中文摘要：

目的初步研究肠道病毒71型（EV71）诱导人恶性胶质瘤细胞U251的自噬现象及其对微管相关蛋白1轻链3（LC3）表达的影响。方法RPMI1640培养U251细胞24h后随机分为实验组和对照组，实验组按感染复数（MOI）值为1加入EV71，EV71感染12h后采用单丹磺酰戊二胺（MDC）染色标志细胞自噬泡，荧光显微镜观察自噬泡的形成。EV71感染24h后细胞免疫荧光检测LC3，荧光显微镜观察LC3在U251细胞内的分布。EV71感染2、4、8、12、24、48h采用Westernblot检On．0LC3-I和LC3-II蛋白的表达，并对LC3-II蛋白进行半定量分析。结果白噬泡经过MDC染色后，荧光显微镜观察发现自噬泡呈点状结构。与对照组比较，实验组在EV71感染12h，U251细胞内自噬泡明显增多；EV71感染24h，U251细胞皱缩、变小、形态不规则，LC3蛋白表达明显增多，主要分布在细胞质和细胞核周围；EV71感染4h后，LC3-II蛋白表达开始增加，明显高于对照组（P〈0．01）。结论EV71能有效诱导U251细胞自噬，发挥其溶瘤作用。

英文摘要：

Objective To show evidences that autophagy is induced in human malignant glioma cell line U251 by enterovirus （ EV71 ） and its effect on the expression of microtubule-associated protein 1 lightehain 3 （ LC3 ） in vitro. Methods /5251 cells were cultured in RPMI 1640 for 24 hours, then randomly divided into experimental group and control group. In experimental group, EV71 were added in cell culture holes at multiplicity of infection （MOI） equal to 1,and cultured continuously. After 12 hours post infection of U251 cells with EV71, autophagic vacuoles of U251 cells were marked by monodansyleadaverine staining and observed under fluorescence microscope. After 24 hours post infec- tion, expression and intraeellular distribution of LC3 protein in U251 ceils were observed under fluorescence microscope by immunofluorescenee. Expressions of LC3-I and LC3-II protein were measured by Western blot and analysis of LC3- II protein expression was performed with semi-quantitative calculation at 2,4,8,12,24 and 48 hours post infection of U251 cells with EV71 ,respectively. Results Autophagic vacuoles stained by MDC in U251 cells appeared as dis- tinct dot-like structures distributed under fluorescence microscope. The number of autophagic vacuoles were increased significantly at 12 hours post infection of U251 cells with EV71 when compared with control group. The morphological features of these cells became significantly shrunken, smaller and irregular shape at 24 hours post infection of U251 cells, and the expressions of LC3 protein were significantly higher in experimental group than those in control group. Un- der a fluorescence microscope, LC3 protein distributed within the cytoplasm or localizing in the perinuclear regions. At 4 hours post infection of U251 cells with EV71, the expression of LC3-1I protein started to increase, and was significantly higher than that in control group （P 〈 0.01 ）. Conclusion These results indicate that EV71 can effectively induce au- tophagy of human malignant glioma cell line U