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A system capable of stably expressing cell cycle factor FoxM1 and its medical use

A cell cycle and expression vector technology, applied in the biological field, can solve problems such as difficulties

Active Publication Date: 2010-03-24
引加苏州生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is very difficult to find a method that can significantly enhance the proliferation ability of donor hepatocytes without potential toxic side effects

Method used

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  • A system capable of stably expressing cell cycle factor FoxM1 and its medical use
  • A system capable of stably expressing cell cycle factor FoxM1 and its medical use
  • A system capable of stably expressing cell cycle factor FoxM1 and its medical use

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] The construction of embodiment 1.FoxM1 non-viral vector

[0075] 1. Select the appropriate promoter

[0076] General promoters such as CMV, Caggs (CMV enhancer / chicken β-actin promoter / β-globin intron fusion sequence), liver-specific promoters such as albumin (Albumin), thyroxine binding protein ( TTR) promoters are optional promoters that regulate gene expression. In order to obtain efficient and stable expression of the FoxM1 gene, the inventors conducted a series of experiments to select the promoter.

[0077] By detecting the differences in the expression levels of the luciferase gene driven by different promoters in the liver of the host, the inventors compared the differences of several promoters in liver gene therapy. Select different specific promoters and vector pKT2-CMV-FAHIL-SB ( figure 2 A, Obtained from Oregon Health and Science University; As the replacement of the CMV promoter in the expression vector 1), three other expression vectors were obtained, ...

Embodiment 2

[0085] Example 2. Obtaining hepatocytes stably expressing the FoxM1 gene

[0086] 1. In vitro transfection of liver cells with non-viral vector carrying FoxM1 gene

[0087] The present inventors isolated hepatocytes or mouse embryonic stem cells hepatically differentiated cells from adult livers of wild-type mice with a 129S4 background (obtained from Oregon Health and Science University) or fetal livers of 14 days of pregnancy, and digested the above cells into single Cell suspension, count 5×10 6 were inoculated on 10 cm culture dishes covered with type I collagen. The constructed pKT2-Caggs-mFoxM1-IRES-dsRed2-SB expression vector was used to transfect hepatocytes with Qiagen high-efficiency transfection reagent, and the transfected hepatocytes were screened by Ds-Red reporter gene and flow cytometry.

[0088] The work of the present inventors shows that the proportion of Ds-Red positive liver cells is between 5 and 10% through transposon transmission in cis. Ds-Red posit...

Embodiment 3

[0092] Example 3. Study on promoting liver repopulation after liver cell transplantation stably expressing FoxM1

[0093] The following mice were used as test materials:

[0094] Rosa-26β-gal transgenic mice: Obtained from Oregon Health and Science University, USA; the β-gal-labeled liver cells were used as control cells.

[0095] Immunodeficient Fah - / - mouse strain (Fah - / - Rag-2 - / - ): Obtained from Oregon Health and Science University, USA.

[0096] The following two evaluation systems for liver cell transplantation were selected:

[0097] Fah - / - Knockout mouse model: Wild-type hepatocytes also have a selective advantage in this model and can completely repopulate the liver. This mouse model was obtained from Oregon Health and Science University.

[0098] Wild-type mouse hepatectomy model: wild-type hepatocytes have no selective advantage in this model. The mouse model was purchased from Shanghai Experimental Animal Center, Chinese Academy of Sciences.

[0099] 1...

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Abstract

The invention relates to a system capable of stably expressing cell cycle factor FoxM1 and its medical use. The invention discloses a use of the cell cycle factor FoxM1 gene or protein in preparing combination or system for reinforcing liver cell proliferation. The invention discloses a recombined express vector containing the cell cycle factor FoxM1 gene. The invention also discloses a cell capable of expressing the cell cycle factor FoxM1 protein and a combination containing the cell.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to a system for stably expressing cell cycle factor FoxM1 and its medical application. Background technique [0002] Liver transplantation is currently the only effective treatment for end-stage liver disease, but the lack of donor liver sources greatly limits its clinical application. In the past two decades, as an alternative to orthotopic liver transplantation, hepatic cell transplantation technology has been clinically researched and achieved certain curative effects. However, due to the slow proliferation of transplanted hepatocytes in the host liver, therapeutic liver repopulation is still difficult to achieve, which has become a bottleneck problem hindering the development of current hepatocyte transplantation technology. [0003] Liver Repopulation refers to the phenomenon of meaningful implantation of hepatocytes in the recipient liver, that is, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10A61K48/00A61P1/16
Inventor 王欣何志颖
Owner 引加苏州生物医药科技有限公司
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