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Method for detecting C-MET gene mutation of hepatocellular carcinoma patient through peripheral blood circulating tumor cells

A technique for peripheral blood circulation and hepatocellular carcinoma, applied in separation methods, chemical instruments and methods, filtration and separation, etc., can solve problems such as false negatives, false positives of C-MET real-time dynamic status, inability to evaluate patients, etc., to reduce cell death loss, avoidance of edge effects, avoidance of disturbing effects

Inactive Publication Date: 2020-09-08
山东凯歌智能机器有限公司
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Problems solved by technology

[0007] Aiming at the disadvantages in the prior art of detecting patients with advanced or recurrent hepatocellular carcinoma that tissue samples cannot be obtained by real-time or repeated puncture, and thus cannot evaluate the real-time dynamic status of C-MET in patients, and that existing detection methods are prone to false positives and false negatives, this paper The invention provides a kit and detection method for detecting C-MET gene mutations in peripheral blood circulating tumor cells of patients with hepatocellular carcinoma: the circulating tumor cells (CTCs) in the peripheral blood of patients with advanced hepatocellular carcinoma are separated by using a membrane filtration device, and further used Detection of C-MET expression on CTC by immunohistochemical technique

Method used

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  • Method for detecting C-MET gene mutation of hepatocellular carcinoma patient through peripheral blood circulating tumor cells
  • Method for detecting C-MET gene mutation of hepatocellular carcinoma patient through peripheral blood circulating tumor cells
  • Method for detecting C-MET gene mutation of hepatocellular carcinoma patient through peripheral blood circulating tumor cells

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Embodiment 1

[0048] 1. Use a membrane filtration device to separate and obtain CTCs in the peripheral blood of patients with advanced or recurrent hepatocellular carcinoma whose tissue samples cannot be obtained, and determine whether CTCs exist:

[0049] Collect 5ml of fasting blood from the median vein of the elbow for 8-12 hours, dilute the peripheral blood with 45ml of diluent, and then add 3ml of 4% paraformaldehyde to fix the diluted blood sample for 10 minutes;

[0050] At fixed intervals, assemble the membrane filtration device: as attached figure 1 , figure 2 , image 3 As shown, the filtering device is composed of a filter 3, a filter membrane 7, a blood sample container 2, a waste liquid tank 5, and an iron stand 1;

[0051] Wet the filter 3 with 10ml of PBS, and then add the fixed peripheral blood sample into the blood sample container 2 of the membrane filtration device, so that it is naturally filtered by gravity, and the CTC is trapped on the filter membrane 7;

[0052] ...

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Abstract

The invention provides a kit and method for detecting C-MET gene mutation of a hepatocellular carcinoma patient through peripheral blood circulating tumor cells. The kit comprises a diluent, a decolorizing solution, a staining solution A, a staining solution B, a rabbit anti-human C-MET primary antibody, a biotin labeled mouse anti-rabbit secondary antibody, 0.1% Triton X-100, 0.3% H2O2 and a reagent A. The detection method comprises the following steps: separating and obtaining peripheral blood circulating tumor cells CTC by using a membrane filtration device, and detecting the C-MET expression condition of the peripheral blood CTC by using an immunohistochemical technology. By utilizing the detection method provided by the invention, the C-MET expression condition of the patient with advanced or recurrent hepatocellular carcinoma needle biopsy can be obtained needless of obtaining specimen by puncture, and real-time dynamic detection can be realized by utilizing a minimally invasivetechnology; by adopting the separation device and the diluent, the circulating tumor cells are well separated, the interference of blood cells is avoided, the false positive result caused by the edgeeffect possibly generated in the dyeing process is avoided, the stability is high, the cell loss is reduced, and the detection accuracy is improved.

Description

technical field [0001] The invention provides a kit and a detection method for detecting C-MET gene mutation of peripheral blood circulating tumor cells of patients with hepatocellular carcinoma, belonging to the technical field of molecular biology. Background technique [0002] Liver cancer is one of the common malignant tumors in my country, with a high mortality rate. It ranks third in the death order of malignant tumors after stomach and esophagus; it ranks second in rural areas in some areas, second only to gastric cancer. About 110,000 people die from liver cancer every year in my country, accounting for 45% of the world's liver cancer deaths. The average age of liver cancer is 44 years old. The pathological types of liver cancer are hepatocellular carcinoma, cholangiocarcinoma, and mixed liver cancer, among which hepatocellular carcinoma accounts for 70-80%, which is highly malignant and develops rapidly. If the treatment is not timely or the treatment plan is not se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/573G01N33/569B01D29/05
CPCB01D29/05G01N33/56966G01N33/573G01N33/57438G01N33/57492G01N2333/71G01N2333/912
Inventor 李琛琛洪瑛侯景阳李文涛
Owner 山东凯歌智能机器有限公司
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