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A method for identifying liver cancer stem cells

A liver cancer stem cell and identification method technology, applied in instruments, analytical materials, fermentation, etc., can solve the problems of low accuracy and cumbersome operation of detection methods, and achieve the effects of high accuracy, good application prospects, and convenient operation

Inactive Publication Date: 2020-05-08
杭州爱唯生命科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims at the technical defects of the prior art, and provides an identification method for liver cancer stem cells, so as to solve the cumbersome operation of the detection method based on cell surface markers in the prior art
[0006] The present invention aims at the technical defects of the prior art, and provides a rapid detection kit for liver cancer stem cells to solve the problem of the low accuracy of the detection method based on cell surface markers in the prior art

Method used

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  • A method for identifying liver cancer stem cells
  • A method for identifying liver cancer stem cells
  • A method for identifying liver cancer stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] 1. Materials and methods

[0035] 1.1. Construction of LCSC cell lines

[0036] Take HepG2 cells, a human liver cancer cell line, and culture them in DMEM supplemented with 10% fetal bovine serum and 1% double antibody based on 5% CO 2 Incubate at 37°C in an incubator. HepG2 cells cultured to the exponential phase were digested with trypsin, and then treated with 20ng / mL basic fibroblast growth factor, 20ng / mL epidermal growth factor, 10ng / mL hepatic growth factor, b27, 1% methylcellulose Resuspended in DMEM / F12 medium with 1% double antibody, seeded the cell suspension into a low-adhesion 12-well plate at 2000 cells / mL, and placed in 5% CO 2 Incubate at 37°C in an incubator. Microcapsules with a diameter of about 200 μM are formed in 10 to 15 days. Obtain enriched cancer stem cell models.

[0037] Cells were sorted by flow cytometry using a flow cytometer. Liver cancer cells were first labeled with anti-CD133 monoclonal antibody, then washed with PBS and resuspende...

Embodiment 2

[0055] A method for identifying liver cancer stem cells, comprising the following steps:

[0056] 1) Take the MEM medium that has eliminated L-asparagine, L-aspartic acid and glycine as the cell culture medium, and in CO 2 In an incubator, culture the cell mass to be tested in the cell culture medium at a temperature of 28° C. in the dark for 16 hours;

[0057] 2) Prepare an aqueous solution containing the following components as an inducer: HuEPO 25 μg / L, polyoxyethylene lauryl ether 80 μg / L, polyvinylpyrrolidone 40 μg / L, chitosan 200 μg / L, sodium chloride 1 mg / L, p-hydroxyl Ethyl benzoate mg / L, BSA 150 μg / L, phosphate buffer 1 mg / L; take the product obtained in step 1), collect the cell mass, insert it into the inducer, and place it under anaerobic and light-proof conditions at a temperature of 35°C. Keep 24h under the condition;

[0058] 3) Take the product obtained in step 2), ultrasonically break it at a frequency of 15kHz for 8min, collect the product and centrifuge at...

Embodiment 3

[0061] A method for identifying liver cancer stem cells, comprising the following steps:

[0062] 1) Take the MEM medium that has eliminated L-asparagine, L-aspartic acid and glycine as the cell culture medium, and in CO 2 In an incubator, culture the cell mass to be tested in the cell culture medium at a temperature of 28° C. in the dark for 16 hours;

[0063] 2) Prepare an aqueous solution containing the following components as an inducer: HuEPO 25 μg / L, polyoxyethylene lauryl ether 80 μg / L, polyvinylpyrrolidone 40 μg / L, chitosan 200 μg / L, sodium chloride 1 mg / L, p-hydroxyl Ethyl benzoate mg / L, BSA 150 μg / L, phosphate buffer 1 mg / L; take the product obtained in step 1), collect the cell mass, insert it into the inducer, and place it under anaerobic and light-proof conditions at a temperature of 35°C. Keep 24h under the condition;

[0064] 3) Take the product obtained in step 2), ultrasonically break it at a frequency of 15kHz for 8min, collect the product and centrifuge at...

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Abstract

The invention provides an identification method for liver cancer stem cells. According to research finding, LCSC (liver cancer stem cells) can express the unique protective proteins intracellularly orextracellularly under the certain induction condition; based on the beneficial finding, the invention further studies the specificity and the sequence signature of the proteins, and obtains two protein segments which have the good specificity on liver cancer cells and common liver cells through screening, and the test verifies that the proteins are only stably expressed in LCSC while do not existin common liver cancer cells, therefore the proteins can serve as a marker for identifying LCSC. On the basis, the invention optimizes the cell induction method aiming at ensuring the expression quantity and the stability of the protein segments, and by using the method to induce cell samples, not only can the stable expression of the marker protein be ensured, but also the expression quantity and the LCSC cell content present a good linear relation; and based on the rule, relative quantitative analysis of the LCSC cell content can be achieved.

Description

technical field [0001] The invention relates to the technical field of stem cells, and further relates to a molecular biology detection technology of tumor stem cells, in particular to an identification method for liver cancer stem cells. Background technique [0002] Stem cells are a kind of pluripotent cells with self-replication ability, which can differentiate into various functional cells under certain conditions. Stem cells have high hopes in cell replacement and tissue regeneration therapy due to their proliferation and differentiation characteristics. They can not only be used for the treatment of diseases caused by definite cell type lesions such as neurodegeneration and diabetes, but also can be used in tissue engineering and organ regeneration. Reengineering technology is used in tissue repair and organ transplantation, as well as new drug development, gene therapy, immune regulation therapy and other fields. Cancer stem cells, also known as cancer stem cells, re...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/569C12P21/06
CPCC12P21/06G01N33/56966G01N33/6848
Inventor 方苏萍邢继亮江志华
Owner 杭州爱唯生命科技有限公司
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