背景 研究证实,羟喜树碱可通过蛋白激酶R样内质网激酶(PERK)途径引起人Tenon囊成纤维细胞(HTFs)凋亡,细胞自噬与凋亡均为细胞在外界压力下产生的程序性死亡反应,二者关系密切,但羟喜树碱与HTFs自噬之间的关系尚不明确. 目的 探讨羟喜树碱对HTFs自噬的作用及其机制.方法 从健康成年眼球供体中取人Tenon囊组织,经组织块培养法培养细胞,并用免疫组织化学法检测细胞中波形蛋白和角蛋白的表达以鉴定培养的细胞.利用293T细胞包装pLVX-PERK慢病毒,将慢病毒转染入HTFs,通过嘌呤霉素筛选出稳定的PERK敲除细胞系.用质量浓度为0.10 g/L的羟喜树碱处理细胞5 min后继续培养24h作为实验组,未用羟喜树碱处理的细胞作为对照组.采用Western blot法检测各组HTFs中自噬相关蛋白Beclin-1、自噬相关基因5(ATG-5)、微管相关蛋白1轻链3(LC-3)表达的灰度值;采用Cyto-ID荧光染色法检测各组HTFs中自噬小体的荧光强度及数量,将各实验结果进行分析比较. 结果 0.10 g/L羟喜树碱处理组HTFs中Beclin-1、ATG-5、LC-3-Ⅰ和LC-3-Ⅱ蛋白表达的灰度值分别为0.980±0.070、1.495±0.095、0.585±0.025和0.785±0.055,明显高于对照组的0.365 ±0.045、0.765 ±0.055、0.120±0.030和0.215±0.035,差异均有统计学意义(Beclin-1:P=0.018;ATG-5:P=0.022;LC-3-Ⅰ:P=0.007;LC-3-Ⅱ:P=0.013).荧光显微镜检测发现,羟喜树碱处理组中自噬小体的绿色荧光强于对照组.Western blot检测结果显示,PERK敲除的细胞中PERK蛋白灰度值为0.130±0.030,明显低于对照组的0.765 ±0.055,差异有统计学意义(P=0.010),而2个组间细胞中Beclin-1、ATG-5和LC-3蛋白反应条带强度无明显差别.0.10 g/L羟喜树碱处理组HTFs中PERK蛋白表达的灰度值为1.790±0.060,较对照组的0.880±0.070明显升高,差异有统计学意义(P=0.010).PERK敲除±0.10 g/L羟喜树碱处理组HTFs中Beclin-1、ATG-5、LC-3-Ⅰ和LC-3-Ⅱ蛋白表达的灰度值分别为0.475 ±0.045、0.390±0.040、0.055±0.015和0.075±0.025,明显低于对照±0.10 g/L羟喜树碱处理组的0.955±0.065、0.765±0.055、0.155±0.015和0.280±0.030,差异均有统计学意义(Beclin-1:P=0.026;ATG-5:P=0.031;LC-3-Ⅰ:P=0.042;LC-3-Ⅱ:P=0.034).PERK敲除+0.10 g/L羟喜树碱处理组细胞中自噬小体的绿色荧光强度明显低于对照+0.10 g/L羟喜树碱处理组. 结论 羟喜树碱可能通过PERK途径诱导HTFs的自噬.%Background Studies confirmed that hydroxycamptothecin cause the apoptosis of human Tenon capsule fibroblasts (HTFs) by protein kinase R-like endoplasmic reticulum stress kinase (PERK) single pathway.Autophagy and apoptosis are programmed cell death following stress reaction,so they remain a close association.However,the effect of hydroxycamptothecin on the autophagy of HTFs and its mechanism are still unclear.Objective This study was to explore the promoting effect of PERK signal pathway on hydroxycamptothecin inducing the autophagy of HTFs.Methods This study procedure was approval by Ethic Committee of Nanjing Medical University.Human Tenon capsule tissue was obtained from fresh adult donors.HTFs were cultured and passaged by explant-culture method and identified by immunofluorescence for vimentin and keratin.pLVX-PERK lentiviral packed by 293T cells was transfected into HTFs to obtain stable PERK-knockout cell line by puromycin selection.Then the HTFs were treated with 0.10 g/L of hydroxycamptothecin for 5 minutes and consecutively cultivated for 24 hours,and the untreated cells were used as the control group.Western blot assay was used to detect the expressions of autophagy specific proteins in the cells,including autophagy related gene 5 (ATG-5),Beclin-1,light chain 3 (LC-3).Cyto-ID staining was used to identify the autophagosome in the cells.The experimental results were analyzed and compared between different treating groups.Results The gray scales for the expressions of Beclin 1,ATG-5,LC-3-Ⅰ and LC-3-Ⅱ proteins in HTFs were 0.365:±0.045,0.765 ±0.055,0.120±0.030 and 0.215 ±0.035 in the control group,and those in the hydroxycamptothecin treated group were 0.980±0.070,1.495±0.095,0.585±0.025 and 0.785±0.055,showing a significant decline in the hydroxycamptothecin treated group(P=0.018,0.022,0.007,0.013).The green fluorescence of the autophagosome was stronger in the hydroxycamptothecin treated group compared with the control group.Western blot revealed that the gray scale of PERK expression in the cells was 0.130±0.030 in the PERK-knockout group,with a significant reduce in comparison with 0.765 ±0.055 of the control group (P =0.010).However,no obvious distinctions were seen in the band intensities of the expressions of Beclin-1,ATG-5 and LC-3 proteins between the two groups.Western blot indicated that the grey scale of the PERK expression in the cells was 1.790± 0.060 in the 0.10 g/L hydroxycamptothecin group,which was significantly higher than 0.880 ± 0.070 of the control group (P =0.010).Expression levels (gray scales) of Beclin-1,ATG-5,LC-3-Ⅰand LC-3-Ⅱ in the PERK-knockout+ 0.10 g/L hydroxycamptothecin group were 0.475 ± 0.045,0.390 ± 0.040,0.055 ± 0.015 and 0.075 ± 0.025,which were significantly lowed in comparison with 0.955 ± 0.065,0.765 ± 0.055,0.155 ± 0.015 and 0.280 ± 0.030 of the control+ 0.10 g/L hydroxycamptothecin group (P =0.026,0.031,0.042,0.034).In addition,the fluorescence intensity of autophagosomes was weaker in the PERK-knockout+0.10 g/L hydroxycamptothecin group compared with the control+0.10 g/L hydroxycamptothecin group.Conclusions Hydroxycamptothecin induces the autophagy of HTFs by PERK signal pathway.
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