Gene combination for inducing hepatocytes into live cancer cells and application of gene combination

A technology of gene combination and liver cancer cells, applied in the field of genetic engineering, can solve the problems of low transplantation efficiency, affecting application, unable to simulate liver cancer models, etc., and achieve the effect of high cancer rate.

Pending Publication Date: 2020-09-08
SHENZHEN IN VIVO BIOMEDICINE TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the liver cancer model of orthotopic transplantation of cell lines cannot simulate the microenvironment of human liver cancer cells, and the mice with orthotopic transplantation of human liver cancer cells have problems such as low transplantation efficiency and long modeling period, which affect its application in research

Method used

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  • Gene combination for inducing hepatocytes into live cancer cells and application of gene combination
  • Gene combination for inducing hepatocytes into live cancer cells and application of gene combination
  • Gene combination for inducing hepatocytes into live cancer cells and application of gene combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] In this example, the TP53 / KRAS / c-MYC triple transgene was used to induce #1 primary liver cells to transform into liver cancer cells in vitro

[0096] (1) Culture of primary human hepatocytes

[0097] Adjust the hepatocyte concentration to 5 x 10 5 / cm 3 , inoculated in a 10cm culture dish, the medium was added with InVitroGRO CP hepatocyte medium, placed in 5% CO 2 After culturing in an incubator at 37°C for 4 hours, replace with fresh medium and remove unattached cells, and replace with fresh medium every two days.

[0098] (2) Lentiviral packaging

[0099] Inoculate 293T cells in a good state in a 10cm dish, and when the 293T cells grow to 70% confluence, starve and culture them with DMEM medium containing 1% FBS and 1% double antibody for 2 hours;

[0100] Add PEI (Polyethyleneimine, polyethyleneimine) (72 μg / dish) into opti-MEM (serum-free medium), mix well, and let stand at room temperature for 5 minutes;

[0101] Add the plasmids pWPXLd-TP53-2A-KRAS-2A-MYC-...

Embodiment 2

[0107] TP53 / KRAS / c-MYC triple transgene induces #2 primary liver cells to transform into hepatocellular carcinoma cells in vitro

[0108] Divide #2 liver cells into two groups, the TP53 / KRAS / c-MYC transduction experimental group and the GFP blank control group, and set up 3 replicate wells in each group, with the number of cells per well 1×10 6 Cultured in a 6-well plate culture dish, medium 2mL: DMEM containing 10% FBS and 1% double antibody, placed at 37°C, 5% CO 2 After culturing for 24 hours, the TP53 / KRAS / c-MYC and GFP lentivirus solutions obtained in step (2) in Example 1 were added respectively.

[0109] After 10 days, the cell proliferation and growth morphology of different experimental groups were observed, and the results were as follows: figure 2 shown.

[0110] It was found that the liver cells of the blank control group transfected with GFP grew slowly, the cell shape was irregular, did not grow in clusters, and there was contact inhibition; while the liver ce...

Embodiment 3

[0112] In this example, #3 primary liver cells were induced to transform into liver cancer cells in vitro by TP53 / c-MYC double transgene and TP53 / KRAS / c-MYC triple transgene.

[0113] (1) Lentiviral packaging

[0114] Inoculate 293T cells in a good state in a 10cm dish, and when the 293T cells grow to 80% confluence, starve and culture them with DMEM medium containing 1% FBS and 1% double antibody for 2 hours;

[0115] Add PEI (72 μg / dish) to opti-MEM, mix well, and let stand at room temperature for 5 minutes; mix 9 μg / dish of the plasmids in Table 1 with psPAX2 (12 μg / dish) and pMD2.G (3 μg / dish) respectively Add to opti-MEM and mix well;

[0116] Table 1

[0117]

[0118] Add the pre-mixed opti-MEM PEI medium to each plasmid medium at a ratio of 1:1, shake gently to mix, and then stand at room temperature for 20 minutes;

[0119]Add drop by drop to starved 293T cell culture dish, shake gently to mix, culture in 37°C incubator, change medium after 8 hours to continue cu...

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Abstract

The invention provides a gene combination for inducing liver cells into liver cancer cells and application of the gene combination. The gene combination comprises a TP53 mutant gene and a c-MYC gene.Specifically, the gene combination is overexpressed in hepatocytes by utilizing lentivirus, so that normal hepatocytes can be induced to form live cancer cells. A primary live cancer humanized mouse model can be obtained by transplanting the human primary hepatocytes subjected to transfer of the gene combination into Fah gene mutation immunodeficient animals such as mice for in-vivo culturing. Liver cancer cells of the model are transformed from normal liver cells, and the model is an in-vivo model in which normal liver cells and liver cancer cells are jointly embedded, so that the human livercancer forming process and the liver cancer microenvironment can be better simulated, researchers can conveniently utilize the model to research occurrence, development and evolution process of livercancer, and a favorable model is provided for development of novel liver cancer drugs, pharmacological toxicology research of the drugs and drug resistance mechanism exploration of liver cancer treatment drugs.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a gene combination for inducing liver cells into liver cancer cells and its application, in particular to a gene combination for inducing liver cells into liver cancer cells, human liver cancer cells and human liver cancer cells prepared by using it. Primary liver cancer humanized animal model and its construction method and application. Background technique [0002] Liver cancer is mainly divided into hepa-tocellular carcinoma (HCC), intra-hepatic cholangiocarcinoma (ICC) and mixed liver cancer. It is one of the tumors with high incidence and high mortality in my country. 85%-90% of the total liver cancer. Because the symptoms of liver cancer are not obvious in the early stage, it is easy to be ignored, and more than half of the patients are already in the middle and late stages once discovered. Therefore, liver cancer is one of the malignant tumors with ...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10A01K67/027A61K49/00C12Q1/18
CPCC12N15/86C12N5/0693C12N5/067C07K14/82C07K14/4746A01K67/0271A61K49/0008G01N33/5011G01N33/5044C12N2740/15043C12N2510/00C12N2503/02A01K2207/12A01K2227/105A01K2267/0331G01N2500/10
Inventor 不公告发明人
Owner SHENZHEN IN VIVO BIOMEDICINE TECH LTD
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