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Preparation method of immortalization pig liver cell

An immortalization, cell technology, applied in the field of medical biology, can solve problems such as no application report, loss of secreted albumin, etc., and achieve the effect of fast cell proliferation

Inactive Publication Date: 2006-11-29
ZHEJIANG UNIV
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AI Technical Summary

Problems solved by technology

However, since the cells lost the ability to secrete albumin during passage, there was no report on the basic or clinical application of the cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0010] 1. Construction of recombinant retroviral vector containing SV40 large T antigen

[0011] Using the upstream primer: 5'-GCCCAGGATCCTTAACAACAACAACAAT-3', the downstream primer: 5'-ACGCTGAATTCCCCTCTGAGCTAT-3' as the primer, and the SV40 DNA (strain 776) as the template, the high-fidelity long-fragment PCR method was used to amplify about 2400 bp of SV40 large T DNA The fragments were respectively digested with EcoR I / BamH I endonuclease and inserted into the EcoR I / BamH I site of the pLXSN retroviral vector; then, ligated with T4 ligase at 22°C and transformed into competent E.coli DH5-alpha Escherichia coli, cultured overnight at 37°C, screened by x-gal, selected colorless colonies and inoculated in 3ml LB solution, cultured overnight at 37°C, purified the recombinant plasmid with a plasmid purification kit; identified by enzyme digestion and sequencing, the sequence After the accuracy is correct, the Escherichia coli containing the SV40 LT recombinant plasmid is expande...

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Abstract

This invention relates to pork liver cell immortalities and its producing method. Procedures are showed: high live ratio fresh original pork liver cell is got by dispase-collagenase perfusion method; it is cultured for 24 hours. Then cell upper heat removing of recombination retronituse that contains SV40 big T antigen is used to infect the pork cell under condition of polybrene which concentration is 8ug / ml. Then it is medicine pressure filtrated by 500ug / ml G418 after one week, single clone cell is picked for large culture when the cell clone appears and grows to 1.0-2.0cm to get pork cell that can passage. Reinfection is done under condition of 8ug / ml polybrene. Then it is medicine pressure filtrated by 2ug / ml puromycin after one week. Single clone cell is picked for large culture when the cell clone appears and grows to 1.0-2.0cm to get immortality pork cell.

Description

technical field [0001] The invention belongs to the field of medical biotechnology, and relates to a preparation method of immortalized pig hepatocytes used in bioartificial livers. Background technique [0002] Artificial liver is recognized as one of the effective ways to treat liver failure and has been widely used clinically; moreover, bioartificial liver is the hot spot and development direction of artificial liver research. Since the size and structure of porcine hepatocytes are similar to those of human hepatocytes, and their biological and immune functions are similar to those of human hepatocytes, they have high metabolic activity. Therefore, most animal and clinical studies use primary porcine hepatocytes. Although the source of primary pig hepatocytes is abundant and easy to obtain in large quantities, the development of bioartificial liver is limited due to the shortcomings of primary hepatocytes such as short survival time in vitro, difficulty in passage, and di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12N5/10
Inventor 李兰娟潘小平
Owner ZHEJIANG UNIV
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