Method for separating and identifying disseminated hepatoma cells

A separation method and technology of liver cancer cells, applied in the direction of tumor/cancer cells, biochemical equipment and methods, animal cells, etc., can solve the problem of lack of monoclonal antibodies on the surface of liver cancer cells, etc., and achieve simple operation, high sensitivity and specificity strong effect

Inactive Publication Date: 2008-11-12
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The inventors used ASGPR ligands or monoclonal antibodies against the extracellular region of ASGPR to create a technique for separating disseminated liv

Method used

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  • Method for separating and identifying disseminated hepatoma cells
  • Method for separating and identifying disseminated hepatoma cells
  • Method for separating and identifying disseminated hepatoma cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Preparation of biotinylated asialofetuin

[0056] Main materials: amino biotinylation reagent Sulfo-NHS-lC-Biotin (Pierce), asialofetuin (Sigma), BCA protein quantification kit (Pierce), L-lysine (Sinopharm), centrifugal ultrafiltration tube (MW cut-off 50,000) (Millipore).

[0057] Experimental procedure: The reaction between asialofetuin and aminobiotinylation reagent was carried out according to the product instructions. Amino biotinylation reagent Sulfo-NHS-lC-Biotin can selectively react with free amino groups in proteins, the reaction formula is as follows figure 1 shown.

[0058] After the reaction, the reaction solution was transferred into a centrifugal ultrafiltration tube, and centrifuged at 25° C. at 4000 g for 20 min. Then add double-distilled water and L-lysine to the centrifuge tube to make the total volume 3ml, the final concentration of L-lysine is 0.08mg / ml, and place it at room temperature for 1h. Then centrifuge the centrifugal ultrafil...

Embodiment 2

[0059] Example 2 Determining the ideal flow concentration of ASGPR ligands and antibodies

[0060] 1. The following experiments use flow cytometry to verify the reactivity of biotinylated asialofetuin with ASGPR, and determine the ideal concentration of biotinylated asialofetuin for flow cytometry.

[0061] Main materials: PE-labeled mouse anti-human CD45 monoclonal antibody (IgG1), PE-labeled mouse isotype monoclonal antibody (IgG1), FITC-labeled streptavidin, all purchased from Jingmei Company. Biotinylated asialofetuin, homemade. PBS washing solution (PBS+1%FBS), self-made.

[0062] Experimental steps:

[0063] 1) Count 5 million Hep3B liver cancer cells (Cell Bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences), centrifuge at 20°C, 300g for 5min, discard the supernatant, resuspend in 550μl serum-free DMEM culture medium, mix well, 100μl / tube , divided into 5 EP tubes, numbered 1-5 in turn. Tube No. 1 is a blank control tube, Tube No. 2 i...

Embodiment 3

[0102] Example 3 Isolation and Identification of Disseminated Hepatocellular Carcinoma Cells Using ASGPR Ligands

[0103] Main materials: 5ml of peripheral blood from liver cancer patient volunteers, anticoagulated with heparin. Ficoll-Paque Plus, available from GE Healthcare. Magnetic separator, anti-biotin magnetic beads, MS magnetic separation column, 30 μm filter, and CK3-6H5 antibody (tumor cell marker antibody derived from epithelial tissue) were purchased from Miltenyi, Germany. 400R desktop centrifuge (with cytocentrifuge smear attachment), purchased from Heraeus, Germany. Polylysine slides were purchased from Fujian Maixin Company. PV9000 two-step immunohistochemistry kit was purchased from GBI, USA. Biotinylated asialofetuin, homemade. PBS washing solution (containing 0.5% BSA, 80U / ml heparin), prepared by yourself.

[0104] Reagent 1: PBS washing solution (containing 0.5% BSA, 80U / ml heparin)

[0105] Reagent 2: Serum-free DMEM medium

[0106] Reagent 3: Bio...

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Abstract

The invention provides a method for isolating and identifying disseminated liver cancer cells. The method is based on a de-ASGPR on a liver cancer cell membrane and in the method, a ligand or antibody of the de-ASGPR is combined with a liver cancer cell, a bead is used to mark the liver cancer cell indirectly for positive isolation and enrichment, a immunocytochemistry method or a flow cytometry is adopted to identify and inspect to judge whether any liver cancer cells exist in the sample. The method can be used for the diagnosis, metastasis, recurrent forecasting and efficacy monitoring of liver cancer, can be used to detect disseminated liver cancer cells and is high in sensibility, good in specificity and convenient to operate.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to a method for separating and identifying disseminated liver cancer cells. Background technique [0002] Tumor metastasis is a multi-stage, multi-step complex process. Tumor cells that exist outside the primary tumor and metastases are collectively called disseminated tumor cells (DTC), also known as isolated tumor cells (ITC), including individual tumor cells in tissues such as lymph nodes, bone marrow, and blood. or small clusters of tumor cells. Among them, those entering the bloodstream are also called circulating tumor cells (Circulating tumor cells, CTCs). [0003] Primary hepatocellular carcinoma is one of the most common and frequently occurring malignant tumors in my country. Although the treatment methods have been increasing in recent years, it has not fundamentally changed the high mortality rate of liver cancer. The reason is that the high rate of metastasis and recurren...

Claims

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Application Information

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IPC IPC(8): C12N5/08G01N33/577G01N33/48C12N5/09
Inventor 殷正丰徐文康晓燕
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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