\u8840\u7ba1\u751f\u6210\u4e0d\u4ec5\u6d89\u53ca\u5305\u62ec\u764c\u75c7\u751f\u7269\u5b66\u548c\u975e\u80bf\u7624\u75be\u75c5\u5728\u5185\u7684\u75c5\u7406\u6761\u4ef6\uff0c\u8fd8\u6d89\u53ca\u5305\u62ec\u751f\u6b96\u3001\u53d1\u80b2\u548c\u4fee\u590d\u5728\u5185\u7684\u8bb8\u591a\u751f\u7269\u5b66\u8fc7\u7a0b\u3002\u5728\u8840\u7ba1\u751f\u6210\u8fc7\u7a0b\u4e2d\uff0c\u5185\u76ae\u7ec6\u80de\uff08EC\uff09\u5728\u8840\u7ba1\u751f\u6210\u56e0\u5b50\u4e0e\u5176\u53d7\u4f53\u7ed3\u5408\u3001\u91ca\u653e\u86cb\u767d\u9176\u4ee5\u6eb6\u89e3\u57fa\u5e95\u819c\u3001\u5411\u8840\u7ba1\u751f\u6210\u4fe1\u53f7\u8fc1\u79fb\u3001\u589e\u6b96\u548c\u7ec6\u80de\u6570\u91cf\u589e\u52a0\u4ee5\u5f62\u6210\u65b0\u8840\u7ba1\u540e\u53d1\u751f\u6fc0\u6d3b\u3002\u6700\u540e\uff0c\u5185\u76ae\u7ec6\u80de\u7684\u91cd\u7ec4\u5f62\u6210\u4e86\u4e09\u7ef4\u8840\u7ba1\u7cfb\u7edf\u3002HUVEC\u7ba1\u5f62\u6210\u6d4b\u5b9a\u6cd5\u662f\u4e00\u79cd\u7b80\u5355\u4f46\u516c\u8ba4\u7684\u4f53\u5916\u8840\u7ba1\u751f\u6210\u6d4b\u5b9a\u6cd5\uff0c\u57fa\u4e8e\u5185\u76ae\u7ec6\u80de\u5728\u751f\u957f\u56e0\u5b50\u51cf\u5c11\u7684\u57fa\u5e95\u819c\u63d0\u53d6\u7269\u51dd\u80f6\u4e0a\u57f9\u517b\u65f6\u5f62\u6210\u4e09\u7ef4\u6bdb\u7ec6\u7ba1\u72b6\u7ba1\u72b6\u7ed3\u6784\u7684\u80fd\u529b\u3002\u5728\u6d4b\u5b9a\u8fc7\u7a0b\u4e2d\uff0c\u5185\u76ae\u7ec6\u80de\u5206\u5316\u3001\u5b9a\u5411\u8fc1\u79fb\u4ee5\u6392\u5217\u3001\u5206\u652f\u5e76\u5f62\u6210\u8840\u7ba1\u7684\u7ba1\u72b6\u591a\u8fb9\u5f62\u7f51\u7edc\u3002<\/p>\n
\u9700\u8981\u51c6\u5907\u7684\u8bd5\u5242\uff1a<\/p>\n
\u57fa\u8d28\u80f6\uff08\u542f\u8fbe\u751f\u7269\uff0c\u8d27\u53f7\uff1aSD0054\uff09<\/p>\n
Dulbecco\u6539\u826fEagle\u57f9\u517b\u57fa\uff08DMEM\uff09\uff08\u9ad8\u7cd6L-\u8c37\u6c28\u9170\u80fa\uff0c500\u6beb\u5347\uff09<\/p>\n
\u80ce\u725b\u8840\u6e05\uff08\u542f\u8fbe\u751f\u7269, \u8d27\u53f7\uff1aSD0200\uff09<\/p>\n
\u539f\u4ee3\u4eba\u8110\u9759\u8109\u5185\u76ae\u7ec6\u80de\uff08HUVEC\uff09\uff08\u542f\u8fbe\u751f\u7269, \u8d27\u53f7\uff1aCD0290\uff09<\/p>\n
ECGM\u5185\u76ae\u7ec6\u80de\u57f9\u517b\u57fa\/\u5957\u88c5\uff08\u542f\u8fbe\u751f\u7269, \u8d27\u53f7\uff1aP1001\uff09<\/p>\n
ECGS\u5185\u76ae\u7ec6\u80de\u751f\u957f\u56e0\u5b50\uff08\u542f\u8fbe\u751f\u7269\uff0c\u8d27\u53f7\uff1aP1002\uff09<\/p>\n
\u6761\u4ef6\u57f9\u517b\u57fa\uff08CM\uff09<\/p>\n
\u5907\u6ce8\uff1a\u9776\u7ec6\u80de\u7cfb\u4e5f\u53ef\u4ee5\u662f\u90a3\u4e9b\u6709\u6216\u6ca1\u6709\u836f\u7269\u6cbb\u7597\u6216\u8868\u8fbe\u611f\u5174\u8da3\u57fa\u56e0\u7684\u7ec6\u80de\u7cfb\u3002<\/p>\n
\u9700\u8981\u51c6\u5907\u7684\u8017\u6750\uff1a<\/p>\n
\u7ec6\u80de\u57f9\u517b\u7bb1<\/p>\n
\u5e26\u6570\u7801\u76f8\u673a\u7684\u5012\u7f6e\u663e\u5fae\u955c\uff08\u5c3c\u5eb7TMS\uff09<\/p>\n
Scion Image\u8f6f\u4ef6<\/p>\n
96\u5b54\u677f<\/p>\n
\u79bb\u5fc3\u673a<\/p>\n
\u7ec6\u80de\u8ba1\u6570\u5668<\/p>\n
T25\u7ec6\u80de\u57f9\u517b\u74f6<\/p>\n
HUVEC\u6210\u7ba1\u64cd\u4f5cprotocol<\/p>\n
A.<\/p>\n
\u4ece\u9776\u7ec6\u80de\u7cfb\u5236\u5907\u6761\u4ef6\u57f9\u517b\u57fa<\/p>\n
1.\u4e3a\u4e86\u5236\u5907\u6761\u4ef6\u57f9\u517b\u57fa\uff08CM\uff09\uff0c\u63a5\u79cd\u9776\u7ec6\u80de\u5e76\u751f\u957f\u81f330-40%\u6c47\u5408\uff08\u53d6\u51b3\u4e8e\u7ec6\u80de\u7cfb\u7684\u751f\u957f\u901f\u7387\uff09\uff0c\u7528\u65e0\u8840\u6e05DMEM\uff08\u4f8b\u5982\uff0c\u5bf9\u4e8eT75\u7ec4\u7ec7\u57f9\u517b\u74f6\u4e3a10ml\uff1b\u6297\u751f\u7d20\u7684\u5b58\u5728\u4e0e\u5426\u65e0\u5173\u7d27\u8981\uff09<\/p>\n
2. \u4ee3\u66ff\u751f\u957f\u57f9\u517b\u57fa24\u5c0f\u65f6\uff0c\u7136\u540e\u5f53\u7ec6\u80de\u5728T75\u7ec4\u7ec7\u57f9\u517b\u74f6\u4e2d\u8fbe\u523060-80%\u6c47\u5408\u65f6\u6536\u83b7CM\u3002<\/p>\n
\u5982\u679c\u6536\u96c6CM\u540e\u672a\u7acb\u5373\u4f7f\u7528\uff0c\u5219\u53d60.5 ml CM\u7b49\u5206\u8bd5\u6837\uff0c\u5e76\u5728-80\u00b0C\u4e0b\u50a8\u5b58\u3002<\/p>\n
B.<\/p>\n
\u4eba\u8110\u9759\u8109\u5185\u76ae\u7ec6\u80de\uff08HUVEC\uff09\u7ec6\u80de\u7684\u5236\u5907<\/p>\n
1.\u6309\u7167\u57f9\u517b\u57fa\u914d\u6bd4\u8981\u6c42\u914d\u597dECGM\u5b8c\u5168\u57f9\u517b\u57fa\u3002<\/p>\n
2.\u6839\u636e\u9700\u8981\u5c06HUVEC\u7ec6\u80de\u63a5\u79cd\u5728T25\u6216T75\u57f9\u517b\u74f6\u4e2d\uff0c\u4f7f\u5176\u8fbe\u523070-80%\u7684\u878d\u5408\u3002<\/p>\n
3.\u5728\u4e0d\u542b\u6297\u751f\u7d20\u7684\u57f9\u517b\u57fa200PRF\u4e2d\u8fdb\u884c\u8bd5\u7ba1\u5f62\u6210\u6d4b\u5b9a\u4e4b\u524d\uff0c\u53bb\u9664\u8840\u6e05\u4f7fHUVEC\u7ec6\u80de\u9965\u997f3-6\u5c0f\u65f6\uff08\u4f8b\u5982\uff0c\u5bf9\u4e8eT25\u7ec4\u7ec7\u57f9\u517b\u74f6\u4e3a5\u6beb\u5347\uff09\u3002<\/p>\n
\u6ce8\uff1a\u5728\u8840\u6e05\u9965\u997f\u7ed3\u675f\u65f6\uff0c\u8f6c\u81f3\u7a0b\u5e8fC\u6b65\u9aa41\u3002<\/p>\n
4.\u5728\u7528\u751f\u957f\u56e0\u5b50\u51cf\u5c11\u7684\u57fa\u8d28\u80f6\u6d82\u5e0396\u5b54\u677f\u540e\uff0c\u7528\u80f0\u86cb\u767d\u9176\u4ece\u70e7\u74f6\u8868\u9762\u53bb\u9664\u7ec6\u80de\uff0c\u7528DMEM\u4e0e\u8840\u6e05\u4e2d\u548c\uff0c\u5728\u5ba4\u6e29\u4e0b\u4ee51200rpm\uff08276xg\uff09\u79bb\u5fc33\u5206\u949f\u4f7f\u7ec6\u80de\u6c89\u6dc0\uff0c\u91cd\u60ac\u4e8e2-3ml\u65e0\u8840\u6e05DMEM\u4e2d\uff0c\u901a\u8fc7\u8ba1\u6570\u7ec6\u80de\u6765\u6d4b\u5b9aHUVEC\u7684\u6d53\u5ea6\uff0c\u5e76\u901a\u8fc7\u5411\u4e0a\u548c\u5411\u4e0b\u5438\u79fb\u51e0\u6b21\u4ee54\u00d7105\/ml\u65e0\u8840\u6e05DMEM\u91cd\u60acHUVEC\u7ec6\u80de\uff0c\u4ee5\u786e\u4fdd\u5747\u5300\u7684\u5355\u7ec6\u80de\u60ac\u6d6e\u6db2\u3002<\/p>\n
\u6ce8\uff1a\u5bf9\u4e8e\u751f\u957f\u56e0\u5b50\u51cf\u5c11\u7684\u57fa\u8d28\u80f6\uff0c\u5e94\u5c3d\u53ef\u80fd\u51cf\u5c11\u89e3\u51bb\/\u51b7\u51bb\u5468\u671f\u3002<\/p>\n
5.\u5728\u5c06500\u03bcl HUVEC\u7ec6\u80de\u60ac\u6d6e\u6db2\u79fb\u6db2\u52301.5 ml\u8bd5\u7ba1\u4e2d\u7684\u540c\u65f6\u5145\u5206\u6df7\u5408\u3002\u4f7f\u7528\u53f0\u5f0f\u79bb\u5fc3\u673a\u4ee54000rpm\uff081100rcf\uff09\u7684\u8f6c\u901f\u5c06\u7ec6\u80de\u964d\u901f3\u5206\u949f\u3002\u5728\u4e0d\u5e72\u6270\u7ec6\u80de\u6c89\u6dc0\u7684\u60c5\u51b5\u4e0b\u5c0f\u5fc3\u5730\u5438\u51fa\u4e0a\u6e05\u6db2\u3002\u5c3d\u53ef\u80fd\u591a\u5730\u53bb\u9664\u4e0a\u6e05\u6db2\u3002\u6839\u636e\u8981\u4f7f\u7528\u7684\u9776\u7ec6\u80de\u7cfb\u7684\u6570\u91cf\uff0c\u57281.5ml\u8bd5\u7ba1\u4e2d\u5236\u5907\u9002\u5f53\u6570\u91cf\u7684HUVEC\u7ec6\u80de\u3002<\/p>\n
\u6ce8\uff1a\u6bcf\u4e2a\u9776\u7ec6\u80de\u7cfbCM\u5c06\u60ac\u6d6e\u4e00\u7ba1HUVEC\uff0c\u6bcf\u4e2a\u60ac\u6d6e\u7684HUVEC\u5c06\u5206\u914d3\u4e2a\u4e00\u5f0f\u4e09\u4efd\u7684\u5b54\u3002\u56e0\u6b64\uff0c\u6bcf\u4e2a\u9776\u7ec6\u80de\u7cfb\u603b\u5171\u5c06\u4f7f\u75283\u4e2a\u5b54\u3002<\/p>\n
6.\u89e3\u51bb\u4ece\u7a0b\u5e8fa\u6b65\u9aa42\u6536\u96c6\u76840.5ml\u7b49\u5206\u7684\u76ee\u6807\u7ec6\u80de\u7cfbCM\uff0c\u5e76\u7528\u80ce\u725b\u8840\u6e05\u5c06\u5176\u8865\u5145\u81f31%\u7684\u6700\u7ec8\u6d53\u5ea6\uff0c\u4ee5\u5728\u7a0b\u5e8fB\u6b65\u9aa45\u4e2d\u91cd\u60acHUVEC\u7ec6\u80de\u9897\u7c92\u3002<\/p>\n
\u6ce8\uff1a\u6bcf\u4e2a\u9776\u7ec6\u80de\u7cfb\u5e94\u4e00\u5f0f\u4e09\u4efd\uff08\u6bcf\u4e2a\u5b54\u9700\u8981100\u03bcl HUVEC\u7ec6\u80de\u60ac\u6d6e\u6db2\uff09\u3002<\/p>\n
C.<\/p>\n
\u7528\u751f\u957f\u56e0\u5b50\u51cf\u5c11\u7684\u57fa\u8d28\u80f6\uff08\u542f\u8fbe\u751f\u7269\uff0c\u8d27\u53f7 SD0054 \uff09\u6d82\u898696\u5b54\u677f<\/p>\n
1.\u4f7f\u7528\u524d\u4e00\u5929\u57284\u00b0C\u4e0b\u89e3\u51bb\u9002\u5f53\u4f53\u79ef\u7684\u57fa\u8d28\u80f6\u3002<\/p>\n
2.\u5c0696\u5b54\u677f\u548c\u79fb\u6db2\u7ba1\u5c16\u7aef\u5728-20\u00b0C\u4e0b\u9884\u51b72-3\u5c0f\u65f6\u3002<\/p>\n
3.\u5728HUVEC\u7ec6\u80de\u8840\u6e05\u9965\u997f\u63a5\u8fd1\u5c3e\u58f0\u65f6\uff0c\u5728\u8ba1\u6570HUVEC\u4e4b\u524d\uff0c\u5728\u51b0\u4e0a\u9884\u51b796\u5b54\u677f\u7684\u6bcf\u5b54\u4e2d\uff0c\u7b49\u5206\u8bd5\u683750\u03bcl\u7684\u57fa\u8d28\u80f6\u3002\u65cb\u8f6c\u94fa\u677f\uff0c\u76f4\u5230\u51dd\u80f6\u5747\u5300\u5730\u5206\u5e03\u5728\u6574\u4e2a\u5b54\u4e0a\u3002\u907f\u514d\u6c14\u6ce1\u7684\u5f62\u6210\u662f\u975e\u5e38\u91cd\u8981\u7684\u3002\u4f7f\u5176\u5728\u5ba4\u6e29\u4e0b\u5728\u6c34\u5e73\u8868\u9762\u4e0a\u805a\u54081\u5c0f\u65f6\u3002<\/p>\n
D.<\/p>\n
\u5c06HUVEC\u7ec6\u80de\u5206\u914d\u5230\u6d82\u5e03\u768496\u5b54\u677f\u4e0a<\/p>\n
1.\u5c06100\u03bcl\u7a0b\u5e8fB\u6b65\u9aa46\u4e2d\u83b7\u5f97\u7684HUVEC\u7ec6\u80de\u60ac\u6d6e\u6db2\u5b8c\u5168\u6df7\u5408\u523096\u5b54\u677f\u7684\u6807\u8bb0\u5b54\u4e2d\u3002\u5c06\u5e73\u677f\u572837\u00b0C\u30015%CO2\u4e0b\u57f9\u517b4-6\u5c0f\u65f6\u3002<\/p>\n
2.\u7528\u5149\u5b66\u663e\u5fae\u955c\u89c2\u5bdf\u7ec6\u80de\u3002\u62cd\u6444\u6bdb\u7ec6\u7ba1\u7f51\u7edc\u7684\u56fe\u50cf\uff0c\u5e76\u4f7f\u7528Scion Image\u8f6f\u4ef6\u8ba1\u7b97\u7ba1\u9053\u957f\u5ea6\u3002<\/p>\n
\u4e0eCM\u5b75\u80b26\u5c0f\u65f6\uff08100x\uff0c100\u03bcm\uff09\u540e\uff0cHUVEC\u5728\u57fa\u8d28\u51dd\u80f6\u4e0a\u6210\u7ba1\u3002<\/p>\n
\u53c2\u8003\u6587\u732e<\/p>\n
1. Ko, J. M. and Lung, M. L. (2012). In vitro Human Umbilical Vein Endothelial Cells (HUVEC) Tube-formation Assay. Bio-protocol 2(18): e260. DOI: 10.21769\/BioProtoc.260.<\/p>\n
2. Chan, K. C., Ko, J. M., Lung, H. L., Sedlacek, R., Zhang, Z. F., Luo, D. Z., Feng, Z. B., Chen, S., Chen, H., Chan, K. W., Tsao, S. W., Chua, D. T., Zabarovsky, E. R., Stanbridge, E. J. and Lung, M. L. (2011). Catalytic activity of Matrix metalloproteinase-19 is essential for tumor suppressor and anti-angiogenic activities in nasopharyngeal carcinoma. Int J Cancer 129(8): 1826-1837.<\/p>\n
3. Kong, D., Li, Y., Wang, Z., Banerjee, S. and Sarkar, F. H. (2007). Inhibition of angiogenesis and invasion by 3,3′-diindolylmethane is mediated by the nuclear factor-kappaB downstream target genes MMP-9 and uPA that regulated bioavailability of vascular endothelial growth factor in prostate cancer. Cancer Res 67(7): 3310-3319.<\/p>\n
\u6e29\u99a8\u63d0\u793a\uff1a<\/p>\n
\u96c6\u6587\u732e\u9886\u7ea2\u5305\u6d3b\u52a8\u5f00\u59cb\u5566~<\/p>\n
\u4f7f\u7528\u6211\u4eec\u7684\u4ea7\u54c1\u53d1\u8868\u5728\u5404\u5927\u671f\u520a\u4e0a\u7684\u6587\u7ae0\u5907\u6ce8\uff1aShanghai QiDa Biotechnology Co.,LTD\u6587\u5b57\u7684\uff0c\u9886\u53d6\u73b0\u91d1\u7ea2\u5305\u3002<\/p>\n
\u6216\u53c2\u52a0\u516c\u53f8\u5e74\u5ea6\u52a9\u5b66\u8ba1\u5212\uff0c\u6700\u9ad8\u5206\u53ef\u5f97\u5956\u52b1\u73b0\u91d13000\u5143\u3002\u6bcf\u5e74\u8bc4\u90092\u4eba\uff01\u6709\u6587\u732e\u7684\u8001\u5e08\u8bf7\u52a0\u5fae\u4fe1\u5ba2\u670d\uff1a17521589219 \u9886\u53d6\u54e6\uff0c\u6bcf\u7bc7\u6587\u7ae0\u9650\u53c2\u52a0\u4e00\u6b21\uff01<\/p>\n<\/p>\n
\u70b9\u51fb\u300c\u4e0a\u65b9\u4e8c\u7ef4\u7801\u300d\u53ef\u5feb\u901f\u5173\u6ce8\u5b98\u65b9\u7f51\u7ad9: http:\/\/\/<\/p>\n
\u70b9\u4e00\u4e0b\u53f3\u4e0b\u89d2\u201c\u5728\u770b\u201d\uff0c\u8ba9\u6211\u4eec\u505a\u7684\u66f4\u597d<\/p>\n","protected":false},"excerpt":{"rendered":"
\u8840\u7ba1\u751f\u6210\u4e0d\u4ec5\u6d89\u53ca\u5305\u62ec\u764c\u75c7\u751f\u7269\u5b66\u548c\u975e\u80bf\u7624\u75be\u75c5\u5728\u5185\u7684\u75c5\u7406\u6761\u4ef6\uff0c\u8fd8\u6d89\u53ca\u5305\u62ec\u751f\u6b96\u3001\u53d1\u80b2\u548c … \u7ee7\u7eed\u9605\u8bfb