目的 观察大鼠OPTN基因的低表达对视网膜神经节细胞系(RGC5)亚细胞形态结构及存活的影响.方法 实验研究.设计并合成特异性针对Sprague-Dawley(SD)大鼠OPTN的3对RNA干扰片段(siRNA),体外培养SD大鼠星型胶质细胞,取对数生长期的细胞做实验,分别将3对siRNA(si-OPTN-001、si-OPTN-002、si-OPTN-003)通过脂质体Lipofectemine 2000转染入星型胶质细胞,24 ～ 48 h后采用实时PCR和Western Blot检测siRNA的干扰效果,筛选出抑制效果最佳的序列;以筛选出来的siRNA序列构建绿色荧光蛋白EGFP标记的si-OPTN,并通过Lipofectemine 2000包裹转染RGC5,将RGC5分为4组:空白对照组,绿色荧光蛋白真核细胞表达载体(pEGFP)阳性对照组,si-OPTN组,脂质体阴性对照组.转染24～48 h后用细胞器特异荧光染料标记细胞内不同细胞器结构,通过共聚焦荧光显微镜来观察表达的si-OPTN在RGC5内的分布、定位及其对细胞器形态结构的影响;通过形态学观察和Hoechst33342-PI荧光染色观察si-OPTN对RGC5存活的影响.实验中不同组间的总体比较采用单因素方差分析.结果 实时PCR及Western Blot的结果显示:转染si-OPTN-001、si-OPTN-002和si-OPTN-003 24 h后星型胶质细胞内OPTN的mRNA表达下降,分别为阴性对照组的(61.71±0.84)％、(48.13±0.92)％和(46.22±0.73)％,而转染si-OPTN-001、si-OPTN-002和si-OPTN-003 48 h后星型胶质细胞内OPTN的蛋白表达水平下降,分别为阴性对照组的(64.44±2.01)％、(57.78±1.97)％和(37.78±1.84)％,其中si-OPTN-003与阴性对照组相比降低显著;si-OPTN转染RGC5细胞24 h后的转染效率为(17.43±0.94)％;转染48 h后的转染效率为(20.13±1.24)％;表达的si-OPTN在细胞内基本上均匀分布于整个细胞,覆盖胞质和胞核,与阴性对照组细胞器染色情况比较,转染细胞内的肌丝蛋白、线粒体、溶酶体及高尔基体形态结构未见明显损伤性改变;Hochest33342/PI荧光染色结果显示:转染24 h,与空白对照组(0.74±0.34)％和阴性对照组(0.96±0.41)％比较,EGFP阳性对照组及si-OPTN组整组的RGC5细胞凋亡率均增加,差异有统计学意义(F=5.457,P＜0.05),但si-OPTN组转染质粒的细胞凋亡率明显低于阳性对照组,差异有统计学意义(F=6.541,P＜0.05).随转染时间延长,在转染48 h时,阳性对照组凋亡率较24 h时下降,si-OPTN组凋亡率稍升高但无统计学意义(F=3.212,P＞0.05),且仍低于阳性对照组.结论 相较于另2对si-RNA,si-OPTN-003更能有效地抑制OPTN的表达;si-OPTN转染RGC5后均匀分布于全细胞,对细胞内的亚细胞器结构未见明显损伤性改变;通过转染si-OPTN使OPTN表达降低可能对RGC5的存活有保护作用.%Objective To investigate the effects of decreased OPTN expression on the morphological alteration of sub-cellular structure and the survival of the rat retinal ganglion cell line RGC5.Methods Experimental study.OPTN specific siRNA was designed,synthesized and transfected into the astrocyte cell by Lipofectemine 2000.The mRNA and the protein of OPTN were assessed by real time-polymerase chain reaction (real time-PCR) and Western Blot.Eukaryotic expressing plasimid of OPTN specific siRNA (siOPTN) was constructed with the most effective RNA interference sequence and transfected into RGC5 by Lipofectemine 2000.The RGC5 was divided into 4 groups:blank control group,pEGFP positive group,siOPTN group,and liposome negative group.Specific fluorescent labeling dyes were used to mark the corresponding cell organelle.The distribution of optineurin and the morphology alteration of sub-cellular structure induced by the abnormal expression of optineurin in RGC5 were observed with confocal fluorescence microscopy.The effect of si-OPTN on cell survival was monitored by morphologic observation and PIHoechst33342 fluorescence staining of cells.One-way ANOVA was used between groups.Results After a 24 h transfection,the expression of OPTN mRNA was significantly inhibited by transfection of si-OPTN-001,si-OPTN-002 and si-OPTN-003 which was (61.71 ±0.84)％,(48.13 ±0.92)％ and (46.22 ±0.73)％ respectively comparing with negative control.Forty-eight hours after transfection,the protein expression of OPTN decreased to (64.44 ± 2.01) ％,(57.78 ± 1.97) ％ and (37.78 ± 1.84) ％ comparing with negative control.When cells were transfected after 24 h and 48 h,the transfecting efficiency of plasmids to RGC5 was (17.43 ± 0.94)％ and (20.13 ± 1.24)％ respectively.The distribution of si-OPTN was similar to EGFP.The green fluorescence distributed evenly in the cytoplasm and the nucleus.Compared with negative control,the morphology of filaments,mitochondria,lysosomes,and Golgi body had no significant alteration in siOPTN group.By using fluorescent double staining of Hoechst33342 and PI and comparing with blank control group (0.74 ±0.34) ％ and negative control group (0.96 ±0.41) ％,increased cell apoptosis was observed in positive control group and si-OPTN group obviously after a 24 h transfection (F =5.457,P ＜0.05),but the apoptosis rate decreased in si-OPTN group comparing with that of positive control group (F =6.541,P ＜ 0.05).As the transfecting time extended,the apoptosis rate of positive control group increased when tranfecteing cultures reached 48 h.The apoptosis rate in si-OPTN group increased slightly with no significance (F =3.212,P ＞0.05).Conclusions Si-OPTN-003 is the most effective siRNA in inhibiting the OPTN expression.The expression of si-OPTN distributed evenly in the whole cell with no obvious injuries in the transfected cell.The down regulation of optineurin induced by si-OPTN could be decreased RGC5 cell death.
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