{"id":22259,"date":"2019-10-03T11:59:12","date_gmt":"2019-10-03T03:59:12","guid":{"rendered":"http:\/\/biopioneer.com.tw\/?p=22259"},"modified":"2025-03-21T14:39:49","modified_gmt":"2025-03-21T06:39:49","slug":"%e6%b0%b8%e7%94%9f%e5%8c%96%e7%9a%84%e8%a1%80%e7%ae%a1%e5%b9%b3%e6%bb%91%e8%82%8c%e7%b4%b0%e8%83%9e-sv40-immortalized-human-aortic-smooth-muscle-cells-sv40-%e8%b2%a8%e8%99%9ft0549","status":"publish","type":"post","link":"https:\/\/biopioneer.com.tw\/?p=22259","title":{"rendered":"\u6c38\u751f\u5316\u7684\u8840\u7ba1\u5e73\u6ed1\u808c\u7d30\u80de-SV40| Immortalized Human Aortic Smooth Muscle Cells – SV40 \u8ca8\u865fT0549"},"content":{"rendered":"

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

Immortalized Human Aortic Smooth Muscle Cells – SV40\u00a0<\/strong> | \u8ca8\u865fT0549\u00a0 <\/a><\/span>\u7d30\u80de\u6578\u91cf1×106<\/sup> cells \/ 1.0 ml<\/p>\n

\u7522\u54c1\u4ecb\u7d39:<\/p>\n

\u8840\u7ba1\u5e73\u6ed1\u808c\u7d30\u80de\uff08Vascular Smooth Muscle Cells , VSMC\uff09\u53ef\u4ee5\u57f7\u884c\u6536\u7e2e\u529f\u80fd\u4ee5\u53ca\u5408\u6210\u529f\u80fd\uff0c\u9019\u4e9b\u529f\u80fd\u8207\u5f62\u614b(morphology)\uff0c\u589e\u6b96\u7387(proliferation rates)\u4ee5\u53ca\u4e0d\u540c\u6a19\u8a18\u86cb\u767d\uff08marker proteins, \u4f8b\u5982TGF\u03b2\uff09\u7684\u8868\u9054\u6709\u95dc\u3002 \u6c38\u751f\u7684\u4eba\u985e\u5347\u4e3b\u52d5\u8108\u8840\u7ba1\u5e73\u6ed1\u808c\u7d30\u80de(Human Ascending Aorta Vascular Smooth Muscle Cells) \u662f\u5f9e\u60a3\u6709\u4e8c\u5c16\u74e3\u4e3b\u52d5\u8108\u74e3\uff08bicuspid aortic valve , BAV\uff09\u7684\u7537\u6027\u60a3\u8005\u4e2d\u5206\u96e2\u51fa\u4f86\u7684\u3002\u8a72\u8868\u578b\u5f9e\u975c\u6b62\u5230\u53ef\u6536\u7e2e\uff0c\u518d\u5230\u589e\u751f\u548c\u5408\u6210\uff0c\u9019\u53d6\u6c7a\u65bcphenotype\uff0c\u57f7\u884c\u4e00\u7cfb\u5217\u529f\u80fd\u3002 \u6c38\u751f\u5316\u7d30\u80de\u5c0d\u65bc\u57fa\u56e0\u7279\u5225\u662f\u5167\u76ae\u7cd6\u86cb\u767d(endoglin)<\/em>\u7684\u8868\u9054\u548c\u8abf\u63a7\u7684\u7814\u7a76\u975e\u5e38\u6709\u50f9\u503c\u3002Endoglin<\/em>\u00a0is downregulated in the Immortalized Human Ascending Aorta Vascular Smooth Muscle Cells in response to treatment with the drug Losartan (Cozaar) in various concentrations\u3002Immortalized Human Ascending Aorta Vascular Smooth Muscle Cells\uff08SV40 Large T antigen\uff09<\/p>\n\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>\nHeart<\/td>\n<\/tr>\n
Donor Age<\/strong><\/td>\n46<\/td>\n<\/tr>\n
Donor Gender<\/strong><\/td>\nMale<\/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>\nPolygonal<\/td>\n<\/tr>\n
Recommended Seeding Density<\/strong><\/td>\n20,000 \u2013 50,000 cells\/cm2<\/sup><\/td>\n<\/tr>\n
Population Doubling<\/strong><\/td>\n30 \u2013 40 hours<\/td>\n<\/tr>\n
Immortalization Method<\/strong><\/td>\nTransfection with mammalian expression vector carrying the large T SV40 antigen<\/td>\n<\/tr>\n
Description<\/strong><\/td>\nVascular Smooth Muscle Cells (VSMCs) can perform contractile as well as synthetic functions which are associated with changes in morphology, proliferation rates, and the expression of different marker proteins such as TGF\u03b2. Immortalized Human Ascending Aorta Vascular Smooth Muscle Cells are isolated from a male patient with bicuspid aortic valve (BAV). The phenotype varies from quiescent and contractile to proliferative and synthetic which can perform an array of functions depending on the phenotype. The immortalized cells are valuable for research studies of the expression and regulation of genes particularly\u00a0endoglin<\/em>.\u00a0Endoglin<\/em>\u00a0is downregulated in the Immortalized Human Ascending Aorta Vascular Smooth Muscle Cells in response to treatment with the drug Losartan (Cozaar) in various concentrations.<\/td>\n<\/tr>\n
Depositor Information<\/strong><\/td>\nUniversity of L\u00fcbeck<\/td>\n<\/tr>\n
Applications<\/strong><\/td>\nFor Research Use Only<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u6c38\u751f\u5316\u7684\u8840\u7ba1\u5e73\u6ed1\u808c\u7d30\u80de-SV40\uff08Immortalized Human Aortic Smooth Muscle Cells – SV40\uff09\u4e0d\u6747\u65b9\u6cd5 : Transfection with mammalian expression vector carrying the large T SV40 antigen | \u5668\u5b98\u4f86\u6e90Heart | \u7d30\u80de\u57f9\u990a\u689d\u4ef6: \u4f7f\u7528PriCoat\u2122T25\u57f9\u990a\u74f6 (\u8ca8\u865fG299<\/a>)<\/span>\uff0c\u4ee5\u6700\u4f73\u5316\u521d\u4ee3\u548c\u4e0d\u673d\u7d30\u80de\u7684\u751f\u9577\u3001\u589e\u6b96\u548c\u64f4\u589e\u3002\u6216\u5857\u5c64\u7d30\u80de\u5916\u57fa\u8cea(Applied Cell Extracellular Matrix) \u589e\u5f37\u7d30\u80de\u8cbc\u9644 (\u8ca8\u865fG422<\/a><\/span>)\u3002Grow cells in ECM-coated culture vessels with the following conditions. \u57f9\u990a\u6db2\u00a0PriGrowI (<\/u>\u8ca8\u865f<\/u>\u00a0<\/u>TM001,\u00a0Prigrow I Medium\u8ca8\u865fTM001\u00a0|\u5305\u88dd500ml<\/a><\/span><\/u>)<\/u><\/span>\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\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

\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

\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

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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