membrane filtration tumor cell separation device, adding 0.5ml of circulating tumor cell staining solution A into the filter, staining for 3min, and washing with PBS buffer solution; adding 1ml of staining solution B after completely filtering the filtrate, staining for 2min, washing for 2 times by using 1ml of pure water, taking down the filter membrane, placing on a glass slide, drying, and observing under a microscope to determine whether CTC exists;

(5) detecting the expression condition of AR-V7 protein of CTC in peripheral blood by using immunohistochemical technology.

7. The method of claim 6, wherein the specific method for detecting AR-V7 protein expression in peripheral blood CTC in step (5) is as follows:

(1) and (3) decoloring: taking down the filter membrane with CTC from the glass slide, soaking in a decolorizing solution for 4-6 hours, and removing the CTC staining solution;

(2) dropping 100 μ l of 0.1% Triton X-100, incubating at room temperature for 15min, and washing with DI water for 2min × 3 times;

(3) 100 μ l of 0.3% H was added dropwise₂O₂Incubating at room temperature for 10min, washing with PBS for 2min × 3 times;

(4) dripping 100 μ l of mouse anti-human AR-V7 primary antibody, incubating at room temperature for 2h or overnight at 4 deg.C, washing with PBS for 2min × 3 times;

(5) dripping 100 mu l of horseradish peroxidase labeled goat anti-mouse secondary antibody, incubating for 20min at the temperature of 18-26 ℃, and washing for 2min multiplied by 3 times by PBS;

(6) dripping 100 mul of DAB color development solution, incubating at 18-26 ℃, and observing the color development condition under a microscope at any time, wherein the observation time is 3-10 min;

(7) after the color development is finished, discarding DAB color development liquid, flushing with running water for 5min, and dyeing with hematoxylin for 5 min;

(8) the hydrochloric acid alcohol is differentiated for 8 seconds, and tap water is rewound for 5 min;

(9) dehydrating the rewound CTC by using 75% ethanol (1 min), 95% ethanol (1 min) and 100% ethanol (1 min) in a gradient manner, then adding 1mL of reagent A, shaking and mixing uniformly, centrifuging and precipitating, and sealing the precipitate by using neutral resin;

(10) and (5) performing microscopic examination by using an optical microscope.

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