A reversible immortalized liver cell line carrying double suicide genes and its construction method

Abstract

The invention provides an establishing method of a recoverable immortalized hepatic cell line. The establishing method comprises the following steps: separating a hepatic progenitor cell from liver in a mouse which is 12.5-14.5 days old in an embryonic period; guiding a retrovirus containing genes SV40T and HSV-TK into the hepatic progenitor cell; screening a monoclonal cell strain having a hepatic progenitor cell marker and having a function of differing to a mature hepatic cell; then guiding a retrovirus containing CD gene into the cell strain so as to obtain recoverable immortalized hepatic cell carrying double suicide genes. The cell strain can multiply in vitro to obtain the phenotype and functions of a normal hepatic cell, the safety of the immortalized cell becomes adjustable through controllable modes such as locus recombination and drug screening; when the cell is used in a human body, the biological safety of the immortalized cell is ensured to the greatest extent and the dangerousness of the immortalized cell is reduced; therefore, a reliable, safe and ideal hepatic cell material is provided for bioartificial liver technology.

Description

technical field [0001] The invention relates to the field of cell biology, and discloses a method for constructing an immortalized liver cell strain carrying double suicide genes and the constructed mouse embryonic liver progenitor cell strain. [0002] technical background [0003] Liver failure (Liver failure) is the most common clinical liver disease syndrome with high mortality, which seriously threatens human health. According to statistics, the number of deaths caused by severe liver failure in my country is 300,000 to 500,000 every year. The most effective way to treat liver failure is liver transplantation. However, due to the shortage of donor liver sources, less than 10% of patients can get the chance of liver transplantation. Bioartificial liver (Bioartificial liver, BAL) is an in vitro bioartificial organ device based on cultured hepatocytes, which can temporarily replace the liver for detoxification, secretion, synthesis, transformation and other functions. It c...

AI Technical Summary

Problems solved by technology

In the Cre-LoxP protocol, the activity of Cre recombinase and the efficiency of recombination and excision of the LoxP site cannot be controlled, and it cannot be guaranteed that all cells will remove the immortalized gene. After the restoration of immortalization, whether the remaining immortalized gene in the cell inhibits the function of the cell? There are problems such as the risk of tumorigenesis and cell transformation that need to be further verified

Although the suicide gene program increases the safety of cells in vivo, the immortalized cells introduced into the body still have the SV40T gene, which affects the differentiation and functional maturation of cells in vivo

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