Method for immortalizing human periodontal ligament stem cell line by using hTERT (human telomerase reverse transcriptase) lentivirus recombinant

A periodontal ligament stem cell and lentivirus technology, applied in the field of hTERT lentivirus recombinant immortalized human periodontal ligament stem cell line, can solve the problems of loss of cell biological function, low transfection efficiency, poor representation of cell models, etc. Stable properties and good functions

Active Publication Date: 2015-09-23
XI AN JIAOTONG UNIV
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Problems solved by technology

[0004] At present, there is also a method of transfecting periodontal ligament cells with SV40 virus together with other genes to prolong life, but the transfection

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  • Method for immortalizing human periodontal ligament stem cell line by using hTERT (human telomerase reverse transcriptase) lentivirus recombinant
  • Method for immortalizing human periodontal ligament stem cell line by using hTERT (human telomerase reverse transcriptase) lentivirus recombinant
  • Method for immortalizing human periodontal ligament stem cell line by using hTERT (human telomerase reverse transcriptase) lentivirus recombinant

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Embodiment Construction

[0031] The specific implementation process of the present invention is described below. It should be noted that these examples are only illustrative of the present invention, not limiting.

[0032] The method for hTERT lentiviral recombinant immortalized human periodontal ligament stem cell line comprises the following steps:

[0033] Step 1. Culture of primary human periodontal ligament stem cells (hPDLSC):

[0034] Collect the extracted premolars, scrape 1 / 3 of the periodontal ligament tissue from the root, centrifuge at 800rpm for 5min, add 1mL type Ⅰ collagenase to the sedimentation and mix it according to the tissue obtained from one tooth, and place it at 37°C, 5% CO 2 , digested in a standard environmental cell culture box with saturated humidity until the tissue is loose, and replace the culture medium every 3-5 days. The culture medium is DMEM / F12 medium with a volume ratio of 79%, 20% fetal bovine serum with a volume ratio of 79%. Compared with 79% of the 1% three...

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Abstract

The invention relates to a method for immortalizing a human periodontal ligament stem cell line by using an hTERT (human telomerase reverse transcriptase) lentivirus recombinant. The method comprises the following steps: 1, culturing primary human periodontal ligament stem cells (hPDLSC); 2, establishing an immortalized genetic engineering cell line hTERT-PDLSC stably expressing hTERT genes, to be specific, infecting the immortalized genetic engineering cell line with primarily cultured PDLSC by using a lentivirus carrier expressing the hTERT genes under a certain condition, and carrying out resistance screening of puromycin to obtain the immortalized cell line stably expressing the hTERT genes, wherein the service life of the cells is prolonged to 35 generations from 3-8 generations, and thus an immortalized cell model having stable properties, favorable functions and normal phenotype is obtained. The method disclosed by the invention has a beneficial effect on research of periodontal ligament stem cells to periodontal tissue engineering as well as research and development of medicaments for effectively treating periodontitis.

Description

technical field [0001] The invention belongs to the technical fields of cell engineering, genetic engineering and immortalization, and in particular relates to a method for immortalizing human periodontal ligament stem cell line with hTERT lentivirus recombinant, mainly for the primary culture and identification of human periodontal ligament stem cells, through lentivirus The vector constructs a cell line stably expressing the human telomerase reverse transcriptase hTERT gene, and detects the function and transformation of the cell line. Background technique [0002] Chronic periodontitis (chronic periodontitis) is a common and frequently-occurring disease in the human oral cavity. Inflammation and bleeding of periodontal supporting tissues, loss of periodontal attachment, formation of periodontal pockets, alveolar bone destruction and resorption, and eventually tooth loosening will occur sequentially. And fall off, seriously affecting the physical and mental health of human...

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Application Information

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IPC IPC(8): C12N15/867C12N15/54C12N5/10
Inventor 李昂刘瑾苟建重王嗣岑
Owner XI AN JIAOTONG UNIV
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