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目的研究脑源性神经营养因子 (BDNF)对H2 0 2 诱导人成纤维细胞 (hFB)凋亡的作用。方法 将含人Ⅰ型胶原a1链(COLIAl)基因启动子和增强子元件并在其 3′端接BDNFcDNA的微基因pSVCEPBFCAT ,经脂质体介导转染hFB并驱动BDNF异位表达 ,采用四唑盐 (MTT)显色法检测细胞增殖 ,吖啶橙荧光染色显微镜观察细胞形态的改变 ,琼脂糖凝胶电泳检测染色质DNA断裂。结果 经 2 0 0 μmol/LH2 O2 作用 2 4h后 ,pSVCEPBFCAT转染组和对照组包括未转染hFB、空载体pSVCEPCAT转染hFB的细胞生长抑制率分别为 35 %和≥ 84 % ,p <0 .0 0 1;对照组hFB普遍可见凋亡细胞特有的形态学及生物化学改变 ,如胞浆和核固缩、染色质断裂、DNA电泳呈阶梯状条带 ,而pSVCEPBFCAT转染hFB未发现上述凋亡特征。结论 BDNF促进hFB增殖和拮抗H2 O2 诱导凋亡 ,明显抑制H2 O2 对hFB的细胞毒性。
Objective To investigate the effect of brain derived neurotrophic factor (BDNF) on H2O2-induced human fibroblast (hFB) apoptosis. METHODS: The pSVCEPBFCAT, a promoter and enhancer element containing human COLI1 gene promoter, was inserted into pSVCEPBFCAT, which is a 3 ’terminus of BDNF cDNA. Lipofectamine was used to transfect hFB and drive the ectopic expression of BDNF. Cell proliferation was detected by MTT assay. Acridine orange staining was used to observe the changes of cell morphology. Chromatin DNA was detected by agarose gel electrophoresis. Results The cell growth inhibition rate of pSVCEPBFCAT transfected group and control group, including untransfected hFB, transfected hFB with empty vector pSVCEPCAT was 35% and ≥ 84%, respectively, after treated with 200 μmol / LH 2 O 2 for 24 h, p <0 .0 0 1; The morphological and biochemical changes of apoptotic cells such as cytoplasm and nuclear pyknosis, chromatin breaks and DNA electrophoresis showed ladder-like bands in hFB of control group, but not hFB in pSVCEPBFCAT Apoptosis characteristics. Conclusion BDNF can promote the proliferation of hFB and antagonize the H2O2-induced apoptosis, and significantly inhibit the cytotoxicity of H2O2 on hFB.