CN111856022A - Kit and method for detecting expression of peripheral blood circulating tumor cells E-Cadherin of pancreatic cancer patient - Google Patents
Kit and method for detecting expression of peripheral blood circulating tumor cells E-Cadherin of pancreatic cancer patient Download PDFInfo
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Abstract
The invention relates to a kit and a detection method for detecting the expression of peripheral blood circulating tumor cells E-Cadherin of a pancreatic cancer patient, belonging to the technical field of molecular biology. The kit 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 E-Cadherin antibody, 100 muL of rabbit anti-mouse IgG/HRP connected with horseradish peroxidase, 0.1% Triton X-100100 muL, and 0.3% H2O2100. mu.L, 15mL of reagent A, 50mL of reagent B, and 60mL of 6 XPBS buffer. The detection method of the invention utilizes a membrane filtration device to separate and obtain the peripheral blood of the patient with late stage or recurrent pancreatic cancer who can not obtain the tissue specimenThe expression condition of E-Cadherin of the CTC is further detected by using an immunohistochemical technology.
Description
Technical Field
The invention relates to a kit and a detection method for detecting the expression of peripheral blood circulating tumor cells E-Cadherin of a pancreatic cancer patient, belonging to the technical field of molecular biology.
Background
Pancreatic cancer is a common malignancy of the digestive system and one of the worst prognosis, among deaths caused by malignancies, pancreatic cancer ranks fourth, 90% of patients die within one year after diagnosis, and 5-year survival rate is less than 5%. As early symptoms of pancreatic cancer are hidden, the pancreatic cancer is not specific, the pancreatic cancer is highly invasive, the pancreatic cancer is early in metastasis, the pancreatic cancer is mostly in the middle and late stages when the pancreatic cancer is diagnosed, the surgical resection rate is low, and the pancreatic cancer is diagnosed only by 15%, the selection of the treatment method for pancreatic cancer patients is particularly important, and unnecessary surgical trauma and risks can be avoided for part of patients.
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 pancreatic cancer EMT, plays an important role in cell-cell adhesion as a class of cell surface glycoproteins, and is involved in the maintenance of tissues and organs. In recent years, the relationship between EMT and tumor resistance in pancreatic cancer has been increasingly emphasized, and a number of studies have shown that the resistance increases simultaneously among different tumors due to the occurrence of EMT, and is accompanied by changes in E-Cadherin expression. In current clinical practice, samples for detecting E-Cadherin of pancreatic cancer patients mainly comprise tumor tissues, and are from operations or needle biopsies, so that multiple or real-time detection is difficult to achieve.
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.
Disclosure of Invention
In order to overcome the problem of multiple or real-time detection and simultaneously avoid the risks of bleeding, pancreatic fistula, infection and the like in the process of puncture biopsy, the invention provides a kit and a detection method for detecting the expression of E-Cadherin of peripheral blood circulation tumor cells of a pancreatic cancer patient.
The technical scheme adopted by the invention is as follows:
a kit for detecting the expression of E-Cadherin in peripheral blood circulation tumor cells of a pancreatic cancer patient comprises 45mL of diluent, 1mL of destaining solution, 0.5mL of staining solution A, 1mL of staining solution B, 100 muL of mouse anti-human E-Cadherin primary antibody, 100 muL of rabbit anti-mouse IgG/HRP connected with horseradish peroxidase, 0.1% Triton X-100100 muL, 0.3% H2O2100. mu.L, reagent A15mL, reagent B50 mL, and 6 XPBS buffer 60 mL.
The diluent is 19g/L of potassium chloride, 4.8 g/L of ethylene diamine tetraacetic acid dipotassium, 2.6 g/L of pyridinium sodium, 17g/L of monopotassium phosphate, 9g/L of sodium hydroxide, 0.1g/L of trehalose, 2g/L of dihydroxymethyl urea and the balance of purified water.
The destaining solution consists of 0.5 percent hydrochloric acid-70 percent ethanol solution.
The staining solution A is a DAB staining solution; the staining solution B is hematoxylin staining solution.
The reagent A is formaldehyde-xylene, and the volume ratio of the formaldehyde-xylene is 1: 2, preparing a composition; the reagent B is xylene.
The method for detecting the expression of the peripheral blood circulation tumor cells E-Cadherin of a pancreatic cancer patient by using the kit in a non-diagnostic purpose comprises the following steps:
(1) separating and acquiring CTCs in peripheral blood of patients with advanced or recurrent pancreatic cancer, wherein the patients cannot obtain tissue specimens, by using a membrane filtration device: collecting peripheral blood of patients with advanced or recurrent pancreatic cancer who cannot obtain tissue specimens: 5ml of peripheral blood of the median cubital vein;
(2) peripheral blood sample pretreatment: diluting the collected peripheral blood sample by using 45ml of 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 the CTC in the step (5) is as follows:
s1 decolorization: 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;
s2 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;
s3 was added dropwise with 100. mu.l of 0.3% H2O2Incubating at room temperature for 10min, washing with PBS for 2min × 3 times; (4) 100 mul of mouse anti-human E-Cadherin primary antibody is dripped into the chamberIncubating for 2h or overnight at 4 ℃, washing for 2min × 3 times by PBS;
s4, 100 mul of rabbit anti-mouse IgG/HRP connected with horseradish peroxidase is dripped, incubation is carried out for 20min at the temperature of 18-26 ℃, and PBS is washed for 2min multiplied by 3 times;
s5, 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;
s6, after the color development is finished, discarding the DAB color development solution, flushing with running water for 5min, and dyeing with hematoxylin for 5 min;
s7, carrying out alcohol differentiation for 8 seconds by hydrochloric acid, and bluing tap water for 5 min;
s8, dehydrating the rewound CTC by using 75% ethanol, 95% ethanol and 100% ethanol in a gradient manner for 10min respectively, then adding 15 mL of reagent A, oscillating uniformly and filtering; adding reagent B, decolorizing for 30 min, and sealing with neutral resin;
S9 microscopic examination under 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 pancreatic cancer without obtaining a tissue specimen by 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 an image of circulating tumor cells isolated from peripheral blood of a pancreatic 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 invention is shown in table 1:
TABLE 1 kit Specifications
| Components | Content (wt.) |
| 6 XPBS buffer | 60 mL |
| Diluent liquid | 45 mL |
| Decolorizing liquid | 1mL |
| Staining solution A | 0.5 mL |
| Dyeing liquid B | 1mL |
| Mouse anti-human E-Cadherin primary antibody | 100μL |
| Rabbit anti-mouse IgG/HRP connected with horseradish peroxidase | 100μL |
| 0.1% Triton X-100 | 100μL |
| 0.3% H2O2 | 100μL |
| Reagent A | 15mL |
| Reagent B | 50mL |
The method is applied to the embodiment of separating, obtaining and identifying 10 pancreatic cancer patients (detecting 10 normal human samples as negative controls at the same time) peripheral blood circulating tumor cells.
Preparing the diluent: 19g of potassium chloride, 4.8g of ethylene diamine tetraacetic acid dipotassium, 2.6g of pyridinium sodium, 17g of potassium dihydrogen phosphate, 9g of sodium hydroxide, 0.1g of trehalose, 2g of dihydroxymethyl urea and 945.5g of purified water.
Example 1
Firstly, separating and acquiring CTCs in peripheral blood of patients with advanced or recurrent pancreatic cancer, wherein the patients cannot obtain tissue specimens, by using a membrane filtration device, 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.
FIG. 4 is an image of a circulating tumor cell isolated from peripheral blood of a patient with pancreatic cancer, wherein it can be seen that the cell nuclei are abnormal in shape, large in size, irregular in shape, and larger than 15um in cell diameter (long end); high nuclear-to-cytoplasmic ratio, the nuclear-to-cytoplasmic ratio is more than 0.8; nuclear chromatin border shift, large nucleoli, abnormal nuclear division.
By observation, no CTCs were detected in 10 healthy volunteers; CTCs were detected in 3 patients with recurrent pancreatic cancer and 4 patients with advanced pancreatic cancer (Table 2), except that CTCs were not detected in 3 patients with recurrent pancreatic cancer, and the positive rate of this detection was 70%.
It is noted that when the dilution is not added with dihydroxymethylurea or trehalose, the prepared blood sample has poor stability, a part of the blood sample can be layered, 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 the filter membrane 7 carrying the CTC on the glass slide down from the glass slide, soaking in a decolorizing solution of 0.5 percent hydrochloric acid-70 percent ethanol solution for 4-6 hours, and removing the CTC staining solution; 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; 100 μ l of 0.3% H was added dropwise 2O2Incubating at room temperature for 10min, washing with PBS for 2min × 3 times; dripping 100 μ l of mouse anti-human E-Cadherin primary antibody, incubating at room temperature for 2h (or overnight at 4 ℃), washing with PBS for 2min × 3 times; dripping 100 mu l of rabbit anti-mouse IgG/HRP connected with horseradish peroxidase, incubating for 20min at room temperature (18-26 ℃), washing for 2min multiplied by 3 times with PBS; dripping 100 mul DAB color development solution, and incubating at room temperature (18-26℃)Observing the color development condition under a microscope at any time (generally 3-10 min, the time can not exceed 10 min); after the color development is finished, discarding DAB color development liquid, flushing with running water for 5min, and dyeing 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) gradient ethanol, and adding into a solvent with volume ratio of 1: 2, uniformly oscillating formaldehyde and dimethylbenzene, filtering, decoloring the dimethylbenzene, airing, and sealing with neutral resin; 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.
The decolorizing solution prepared from 0.5 percent hydrochloric acid-70 percent ethanol solution has better decolorizing effect than the decolorizing solution prepared from 95 percent ethanol and 100 percent dimethylbenzene according to the volume ratio of 1: 1.
When the reagent A is not adopted and only the dimethylbenzene is used for decolorization, the accuracy of the detection result is 85%, and the reagent A and the reagent B are adopted, namely, the cell concentration of the invention is realized, the form is clear, the dispersion uniformity is good, and the accuracy can reach 99%.
The detected circulating tumor cells are applied to immunohistochemistry to verify the expression of E-Cadherin and are compared with the results of pancreatic cancer operation or puncture tissue specimen E-Cadherin, the difference is observed, the potential of CTCs as noninvasive biopsy for real-time evaluation of pancreatic cancer E-Cadherin expression is verified, and an important basis is provided for evaluation of pancreatic cancer chemotherapy combined immunotherapy prognosis.
Claims (7)
1. A kit for detecting the expression of peripheral blood circulation tumor cells E-Cadherin of a pancreatic 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 E-Cadherin, 100 muL of rabbit anti-mouse IgG/HRP connected with horseradish peroxidase, 0.1% Triton X-100100 muL, 0.3% H2O2100. mu.L, 15mL of reagent A, 50mL of reagent B, and 60mL of 6 XPBS buffer.
2. The kit according to claim 1, wherein the diluent is 19g/L of potassium chloride, 4.8 g/L of dipotassium ethylenediamine tetraacetate, 2.6 g/L of pyridinium sodium, 17g/L of potassium dihydrogen phosphate, 9g/L of sodium hydroxide, 0.1g/L of trehalose, 2g/L of dihydroxymethyl urea, and the balance of purified water.
3. The kit of claim 1, wherein the destaining solution consists of a 0.5% hydrochloric acid-70% ethanol solution.
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 formaldehyde-xylene in a volume ratio of 1: 2, preparing a composition; the reagent B is xylene.
6. A method for non-diagnostic detection of E-Cadherin expression in peripheral blood circulating tumor cells of a pancreatic cancer patient 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 pancreatic cancer, wherein the patients cannot obtain tissue specimens, by using a membrane filtration device: collecting peripheral blood of patients with advanced or recurrent pancreatic cancer who cannot obtain tissue specimens: 5ml of peripheral blood of the median cubital vein;
(2) peripheral blood sample pretreatment: diluting the collected peripheral blood sample by using 45ml of 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 in step (5) is as follows:
s1 decolorization: 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;
s2 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;
s3 was added dropwise with 100. mu.l of 0.3% H2O2Incubating at room temperature for 10min, washing with PBS for 2min × 3 times; (4) dripping 100 μ l of mouse anti-human E-Cadherin primary antibody, incubating at room temperature for 2h or overnight at 4 ℃, washing with PBS for 2min × 3 times;
s4, 100 mul of rabbit anti-mouse IgG/HRP connected with horseradish peroxidase is dripped, incubation is carried out for 20min at the temperature of 18-26 ℃, and PBS is washed for 2min multiplied by 3 times;
S5, 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;
s6, after the color development is finished, discarding the DAB color development solution, flushing with running water for 5min, and dyeing with hematoxylin for 5 min;
s7, carrying out alcohol differentiation for 8 seconds by hydrochloric acid, and bluing tap water for 5 min;
s8, dehydrating the rewound CTC by using 75% ethanol, 95% ethanol and 100% ethanol in a gradient manner for 10min respectively, then adding 15 mL of reagent A, oscillating uniformly and filtering; adding reagent B, decolorizing for 30 min, and sealing with neutral resin;
s9 microscopic examination under optical microscope.
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| CN202010626819.4A CN111856022A (en) | 2020-07-01 | 2020-07-01 | Kit and method for detecting expression of peripheral blood circulating tumor cells E-Cadherin of pancreatic cancer patient |
| PCT/CN2021/102098 WO2022001825A1 (en) | 2020-07-01 | 2021-06-24 | Kit for detecting e-cadherin expression of peripheral blood circulating tumor cells of pancreatic cancer patient and detection method |
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| WO2022001825A1 (en) * | 2020-07-01 | 2022-01-06 | 天津市肿瘤医院（天津医科大学肿瘤医院） | Kit for detecting e-cadherin expression of peripheral blood circulating tumor cells of pancreatic cancer patient and detection method |
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| CN101650364B (en) * | 2009-07-31 | 2012-09-05 | 山东省医学科学院基础医学研究所 | Application of hematoxylin in measuring cellulotoxic effect of cell-mediated immunity effect |
| CN101975850A (en) * | 2010-09-13 | 2011-02-16 | 南京卡博生物科技有限公司 | Diluent for blood cell analyzer |
| AU2015339886B2 (en) * | 2014-09-29 | 2019-05-09 | Cook General Biotechnology Llc | Uses of trehalose in cell suspensions |
| CN104818314A (en) * | 2015-05-13 | 2015-08-05 | 上海海洋大学 | Transparent liquid for rapidly identifying oocyte nuclei of shrimps and crabs |
| CN105588943B (en) * | 2016-01-28 | 2018-03-02 | 山东省肿瘤防治研究院 | A kind of detection method of the genes of peripheral blood from patients with gastric cancer circulating tumor cell Her 2 |
| KR101994370B1 (en) * | 2016-08-24 | 2019-06-28 | 주식회사 제놉시 | Magnetic nanostructure for detecting and isolating circulating tumor cells comprising antibody- and magnetic nanoparticle-conjugated conductive polymer |
| CN109470875A (en) * | 2017-09-07 | 2019-03-15 | 武汉宏强医疗器械有限公司 | A kind of blood cell analysis dilution |
| CN111638355A (en) * | 2020-07-01 | 2020-09-08 | 山东凯歌智能机器有限公司 | Kit and method for detecting expression of circulating tumor cells E-Cadherin in peripheral blood of breast cancer patient |
| CN111856022A (en) * | 2020-07-01 | 2020-10-30 | 山东凯歌智能机器有限公司 | Kit and method for detecting expression of peripheral blood circulating tumor cells E-Cadherin of pancreatic cancer patient |
| CN111638338A (en) * | 2020-07-02 | 2020-09-08 | 山东凯歌智能机器有限公司 | Method for detecting E-Cadherin expression of colorectal cancer patient through peripheral blood circulating tumor cells |
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