Method for screening and constructing primary hepatocyte klotho gene transduction stem cells

Abstract

The invention discloses a method for screening and constructing primary hepatocyte klotho gene transduction stem cells in the technical field of gene transduction stem cell screening and constructing methods. Drugs are screened by taking a T allelic site of rs648202 as a drug design target on the basis of a polymorphic DNA sequence of a klotho gene exon 4 region obtained by PCR (Polymerase Chain Reaction) amplification; and active molecules capable of adjusting the expression level of a klotho gene are screened out, polymorphic typing DNA fragment transduction is adopted based on human hepatocytes, and vector transduction is performed by sequentially adopting hTERT and pLXSN dual vectors, so that transduction of a pLXSN-hTERT expression vector for efficiently expressing hTERT is achieved, and the effect of constructing stem cells for efficiently expressing the klotho gene is achieved. In a low-solubility hygromycin double-screening culture system, the survival ability of the mesenchymal stem cells for tranducing the exogenous klotho gene is improved.

Description

technical field [0001] The invention relates to the technical field of a method for screening and constructing gene-transduced stem cells, in particular to a method for screening and constructing primary hepatocyte klotho gene-transduced stem cells. Background technique [0002] Primary hepatocellular carcinoma (referred to as liver cancer, HCC) is one of the most common malignant tumors worldwide. Hepatitis B virus is a hepatotropic DNA virus that can cause various acute and chronic hepatitis, liver cirrhosis and other diseases. And it has a very close relationship with the occurrence of hepatocellular carcinoma; in addition, Klotho is a gene related to aging discovered in 1997, located on human chromosome 13q12, with a length of about 50kb, consisting of 5 exons and 4 introns, The lack of expression of Klotho gene in mice leads to various phenotypes similar to human aging. In recent years, more and more studies have found that klotho gene can regulate signaling pathways su...

AI Technical Summary

Problems solved by technology

[0003] At present, the existing methods of in vitro expansion of stem cells can be roughly classified into two categories: the first method is to add different combinations of recombinant cell growth factors to the culture system, and the purpose is to use cell growth factors to properly regulate the proliferation and differentiation of stem cells , this kind of technical method can increase the expansion factor of stem cells to a certain extent, but if there are too few types of cytokines in the combination, it will be difficult to achieve the ideal expansion effect, and if there are too many, it will be difficult to be used clinically due to the high cost. application in transplantation; the second type is a method developed in recent years, that is, using bone marrow stromal stem cells to simulate the environment of cell growth and differentiation in vivo to promote its rapid expansion and functional activation, but in this method, due to bone marrow Some cytokines (such as TPO and FL) in stromal stem cells express very little, so it is still necessary to add exogenous cytokines to assist in the expansion culture system. Combined with the application of klotho gene in the treatment of tumors, the integration of klotho gene It is expressed in the nucleus of human target cells, which can lead to changes in cell proliferation and display a variety of tumor-related transformation phenotypes, so that stem cells can acquire the ability to proliferate indefinitely and make them immortal. The most commonly used technology for cell immortalization is through Construct the expression vector containing telomerase catalytic subunit (hTERT), and transfect cells by various methods to support the in vitro expansion ability of stem cells. Therefore, we propose a screening method for stem cells transduced with klotho gene in primary hepatocytes build method