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Preparation method of immortalized human intervertebral disc nucleus pulposus cell system

A technology of nucleus pulposus cells and intervertebral discs is applied in the field of preparation of immortalized human intervertebral disc nucleus pulposus cell system, which can solve the problems of difficulty in meeting the research needs of intervertebral disc degeneration and pathological mechanism, cell degeneration and apoptosis, etc., and achieve the effect of improving the overall prevention and treatment level.

Active Publication Date: 2013-04-17
梁伟国
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Problems solved by technology

However, this method of transfection through viruses, oncogenes, or proto-oncogenes is mainly carried out by inhibiting the mechanism of the M1 phase (the first death phase), and the cells continue to grow after bypassing the M1 phase, after 20 to 30 population doubling times After entering the M2 phase (the second death phase), the cells will undergo degeneration and apoptosis
Therefore, the cell lines constructed by this method are different from germline stem cells, and are transformed cells, which are not truly immortalized, and it is still difficult to meet the research needs of the mechanism of intervertebral disc degeneration

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  • Preparation method of immortalized human intervertebral disc nucleus pulposus cell system

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[0043] Figure 1 to Figure 18 Shown is the embodiment of the preparation method of the immortalized human intervertebral disc nucleus pulposus cell system of the present invention, and its technical process is as follows figure 1 As shown, it is achieved through the steps of cloning the target gene, determining the sequence of the target gene in the eukaryotic expression plasmid, constructing the eukaryotic expression plasmid of the target gene, transfecting hTERT into human nucleus pulposus cells, and performing expression detection. In this example, by constructing a recombinant green fluorescent expression vector of exogenous human telomerase reverse transcriptase (hTERT), it was successfully transfected into normal nucleus pulposus cells to activate telomerase and stably express it in the cells, thereby obtaining immortalized human intervertebral disc marrow Nuclear cell system, and determine its biological characteristics and safety. The specific method is as follows:

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Abstract

The invention discloses a preparation method of an immortalized human intervertebral disc nucleus pulposus cell system. The preparation method is as follows: extrinsic human telomerase reverse transcriptase is transfected into the human nucleus pulposus cell to activate telomerase and construct immortalized human nucleus pulposus cell line through induction immortalized human nucleus pulposus cell line, normal cells not transformed cells are used, wherein the cells have normal growth rate and karyotype, have contact inhibition, anchorage dependence and other safety properties and do not have carcinogenicity and soft agar cloning capability; and as same as the germline stem cells, the cells have real immortalization, thus the standard cell line can be provided for the further research of the pathogenic mechanism of the degenerative disc disease and a new idea is provided to increase the entire control level of the degenerative disc disease.

Description

technical field [0001] The invention relates to the technical field of cell biology, in particular to a preparation method of an immortalized human intervertebral disc nucleus pulposus cell system. Background technique [0002] Intervertebral disc degeneration is an extremely complex physiological and pathological process. Through the study of the mechanism of the change of the nucleus pulposus cell population in the intervertebral disc, it can provide a basis for the clinical use of tissue engineering regeneration methods to repair degenerated intervertebral disc tissue and restore function. However, the proliferation ability of nucleus pulposus cells is weak, and they are prone to aging and growth arrest. Therefore, it is necessary to construct an immortalized nucleus pulposus cell line through genetic engineering technology to establish a standardized in vitro research platform for nucleus pulposus cells. Improve the treatment and overall prevention and treatment of dege...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/52C12N9/00C12N15/85
Inventor 梁伟国叶冬平戴丽冰沈雁陈鸿辉
Owner 梁伟国
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