Method for transdifferentiation of fibroblasts into hepatic stem cells
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A technology of fibroblasts and hepatic stem cells, applied in the field of transdifferentiation of fibroblasts into hepatic stem cells
Inactive Publication Date: 2011-12-21
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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[0006] So far, there has been no report on repr
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Embodiment 1
[0072] Example 1: Transdifferentiation of fibroblasts into hepatic stem cells
[0073] 1. Construction of c-Jun, Foxa2, Hnf1β lentiviral vectors
[0074] Cloning primers were designed for c-Jun (NCBI Reference Sequence: NM_010591.2), Foxa2 (NCBI Reference Sequence: NM_010446.2), Hnf1β (NCBI Reference Sequence: NM 009330.2), and synthesized by Shanghai Sangon Bioengineering Co., Ltd. BamH1 and Sal1 restriction sites were introduced at both ends of the forward and reverse primers respectively, and Kozak sequence (GCCACC) was added before the start codon of the forward primer. Since each gene is expressed in fusion with V5tag, the downstream primer removes the stop codon of the gene itself. The cloning primers are listed in Table 1:
[0075] Table 1: Cloning Primers
[0076]
[0077] Using fetal liver cDNA of mouse E12.5 days as a template, PCR method was used to amplify target gene fragments respectively. The PCR reaction system was 50 μl, containing 1 μl of upstream and d...
Embodiment 2
[0097] Example 2: Identification of Morphological Characteristics and Biological Characteristics of Induced Hepatic Stem Cells (iHSC)
[0098] (1) Morphological characteristics of iHSC under light microscope
[0099] The diameter of iHSC is about 10 μm, the nucleus is large, and there are 2 to 4 obvious nucleoli in the nucleus. When cultured at low density, the cells are triangular; after growing into sheets, they are closely arranged like "paving stones" and uniform in shape, which is a typical epithelial cell ( figure 2 B). In in vitro culture, the morphological characteristics of iHSC indicate that the cell line is a relatively primitive epithelial cell line, which can be continuously passaged with vigorous proliferation ability, and there is contact inhibition between cells, and no cascading growth will occur, indicating that iHSC is not malignant cells.
[0100] (2) RT-PCR detection of molecular phenotype of iHSC cells
[0101] Extract the total RNA of the 4th passag...
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Abstract
The invention relates to the technical field of biomedical engineering. Liver transplantation is the only effective means that can be employed for treating liver diseases in late stage at present and is greatly limited in clinic use due to lack of liver sources. Hepatic stem cells have a self-updating capacity and a bidirectional differentiation capacity of differentiating into hepatocytes and cholangiocytes and therefore can provide an infinite number of donor cells for hepatocyte transplantation treatment in theory. The method for the transdifferentiation of fibroblasts into hepatic stem cells, which is provided by the invention, is to reprogram fibroblasts into hepatic stem cells which have the unique self-updating and bidirectional differentiation capacities of hepatic stem cells by using three transcription factors, namely c-Jun, Foxa2 and Hnf1beta. The inducible hepatic stem cells prepared by the method can be used as ideal cell sources for cell treatment of acute hepatic failure and liver diseases in medium and late stages, can be used as seed cells for medicine screening and tissue engineering livers and can provide an ideal research platform for researching cell biological characteristics of hepatic stem cells, liver development and the like.
Description
technical field [0001] The invention relates to the technical field of medical bioengineering, in particular to a method for transforming fibroblasts into liver stem cells. 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, clinical research on hepatic cell transplantation has been devoted to research, and certain curative effects have been achieved, especially in patients with acute liver failure caused by various reasons. However, this method is still very dependent on donated donor organs, and the problem of insufficient donors has not been effectively resolved. Even if the transplanted patients are successfully implemented, because the obtained organs or cells are allogeneic, they still have to take anti-immune rejection drugs for life. In rece...
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