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Telomerase immortalized skin fibroblast line and construction method thereof

A technology of fibroblasts and construction method, which is applied in the field of telomerase-immortalized skin fibroblasts and its construction, can solve the problems of random integration of gene products interfering with intracellular physiological pathways and the like

Inactive Publication Date: 2010-12-22
柯明哲
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Problems solved by technology

[0009] The purpose of the present invention is to provide a telomerase immortalized skin fibroblast cell line and its construction method, which solves the problems of gene random integration and gene product interference in the prior art when constructing immortalized skin fibroblasts. biological pathways, etc.

Method used

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  • Telomerase immortalized skin fibroblast line and construction method thereof
  • Telomerase immortalized skin fibroblast line and construction method thereof
  • Telomerase immortalized skin fibroblast line and construction method thereof

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0043] Example 1 Construction of telomerase-immortalized skin fibroblast cell line

[0044] Tissue Block Culture of Human Skin Fibroblasts

[0045] The abdominal skin of the patient was surgically excised (the skin of other surgical sites can also meet the conditions), and placed in phosphate buffer containing 0.05% Tween-20 and pH 7.4 containing 100,000 u / L ampicillin and 100 mg / L streptomycin sulfate solution (PBS), after repeated rinsing 3-5 times until the rinse solution is clear and transparent. After cutting away the epidermis, fat and connective tissue, cut the skin as small as possible, and wash the tissue pieces on the scissors with PBS. Place the tissue blocks evenly on the bottom of the bottle with an elbow straw, with a distance of about 5 mm between each small block. After the tissue block is placed, drop a little culture solution into the bottle, slowly place the culture bottle flat in a 37°C incubator, and culture statically, the movement should be gentle so a...

Embodiment 2

[0048] Example 2 Detection and confirmation of stably transfected fibroblast cell lines

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Abstract

The invention discloses a telomerase immortalized skin fibroblast line and a construction method thereof, which belongs to the field of cell modification of genetic engineering. The method comprises subculturing skin fibroblasts; transfecting the skin fibroblasts with pCIneo-hTERT plasmid into normal fibroblasts; and screening the human fibroblasts stably transfected by the pCIneo-hTERT plasmid for positive clone and expanding culture. The method provides a standard safe seed cell close to a physiological process for cell tissue engineering, and solves the problems of random integration of genes, jamming of a gene product to physiological means in a cell and the like in the traditional method.

Description

technical field [0001] The invention belongs to the field of cell modification of genetic engineering, relates to a cell line and its establishment method and application, in particular to a telomerase immortalized skin fibroblast cell line and its construction method and application. Background technique [0002] Cells derived from normal tissues can grow and divide under normal in vitro culture conditions, but after a limited number of passages, they will stop proliferating, and then become senescent and die. This limits the further application of cultured cells in vitro. In order to provide standard cell lines for cell tissue engineering, scholars have proposed a variety of methods for establishing immortalized cell lines. The so-called "immortalization" means that the cells are in a continuous cell cycle and terminate their development process, so that the cells obtain unlimited proliferation ability. The establishment of immortalized cells has at least the following a...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85A61L27/38A61L27/60C12R1/91
Inventor 柯明哲
Owner 柯明哲
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