Method for inducing liver cell cells to be transformed into liver cancer stem cells

A technology for liver cancer stem cells and liver cancer cells, which is applied in the field of inducing the transformation of liver cancer cells to liver cancer stem cells, can solve problems such as difficulty in studying the regulation mechanism of a single factor, and achieve the effects of good drug resistance characteristics, fast aging, and easy operation.

Active Publication Date: 2017-05-17
NANJING DRUM TOWER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The traditional two-dimensional cell culture system is often inconsistent with the in vivo situation because the cells gradually lose their original traits when they grow in vitro. However, animal experiments are affected by multiple complex factors in vivo and in vitro, making it difficult to study the regulation mechanism of a single factor

Method used

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  • Method for inducing liver cell cells to be transformed into liver cancer stem cells
  • Method for inducing liver cell cells to be transformed into liver cancer stem cells
  • Method for inducing liver cell cells to be transformed into liver cancer stem cells

Examples

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Embodiment 1

[0040] Example 1: A method for inducing liver tumor cells to transform into tumor stem cells

[0041] 1) Digest hepatoma cells (HepG2, HUH7 and PLC / PRF / 5) in a good growth state and in the logarithmic growth phase with 0.25% trypsin, and digest them at 37°C for 5 minutes to obtain human hepatoma cells in a single-cell state, remove the After the digestion solution of trypsin, add it to DMEM complete medium containing 10% fetal bovine serum to make cell suspension;

[0042] 2) Spread an appropriate amount of cell suspension on a cell culture dish, place it at 37° C., 5% CO2, 20% oxygen content, and cultivate it in a cell incubator for 7 days, as a control for two-dimensional cultured cells. Replace the DMEM complete medium every other day; wherein, the DMEM complete medium is a high-sugar DMEM medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin and 100 mg / ml streptomycin.

[0043] 3) 60 μl cell suspension (cell density is 10 5 Each / ml) was added dropwise on t...

Embodiment 2

[0045] Example 2 A method for inducing liver tumor cells to transform into tumor stem cells

[0046] 1) Digest hepatoma cells (HepG2, HUH7 and PLC / PRF / 5) in a good growth state and in the logarithmic growth phase with 0.25% trypsin, and digest them at 37°C for 5 minutes to obtain human hepatoma cells in a single-cell state, remove the After the digestion solution of trypsin, add it to DMEM complete medium containing 10% fetal bovine serum to make cell suspension;

[0047]2) Spread an appropriate amount of cell suspension on a cell culture dish, place it at 35° C., 5% CO2, 5% oxygen, and culture it in a cell incubator for 7 days, as a two-dimensional cell culture control. The DMEM complete medium was replaced every other day; wherein, the DMEM complete medium was a high-sugar DMEM medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin and 100 mg / ml streptomycin.

[0048] 3) 60 μl cell suspension (cell density is 10 5 Each / ml) was added dropwise on the porous cel...

Embodiment 3

[0050] Example 3 A method for inducing liver tumor cells to transform into tumor stem cells

[0051] 1) Digest hepatoma cells (HepG2, HUH7 and PLC / PRF / 5) in a good growth state and in the logarithmic growth phase with 0.25% trypsin, and digest them at 37°C for 5 minutes to obtain human hepatoma cells in a single-cell state, remove the After the digestion solution of trypsin, add it to DMEM complete medium containing 10% fetal bovine serum to make cell suspension;

[0052] 2) Spread an appropriate amount of cell suspension on a cell culture dish, place it at 35° C., 5% CO2, 10% oxygen, and cultivate it in a cell incubator for 7 days, as a two-dimensional cell culture control. Replace the serum-free tumor stem cell medium every other day;

[0053] 3) 60 μl cell suspension (cell density is 10 5 Each / ml) was added dropwise on the porous cellulose support (placed in 37°C, 5% CO2, 21% oxygen cell incubator for 4 hours, then added serum-free tumor stem cell culture medium, placed a...

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Abstract

The invention discloses a method for inducing liver cell cells to be transformed into liver cancer stem cells. The method comprises the following steps: adding human liver cancer cells with a single cell state into a culture medium, so as to prepare a cell suspension solution; dropwise adding the cell suspension solution into a porous cellulose bracket; after enabling the cell suspension solution to permeate into the porous cellulose bracket, putting the porous cellulose bracket into a cell culture box and culturing and adhering; then adding the culture medium into the cell culture box and culturing for a plurality of days to obtain three-dimensional cultured cells; detecting changes of expression conditions of liver cancer stem cell marker molecules EpCAM (Epithelial Cellular Adhesion Molecule), OCT4, CD44 and CD133 in the three-dimensional cultured cells through utilizing Western Blot, quantitative PCR (Polymerase Chain Reaction), flow cytometry and IHC (Immunohistochemistry) and (or) a microscope is used for observing; the transformed liver cancer stem cells are detected. The invention further discloses the liver cancer stem cells and application of the liver cancer stem cells. The liver cancer stem cells disclosed by the invention have good drug resistance.

Description

technical field [0001] The invention belongs to the field of oncology and stem cell research, and specifically relates to a method for inducing liver cancer cells to transform into liver cancer stem cells. Background technique [0002] Liver cancer is a kind of end-stage liver disease, the condition is dangerous, and the fatality rate is as high as 80%. It is one of the malignant tumors that seriously endanger human health. my country is a country with a high incidence of liver disease. At present, there are more than 100 million people with various liver diseases. Among the 100 new cases of liver cancer in the world every year, 55 cases are Chinese. Some studies believe that the existence of cancer stem cells is an important reason for the occurrence of malignant tumors. Liver cancer stem cells can resist cancer treatment drugs, resist chemotherapy radiation, and increase the chance of tumor recurrence and metastasis after surgery. In summary, how to develop a suitable ce...

Claims

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Application Information

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IPC IPC(8): C12N5/095
CPCC12N5/0695C12N2533/78
Inventor 巫国谊丁义涛庄永傑詹珊珊
Owner NANJING DRUM TOWER HOSPITAL
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