CN111638344A - Kit and method for detecting E-Cadherin gene mutation of peripheral blood circulating tumor cells of small cell lung cancer patients - Google Patents
Kit and method for detecting E-Cadherin gene mutation of peripheral blood circulating tumor cells of small cell lung cancer patients Download PDFInfo
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Abstract
The invention provides a kit and a method for detecting E-Cadherin gene mutation of peripheral blood circulating tumor cells of a patient with small cell lung cancer, belonging to the technical field of molecular biology. The kit comprises diluent, destaining solution, staining solution A, staining solution B, mouse anti-human E-Cadherin monoclonal antibody, goat anti-mouse IgG/HRP, SABC, 0.1% Triton X-100 and 0.3% H2O2The detection method provided by the invention can detect the E-Cadherin expression condition of a patient with advanced or recurrent small cell lung cancer without obtaining a tissue specimen by a puncture biopsy, belongs to minimally invasive technology, can detect in real time, and can avoid stainingFalse positive result caused by edge effect possibly generated in the process has good stability, reduces loss of cells and improves detection accuracy.
Description
Technical Field
The invention provides a kit and a method for detecting E-Cadherin gene mutation of peripheral blood circulation tumor cells of a patient with small cell lung cancer, belonging to the technical field of molecular biology.
Background
Lung cancer is one of the main malignant tumors causing death of cancer patients, and both the morbidity and mortality of lung cancer are the first in China. Small Cell Lung Cancer (SCLC) accounts for about 15% to 20% of the incidence of lung cancer, and has the characteristics of faster tumor doubling time, rapid growth and easy early metastasis compared to non-small cell lung 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.
At present, Shandong medical university, Shandong medical research institute, Shandong Kaigang intelligent machine company, and Shandong Kege corporation cooperate with circulating tumor cell detection and identification key technology, detection equipment, and kit development and production, Shandong Qixin biological technology company, Shandong Xiaoming biological technology company, Jinan Xin biological technology company, and Shandong discovery biological technology company, and the project is a Shandong major technological innovation project, and the Shandong medical research institute in Jinan school of Shandong medical university is used as a core, a register system is implemented, and the circulating tumor cell detection and identification core diagnosis technology is relied on, and further, the kit is registered, identified and diagnosed, and comprises PD1, PD-L1, ER, PR, Her-2, GPC-3, VEGF, P53, Vimentin, TKI-EGFR, RAS, CK, VEGF, and the other components, ALK-D5F3, CD20, ALK/EML4, Beta-catenin, E-Cadherin, 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, as tracers expressed by CTCs, registered with an ultra-sensitive, ultra-rapid, high-coverage, low-cost, accurate and specific diagnostic kit for identification, through cooperation with Kaykui Intelligent machinery Limited, Shandong Qixin Biotechnology Limited, Shandong Biotechnology Limited, Jinan Xin Biotechnology Limited, Shandong discovery Biotechnology Limited, registered in Jinan.
E-Cadherin (E-Cadherin) is the most classical marker of epithelial cell phenotype, and the down-regulation of E-Cadherin expression marks the reduction of adhesion capacity between cells, so that E-Cadherin is taken as one of the main means for identifying the occurrence of EMT of small cell lung cancer, plays an important role in cell-cell adhesion as a cell surface glycoprotein, and is involved in the maintenance of tissues and organs. In recent years, the relationship between EMT and tumor resistance of small cell lung cancer is increasingly emphasized, and a great deal of research shows that the drug resistance is simultaneously increased due to the occurrence of EMT in different tumors and is accompanied by the change of E-Cadherin expression. At present, the SCLC diagnosis and treatment guidelines at home and abroad are considered as follows: the time points for E-Cadherin mutation detection in SCLC patients include initial diagnosis and striving to detect again at disease progression, and false positives and false negatives should be reduced by repeated detection. In current clinical practice, samples for E-Cadherin detection of SCLC patients mainly comprise tumor tissues, and are from operations or needle biopsies, so that multiple or real-time detection is difficult to achieve.
Disclosure of Invention
In order to overcome the defects that patients with advanced or recurrent small cell lung cancer cannot obtain tissue specimens by real-time or repeated puncture and further cannot evaluate the E-Cadherin state of the patients, the invention provides a detection method for the non-diagnosis purpose of ALK gene mutation of peripheral blood circulating tumor cells of the patients with small cell lung cancer, which comprises the following steps: separating and obtaining CTC in peripheral blood of patients with advanced or recurrent small cell lung cancer, wherein the patients cannot obtain tissue specimens, and further detecting the E-Cadherin expression condition of the CTC by using an immunohistochemical technology.
The technical scheme adopted by the invention is as follows:
the invention provides a kit for detecting E-Cadherin gene mutation of peripheral blood circulating tumor cells of a patient with small cell lung cancer, which comprises 45mL of diluent, 1mL of destaining solution, 0.5mL of staining solution A, 1mL of staining solution B, and mouse100 muL of anti-human E-Cadherin monoclonal antibody, 100 muL of goat anti-mouse IgG/HRP 100 mu L, SABC 100, 0.1% Triton X-100100 muL, 0.3% H2O2100 mu L of the reagent, 0.5mL of the reagent A, 1mL of the reagent B and 60mL of 6 × PBS buffer solution, wherein the pH value of the PBS buffer solution is 7.4.
Further, the diluent is composed of 1mmol/L EDTA +0.1% BSA +0.1% trehalose +0.2% polyoxyethylene polyoxypropylene ether block copolymer, and the base solution is Tris-HCl buffer.
Further, the decoloring solution is composed of 95% alcohol and 100% xylene according to a volume ratio of 1: 1.
Further, the staining solution A is a DAB staining solution; the staining solution B is hematoxylin staining solution.
Further, the reagent A is a hydroxypropyl methyl cellulose xylene mixed solution with the mass fraction of 0.6%; the reagent B is prepared from ethanol and 1, 2-propylene glycol according to a volume ratio of 3: 1.
The invention also provides a method for detecting the E-Cadherin gene mutation of the peripheral blood circulation tumor cells of a small cell lung cancer patient by using the kit for non-diagnosis, which comprises the following steps:
(1) separating and acquiring CTCs in peripheral blood of patients with advanced or recurrent small cell lung cancer, wherein the patients cannot obtain tissue specimens, by using a membrane filtration device: collecting peripheral blood of patients with advanced or recurrent small cell lung cancer who can not obtain tissue specimens: 5ml of peripheral blood of the median cubital vein;
(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 E-Cadherin expression condition of CTC by using an immunohistochemical technology.
The specific method for detecting the E-Cadherin expression of CTC comprises the following steps:
(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 for 10min at room temperature, washing for 2min with PBS for × 3 times, (4) dripping 100 mul of mouse anti-human E-Cadherin monoclonal antibody primary antibody, incubating for 2h at room temperature or overnight at 4 ℃, and washing for 2min with PBS for × 3 times;
(5) dripping 100 mul of goat anti-mouse IgG/HRP, incubating for 20min at the temperature of 18-26 ℃, and washing for 2min multiplied by 3 times by PBS;
(6) dripping 100 mu l of SABC, incubating for 20min at the temperature of 18-26 ℃, and washing for 2min multiplied by 3 times by PBS;
(7) 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;
(8) after the color development is finished, discarding DAB color development liquid, flushing with running water for 5min, and dyeing with hematoxylin for 5 min;
(9) the hydrochloric acid alcohol is differentiated for 8 seconds, and tap water is rewound for 5 min;
(10) 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 0.5mL of reagent A, oscillating uniformly, adding 1mL of reagent B, shaking and mixing uniformly, centrifuging and precipitating, and sealing the precipitate by using neutral resin;
(11) and (5) performing microscopic examination by using an optical microscope.
The device for separating 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 materials, and filter holes with the caliber of 8 microns are uniformly distributed on the filter membrane.
The invention has the beneficial effects that:
(1) the detection method provided by the invention can detect the E-Cadherin expression condition of a patient with advanced or recurrent small cell lung cancer without taking a tissue specimen by a puncture biopsy. The technology belongs to minimally invasive and can detect in real time.
(2) The method provided by the invention can avoid false positive results caused by edge effect possibly generated in the dyeing process, has good stability, reduces the loss of cells and improves the 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 patient with lung cancer;
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 invention is shown in table 1:
TABLE 1
The technical method is applied to the embodiment of separating, obtaining and identifying 8 cases of small cell lung cancer patients (and simultaneously detecting 8 cases of normal human samples as negative controls) peripheral blood circulating tumor cells.
Example 1
Firstly, separating and acquiring CTCs in peripheral blood of a patient with advanced or recurrent small cell lung cancer, wherein a tissue specimen cannot be obtained, by using a membrane filtering device, and determining whether the CTCs exist:
collecting 5ml of fasting 8-12 hr fasting blood from the median cubital vein, diluting peripheral blood with 45ml of diluent (component: 1mmol/L EDTA +0.1% BSA +0.1% trehalose +0.2% polyoxyethylene polyoxypropylene ether block copolymer), and fixing the diluted blood sample with 3ml of 4% paraformaldehyde for 10 min;
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.
By observation, as shown in table 2, CTCs were not found in none of 8 healthy volunteers; CTC is detected by patients with recurrent small cell lung cancer and patients with advanced small cell lung cancer, the positive rate of the detection is 100%, and the positive rate is remarkable in that when 0.1% of trehalose or 0.2% of polyoxyethylene polyoxypropylene ether block copolymer is not added into a diluent, 0.3% of trehalose or 0.3% of polyoxyethylene polyoxypropylene ether block copolymer is singly adopted, the prepared blood sample is poor in stability, a part of the blood sample forms stratification, blood cells are easy to aggregate and adhere, and the final detection effect is influenced.
TABLE 2 results of CTC assay in examples
Secondly, detecting the E-Cadherin expression condition of 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 with PBS, dripping 100 μ l of primary antibody of mouse anti-human E-Cadherin monoclonal antibody for 2h (or overnight at 4 ℃), washing for 2min × 3 times with PBS, dripping 100 μ l of goat anti-mouse IgG/HRP, incubating for 20min at room temperature (18-26 ℃), washing for 2min × 3 times with PBS, dripping 100 μ l of SABC, incubating for 20min at 18-26 ℃, washing for 2min × 3 times with PBS, dripping 100 μ l of DAB developing solution, incubating at room temperature (18-26 ℃) and observing the developing condition under a microscope (generally 3-10 min, the time cannot exceed 10 min), discarding DAB solution after developing, washing for 5min with running water, dyeing for 5min with hematoxylin, dyeing for 8 s with hydrochloric acid and alcohol, returning blue to 5min with tap water, adding 75% ethanol (1 min), 95% ethanol (1 min), dehydrating with 100% ethanol (1 min) gradient, adding 0.6% hydroxypropyl methylcellulose, shaking, adding 1V, mixing the mixture, stirring, precipitating with propylene glycol, and shaking to obtain a precipitate, and centrifuging the precipitate, and adding the mixture of 2V and stirringResin sealing; and (4) microscopic examination is carried out under an optical microscope, and a cytopathologist reads the cell, and the expression condition of the E-Cadherin is judged according to the staining degree of the cell membrane and the cytoplasm.
When the reagent B adopts single ethanol or 1, 2-propylene glycol and is sealed by neutral resin, the detection accuracy of the mixed solvent of the ethanol and the 1, 2-propylene glycol can reach 100 percent after sealing, the accuracy of the single ethanol is 85 percent, and the accuracy of the single 1, 2-propylene glycol is only 70 percent, so that false positive results caused by edge effect possibly generated in the dyeing process can be avoided, the stability is good, the loss of cells is reduced, and the detection accuracy is improved.
FIG. 4 is a diagram of a circulating tumor cell image obtained by separating peripheral blood of a patient with non-small cell lung cancer, wherein the image is 1 CTC cell, the cell nucleus is abnormal, the karyoplasmic ratio is more than 0.8, the cell diameter (long end) is more than 15 μm, the cell nucleus is larger due to deep staining (due to the increase of cancer cell chromatin, coarsening of particles and deep staining), and the shape of the cell nucleus is irregular; nuclear chromatin border shift, abnormal nuclear division.
The detected circulating tumor cells are applied to immunohistochemistry to confirm the expression of E-Cadherin and are compared with the E-Cadherin result of a small cell lung cancer general sample, the difference is observed, the targeted therapy of the small cell lung cancer is guided mainly aiming at the patients with negative E-Cadherin expression in the general sample and positive circulating tumor cell expression, and a new thought is provided for the targeted therapy of the small cell lung cancer.
Claims (7)
1. A kit for detecting E-Cadherin gene mutation of peripheral blood circulation tumor cells of a patient with small cell lung cancer 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 a mouse anti-human E-Cadherin monoclonal antibody, 100 muL of goat anti-mouse IgG/HRP 100 mu L, SABC 100 muL, 0.1% Triton X-100100 muL and 0.3% H2O2100 mu L of the reagent, 0.5mL of the reagent A, 1mL of the reagent B and 60mL of 6 × PBS buffer solution, wherein the pH value of the PBS buffer solution is 7.4.
2. The kit of claim 1, wherein the diluent is composed of 1mmol/L EDTA +0.1% BSA +0.1% trehalose +0.2% polyoxyethylenepolyoxypropylene ether block copolymer and the base is Tris-HCl buffer.
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 a 0.6 mass percent hydroxypropyl methyl cellulose xylene mixture; the reagent B is prepared from ethanol and 1, 2-propylene glycol according to a volume ratio of 3: 1.
6. A method for non-diagnostically detecting E-Cadherin gene mutations in circulating tumor cells in the peripheral blood of patients with small cell lung cancer using the kit of any one of claims 1 to 5, comprising the steps of:
(1) separating and acquiring CTCs in peripheral blood of patients with advanced or recurrent small cell lung cancer, wherein the patients cannot obtain tissue specimens, by using a membrane filtration device: collecting peripheral blood of patients with advanced or recurrent small cell lung cancer who can not obtain tissue specimens: 5ml of peripheral blood of the median cubital vein;
(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 E-Cadherin expression condition of CTC by using an immunohistochemical technology.
7. The assay of claim 6, wherein the specific method for detecting E-Cadherin expression of CTCs 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 for 10min at room temperature, washing for 2min with PBS for × 3 times, (4) dripping 100 mul of mouse anti-human E-Cadherin monoclonal antibody primary antibody, incubating for 2h at room temperature or overnight at 4 ℃, and washing for 2min with PBS for × 3 times;
(5) dripping 100 mul of goat anti-mouse IgG/HRP, incubating for 20min at the temperature of 18-26 ℃, and washing for 2min multiplied by 3 times by PBS;
(6) dripping 100 mu l of SABC, incubating for 20min at the temperature of 18-26 ℃, and washing for 2min multiplied by 3 times by PBS;
(7) 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;
(8) after the color development is finished, discarding DAB color development liquid, flushing with running water for 5min, and dyeing with hematoxylin for 5 min;
(9) the hydrochloric acid alcohol is differentiated for 8 seconds, and tap water is rewound for 5 min;
(10) 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 0.5mL of reagent A, oscillating uniformly, adding 1mL of reagent B, shaking and mixing uniformly, centrifuging and precipitating, and sealing the precipitate by using neutral resin;
(11) microscopic examination under an optical microscope 1.
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| CN113049344B (en) * | 2021-04-20 | 2022-02-01 | 深圳天烁生物科技有限公司 | Preparation method of quality control product for cell staining |
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