{"id":22537,"date":"2019-11-22T11:41:11","date_gmt":"2019-11-22T03:41:11","guid":{"rendered":"http:\/\/biopioneer.com.tw\/?p=22537"},"modified":"2024-07-10T15:18:20","modified_gmt":"2024-07-10T07:18:20","slug":"%e7%89%99%e9%bd%a6%e6%88%90%e7%ba%96%e7%b6%ad%e7%b4%b0%e8%83%9e-immortalized-human-gingival-fibroblasts-htert%e8%b2%a8%e8%99%9ft3104","status":"publish","type":"post","link":"http:\/\/biopioneer.com.tw\/?p=22537","title":{"rendered":"\u7259\u9f66\u6210\u7e96\u7dad\u7d30\u80de | Immortalized Human Gingival Fibroblasts- hTERT\u8ca8\u865fT0026"},"content":{"rendered":"

A<\/strong>bmgood<\/strong>\u85e5\u7269\u7be9\u9078\u5e73\u53f0<\/strong> | <\/strong>\u7d30\u80de\u7cfb\u5217\u5546\u54c1<\/strong> (immortalized cell)<\/strong><\/span><\/p>\n

\u7259\u9f66\u6210\u7e96\u7dad\u7d30\u80de | \u8ca8\u865fT0026<\/span> <\/a><\/span>\u7d30\u80de\u6578\u91cf1×106<\/sup> cells \/ 1.0 ml<\/span><\/p>\n

Immortalized Human Gingival Fibroblasts- hTERT\"\"<\/p>\n

\u7522\u54c1\u4ecb\u7d39: \u7259\u9f66\u6210\u7e96\u7dad\u7d30\u80de(Gingival fibroblasts)\u5728\u53e3\u8154\u50b7\u53e3\u7652\u5408(oral wound healing)\u4e2d\u8d77\u8457\u91cd\u8981\u4f5c\u7528\uff0c\u4e26\u4e14\u662f\u7259\u9f66\u7d50\u7de0\u7d44\u7e54(gingival connective tissue)\u7684\u4e3b\u8981\u6210\u5206\u3002\u9019\u4e9b\u7d30\u80de\u5c0d\u65bc\u6e2c\u8a66\u65b0\u578b\u7259\u79d1\u6750\u6599\u7684\u201c\u751f\u7269\u76f8\u5bb9\u6027 (biocompatibility) \u201d\u6216\u6bd2\u6027 (toxicity) \u5f88\u91cd\u8981\u3002\u904e\u53bb\uff0c\u5df2\u7d93\u4f7f\u7528\u975e\u4eba\u985e\u548c\u4eba\u985e\u60e1\u6027\u7d30\u80de\u682a(human malignant cell lines) \uff08\u4f8b\u5982L929\uff0cCCL163\uff0cCCL171\u548cSaOS2\uff09\u9032\u884c\u4e86\u9ad4\u5916\u6bd2\u6027\u6e2c\u8a66 (toxicity tests)\u3002\u7136\u800c\uff0c\u7531\u65bc\u6240\u8ff0\u7d30\u80de\u682a\u7684\u60e1\u6027\uff0c\u7d50\u679c\u53ef\u80fd\u6216\u53ef\u80fd\u8207\u81e8\u5e8a\u7121\u95dc\u3002\u6c38\u751f\u5316\u4eba\u7259\u9f66\u6210\u7e96\u7dad\u7d30\u80de-hTERT (Human Gingival Fibroblasts- hTERT) \u5728\u751f\u7269\u76f8\u5bb9\u6027\u8a55\u4f30\u4e2d\u8207\u521d\u4ee3\u4eba\u7259\u9f66\u6210\u7e96\u7dad\u7d30\u80de\u5177\u6709\u76f8\u540c\u7684\u5f62\u614b\u5b78\u7d30\u80de\u53cd\u61c9\uff0c\u56e0\u6b64\u53ef\u7528\u65bc\u8a55\u4f30\u7259\u79d1\u6750\u6599\u7684\u6bd2\u6027\u3002<\/p>\n

<\/p>\n

<\/p>\n

Immortalized Human Gingival Fibroblasts- hTERT<\/strong>\"\"<\/p>\n

\"\"<\/p>\n

<\/p>\n

<\/p>\n

<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
Organism<\/strong><\/td>\nHomo sapiens<\/td>\n<\/tr>\n
Source Organ<\/strong><\/td>\nGingiva<\/td>\n<\/tr>\n
BioSafety Level<\/strong><\/td>\nII<\/td>\n<\/tr>\n
Growth Properties<\/strong><\/td>\nAdherent<\/td>\n<\/tr>\n
Morphology<\/strong><\/td>\nSpindle shaped<\/td>\n<\/tr>\n
Recommended Seeding Density<\/strong><\/td>\n7,000-10,000 cells\/cm2<\/sup>; Recommended split ratio is 1:2 to 1:3<\/td>\n<\/tr>\n
Population Doubling<\/strong><\/td>\n50-60 hours<\/td>\n<\/tr>\n
Immortalization Method<\/strong><\/td>\nSerial passaging and transduction with recombinant retroviruses carrying hTERT gene<\/td>\n<\/tr>\n
Description<\/strong><\/td>\nGingival fibroblasts play an important role in oral wound healing and are the major constituents of the gingival connective tissue. These cells are important in testing the \u201cbiocompatibility\u201d, or toxicity of new dental materials. In the past, toxicity tests in vitro have been performed with non-human and human malignant cell lines such as L929, CCL163, CCL171 and SaOS2. However, due to the malignancy of the described cell lines, the results may or may not be clinical relevant. The Immortalized Human Gingival Fibroblasts- hTERT have the same morphological cellular response in biocompatibility evaluation to primary human gingival fibroblasts and can thus be used to evaluate toxicity of dental materials.<\/td>\n<\/tr>\n
Depositor Information<\/strong><\/td>\nYonsei University<\/td>\n<\/tr>\n
Applications<\/strong><\/td>\nFor research only. The Immortalized Human Gingival Fibroblast -hTERT is unique that 1. The cells retained similar cytotoxicity response pattern to their primary counterparts (in response to phenol, gold, titanium and amalgam); 2. The cells retained identical phenotype to that of their primary counterparts; 3. The cells have a diploid status, showing genomic stability.<\/td>\n<\/tr>\n
Markers<\/strong><\/td>\nVimentin and actin<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Immortalized Human Gingival Fibroblasts- hTERT<\/strong>\u7d30\u80de\u682a | \u4e0d\u6747\u65b9\u6cd5 : Serial passaging and transduction with recombinant retroviruses carrying hTERT gene | \u5668\u5b98\u4f86\u6e90Gingiva | \u7d30\u80de\u57f9\u990a\u689d\u4ef6:\u3002Grow cells in\u57f9\u990a\u6db2PriGrowIII (<\/u>\u8ca8\u865f<\/u>\u00a0<\/u>TM003<\/u>)<\/u>\u3002\u70ba\u4e86\u88fd\u5099\u5b8c\u6574\u7684\u751f\u9577\u57f9\u990a\u57fa\uff0c\u5c07\u4e0b\u5217\u7d44\u5206\u6dfb\u52a0\u5230\u57fa\u790e\u57f9\u990a\u57fa\u4e2d\uff1a\u80ce\u725b\u8840\u6e05\uff08TM999\uff09\u81f3\u6700\u7d42\u6fc3\u5ea610\uff05\uff0c\u548cPenicillin\/Streptomycin\u9752\u9ef4\u7d20\/\u93c8\u9ef4\u7d20\u6eb6\u6db2\uff08G255\uff09\u81f3\u6700\u7d42\u6fc3\u5ea6\u70ba1\uff05\u3002\u4e8c\u6c27\u5316\u78b3\uff08CO2\uff09\uff1a5\uff05\uff0c\u6eab\u5ea6\uff1a37.0\u2103\u3002<\/p>\n

\"\"<\/p>\n

\u9ef4\u6f3f\u83ccPCR\u5075\u6e2c<\/a>\u00a0|<\/strong>\u00a0DAPI\u67d3\u6838<\/a>\u00a0|<\/strong>\u00a0\u7d30\u80de\u5167\u9ef4\u6f3f\u83cc\u53bb\u9664\u5291<\/a>\u00a0|<\/strong>\u00a0\u6c34\u6d74\u69fd\u6291\u83cc\u5291<\/a>\u00a0|<\/strong>\u00a0\u5674\u9727\u5f0f\u53bb\u9ef4\u5291 \u7b49\u7cfb\u5217\u5546\u54c1<\/a><\/span><\/p>\n

Abmgood<\/strong>\u4e0d\u673d\u5316\u7cfb\u5217\u5546\u54c1<\/strong>\u00a0(immortalized reagenet)<\/strong>\u00a0Ready-to-use<\/p>\n

Abmgood\u516c\u53f8\u65bc\u4eba\u985e\u8207\u8001\u9f20\u4e0d\u673d\u4e4b\u7d30\u80de\u682a\u4e0a\uff0c\u5177\u6709\u5e7e\u5341\u5e74\u7684\u5be6\u9a57\u7d93\u9a57\uff1b\u63d0\u4f9b\u4e00\u7cfb\u5217\u4e0d\u673d (immortalized)\u5316\u4e4b\u7d30\u80de\u682a\u3001\u53ca\u4e0d\u673d\u5316\u4e4b\u8a66\u5291\u5546\u54c1\u3002ready-to-use\u5b9a\u91cf\u597d\u4e4bretrovirus\u3001lentiviru\u3001 adenovirus\uff0c\u53ef\u76f4\u63a5\u52a0\u65bc\u7d30\u80de\u4e2d\u9032\u884c\u611f\u67d3\u8868\u73fe\uff1b\u75c5\u6bd2\u6db2\u5305\u62ec\u6709SV40\u91cd\u7d44\u75c5\u6bd2\u3001EBV\u91cd\u7d44\u75c5\u6bd2\u3001hTERT\u75c5\u6bd2\u6db2\u3001Myc\u57fa\u56e0\u75c5\u6bd2\u6db2\u3001p53\u57fa\u56e0\u75c5\u6bd2\u6db2\u3001Rb\u57fa\u56e0\u75c5\u6bd2\u6db2\u3001Ras\u57fa\u56e0\u75c5\u6bd2\u6db2\u3002\u63d0\u4f9b106\u53ca109\u5169\u7a2e\u5305\u88dd\u529b\u50f9\u4e4b\u75c5\u6bd2\u6db2\uff1b\u4ea6\u63d0\u4f9b\u4e00\u7cfb\u5217\u75c5\u6bd2\u529b\u50f9\u6fc3\u7e2e\u8207\u5b9a\u91cf\u8a66\u5291\u5546\u54c1\u3002<\/p>\n

\"\"<\/p>\n

\u7d30\u80de\u4e0d\u673d\u5316\u7684\u5de5\u5177\u4e2d\uff0cSV40 T antigen\u662f\u6700\u7c21\u55ae\u4e14\u6700\u5e38\u7528\u4f86\u9020\u6210\u4e0d\u673d\u5316\u7d30\u80de\u751f\u6210\u7684\u65b9\u6cd5\u3002\u591a\u6578\u7684\u75c5\u6bd2\u57fa\u56e0\u548c\u6291\u764c\u57fa\u56e0\uff08p53\u3001Rb\u7b49\uff09\u7684\u4e0d\u6d3b\u5316\u76f8\u95dc\uff0c\u6703\u8a98\u5c0e\u7d30\u80de\u8655\u65bcreplicative senescence\u7684\u968e\u6bb5\u3002\u76ee\u524d\u4e5f\u6709\u7814\u7a76\u986f\u793a\u88ab\u611f\u67d3\u7684\u7d30\u80de\uff0cSV40 T anbtigen\u6703\u8a98\u5c0e\u7aef\u7c92\u9176\uff08Telomerase\uff09\u7684\u6d3b\u6027\u3002\u8207\u7aef\u7c92\uff08telomere\uff09\u9577\u5ea6\u76f8\u95dc\u7684Telomerase Reverse Transcriptase protein (TERT)\u662f\u4e00\u7a2e\u4e0d\u673d\u7d30\u80de\u7684\u7814\u7a76\u61c9\u7528\u3002\u5e73\u5e38\u7d30\u80de\u4e2d\uff0chTERT\u662f\u8655\u65bc\u4e0d\u6d3b\u5316\u614b\uff0c\u5fc5\u8981\u6642\u6703\u9032\u884c\u6d3b\u5316\u4fdd\u6301telomere\u7684\u9577\u5ea6\uff0c\u907f\u514d\u7d30\u80de\u767c\u751freplicative senescence\u7684\u73fe\u8c61\u3002\u67d0\u4e9b\u7d30\u80dehTERT \u904e\u5ea6\u8868\u73fe\uff08over-expression\uff09\u5c0d\u65bc\u8a98\u767c\u7d30\u80de\u4e0d\u673d\u5316\u662f\u7121\u6548\u7684\uff08\u5982\u521d\u4ee3\u4e0a\u76ae\u7d30\u80de\uff09\uff0c\u53cd\u800c\u6703\u9020\u6210\u7d30\u80de\u6b7b\u4ea1\uff1b\u540c\u6642\u8868\u73fehTERT catalytic subunit\u4ee5\u53cap53\u6216RB siRNA\u5728\u4eba\u985e\u521d\u4ee3\u5375\u5de2\u4e0a\u76ae\u7d30\u80de\u6703\u9020\u6210\u7d30\u80de\u4e0d\u673d\u5316\u3002\u904e\u5ea6\u8868\u73feRas\u6216Myc T58A mutants\u4e5f\u6709\u521d\u4ee3\u7d30\u80de\u4e0d\u673d\u5316\u7684\u73fe\u8c61\u7522\u751f\u3002<\/p>\n

 <\/p>\n

Decomplement<\/strong>\u52a0\u71b1\u975e\u6d3b\u5316<\/strong>FBS<\/strong>\uff1a<\/strong><\/p>\n

1.\u8b93FBS\u74f6\u57284\u00b0C\u4e0b\u5b8c\u5168\u89e3\u51cd\u904e\u591c\u3002<\/p>\n

2.\u5c07FBS\u74f6\u7f6e\u65bc56\u00b0C\u7684\u6c34\u6d74\u4e2d30\u5206\u9418\u3002<\/p>\n

Decomplemented FBS\u53ef\u4ee5\u5728-20\u00b0C\u4e0b\u9577\u671f\u4fdd\u5b58\u3002\u907f\u514d\u983b\u7e41\u7684\u51cd\u878d\u5faa\u74b0\u3002\u6bcf2-3\u5929\u66f4\u63db\u4e00\u6b21\u5b8c\u6574\u7684\u5a92\u9ad4\u3002\u4e0d\u8981\u8b93\u4ecb\u8cea\u984f\u8272\u8b8a\u70ba\u9ec3\u8272\u3002<\/p>\n

Thawing<\/strong>\u7d30\u80de\u89e3\u51cd\u65b9\u6cd5\uff1a<\/strong>(<\/strong>\u6bcf\u7a2e\u7d30\u80de\u5efa\u8b70\u689d\u4ef6\u53ef\u80fd\u6709\u8a31\u4e0d\u540c\uff0c\u4f9d\u7167\u539f\u5ee0\u5efa\u8b70\u64cd\u4f5c\uff0c\u4ee5\u4e0b\u70ba\u901a\u7528\u65b9\u5f0f<\/strong>)<\/strong><\/p>\n

1.\u572837\u2103\u6c34\u6d74\u4e2d\u89e3\u51cd\u5c0f\u74f6\uff0c\u76f4\u5230\u53ea\u6709\u4e00\u5c0f\u584a\u51b0\u584a\u3002<\/p>\n

2.\u5c07\u7d30\u80de\u8f49\u79fb\u5230\u542b\u67095ml pre-warmed complete media \u4e4b15ml\u7ba1\u4e2d\u3002<\/p>\n

3.\u5c07\u7d30\u80de\u4ee5290xg\u96e2\u5fc35\u5206\u9418\u3002<\/p>\n

4.\u5c0f\u5fc3\u4e1f\u68c4\u4e0a\u6e05\u6db2\uff0c\u4e0d\u8981\u6253\u64fe\u7d30\u80de\u6c89\u6fb1\uff0c\u8f15\u8f15\u5730\u4e0a\u4e0b\u79fb\u6db2\uff0c\u5c07\u7d30\u80de\u91cd\u65b0\u61f8\u6d6e\u57281ml\u5b8c\u5168\u57f9\u990a\u57fa\u4e2d\u3002<\/p>\n

5.\u5c07\u7d30\u80de\u57f9\u990a\u5728150,000-200,000\u500b\u7d30\u80de\/ cm2\u3002<\/p>\n

Subculture<\/strong>\u7d30\u80de\u7e7c\u4ee3\u65b9\u6cd5<\/strong>:<\/strong>\u00a0(<\/strong>\u6bcf\u7a2e\u7d30\u80de\u5efa\u8b70\u689d\u4ef6\u53ef\u80fd\u6709\u8a31\u4e0d\u540c\uff0c\u4f9d\u7167\u539f\u5ee0\u5efa\u8b70\u64cd\u4f5c\uff0c\u4ee5\u4e0b\u70ba\u901a\u7528\u65b9\u5f0f<\/strong>)<\/strong><\/p>\n

1.\u7576\u7d30\u80de70-90\uff05 confluency\u6642\uff0c\u5efa\u8b70\u7e7c\u4ee3\u57f9\u990a\u3002<\/p>\n

    \n
  1. Aspirate old media\u3002\u61f8\u6d6e\u6db2\u4e2d\u7684\u4e00\u4e9b\u7d30\u80de\u4ecd\u7136\u662f\u6d3b\u7d30\u80de\u3002\u6216\u8005\uff0c\u4e5f\u53ef\u4ee5\u6536\u96c6\u542b\u6709\u61f8\u6d6e\u7d30\u80de\u7684\u4e0a\u6e05\u6db2\u4e26\u96e2\u5fc3\uff08Skip to Step 6 in protocol\uff09\u3002<\/li>\n<\/ol>\n

    3.\u52a0\u51651\uff1a1\u6bd4\u4f8b\u76841X\u7121\u83ccPBS\u548c0.25\uff05\u80f0\u86cb\u767d\u9176-EDTA\uff08TM051\uff09\u3002<\/p>\n

    4.\u5728\u5ba4\u6eab\u4e0b\u5b75\u80b2\u7d30\u80de3-5\u5206\u9418\u4e26\u652a\u62cc\u57f9\u990a\u5bb9\u5668\u76f4\u81f390\uff05\u7684\u7d30\u80de\u812b\u843d\u3002<\/p>\n

      \n
    1. \u4e2d\u548c\u80f0\u86cb\u767d\u9176:\u6dfb\u52a0\u7b49\u91cf\u4e4btrypsin + PBS\u3002<\/li>\n<\/ol>\n

      6.\u6536\u96c6\u7d30\u80de\u4e26\u4ee5290xg\u96e2\u5fc35\u5206\u9418\u3002<\/p>\n

      7.\u4e1f\u68c4\u4e0a\u6e05\u6db2\uff0c\u8f15\u8f15\u5730\u4e0a\u4e0b\u79fb\u6db2\uff0c\u5c07\u7d30\u80de\u91cd\u65b0\u61f8\u6d6e\u5728\u5b8c\u5168\u57f9\u990a\u57fa\u4e2d\u3002<\/p>\n

      8.\u5efa\u8b70split ratio\u6bd4\u4e0d\u8d85\u904e1\uff1a3\u3002<\/p>\n

      Freezing<\/strong>\u7d30\u80de\u51cd\u7d50\uff1a<\/strong>(<\/strong>\u6bcf\u7a2e\u7d30\u80de\u5efa\u8b70\u689d\u4ef6\u53ef\u80fd\u6709\u8a31\u4e0d\u540c\uff0c\u4f9d\u7167\u539f\u5ee0\u5efa\u8b70\u64cd\u4f5c\uff0c\u4ee5\u4e0b\u70ba\u901a\u7528\u65b9\u5f0f<\/strong>)<\/strong><\/p>\n

      Complete growth medium with decomplemented FBS to a final concentration of 50% from PAN Biotech (P40-37500) or from Nucleus Biologics (FBS1824-001) and 5% DMSO.<\/p>\n

      \u5132\u5b58\u6eab\u5ea6\uff1a\u6db2\u6c2e\u6c23\u76f8\u3002\u91cd\u8981\u63d0\u793a\uff1a\u6b64\u7d30\u80de\u4fc2\u5c0dFBS\u6279\u6b21\u975e\u5e38\u654f\u611f\u3002\u6211\u5011\u5f37\u70c8\u5efa\u8b70\u6700\u7d42\u7528\u6236\u4f7f\u7528\u5f9ePAN biotech\u6216Nucleus Biologics \u4e4bFBS\u3002<\/p>\n

      \u6162\u75c5\u6bd2\u5305\u88dd\u7cfb\u5217\u7522\u54c1\u00a0Lentivirus<\/u>\u6162\u75c5\u6bd2\u4f4e\u901f\u7d14\u5316<\/u>(\u7d14\u5316)\u00a0<\/a>| Lentivirus <\/u>\u6162\u75c5\u6bd2\u9ad8\u6fc3\u5ea6\u7d14\u5316<\/u>(\u7d14\u5316)\u00a0<\/a>|\u00a0Lentivirus<\/u>\u75c5\u6bd2<\/u>RT-QPCR<\/u>\u5b9a\u91cf<\/u>(\u5b9a\u91cf)<\/a>\u00a0<\/span><\/p>\n

       <\/p>\n","protected":false},"excerpt":{"rendered":"

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