摘要目的 观察腺相关病毒(AAV)介导小发夹RNA(shRNA)干扰抑制Claudin-3表达对体外培养小鼠视网膜神经节细胞(RGC)的影响.方法 原代培养小鼠RGC分为正常对照组、AAV-shScramble组、AAV-shClaudin3组.细胞接种24 h后,AAV-shScramble组、AAV-shClaudin3组RGC分别转染AAV-shScramble和AAV-shClaudin3.转染96h后,活细胞荧光动态显微镜观察目的病毒转染效率;β-微管蛋白免疫荧光染色检测RGC轴突长度;免疫荧光4',6-二脒基-2-苯基吲哚染色观察RGC凋亡形态;实时聚合酶链反应(RT-PCR)检测各组RGC Claudin-3、血管内皮生长因子(VEGF) mRNA表达;蛋白免疫印迹法(Western blot)检测各组RGC Claudin-3、VEGF、B淋巴细胞瘤/白血病-2基因(Bcl-2)、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)蛋白表达水平.结果 活细胞动态荧光显微镜观察发现,AAV-shScramble组、AAV-shClaudin3组RGC病毒转染效率均＞50％;病毒转染96 h后,AAV-shClaudin3组RGC轴突长度明显低于正常对照组、AAV-shScramble组,差异有统计学意义(F=2 363.274,P＜0.05);AAV-shClaudin3组RGC密度较正常对照组、AAV-shScramble组明显减低,可见细胞核皱缩、趋边以及细胞核裂解形成的凋亡小体.AAV-shClaudin组RGC的Claudin-3、VEGF mRNA表达明显低于正常对照组和AAV-shScramble组,差异有统计学意义(F=257.408、160.533,P＜0.05).AAV-shClaudin3组RGC的Claudin-3、VEGF、Bcl-2蛋白表达明显低于正常对照组和AAV-shScramble组,差异有统计学意义(F=129.671、420.552、62.669,P＜0.05);Caspase-3蛋白表达明显高于正常对照组、AAV-shScramble组,差异有统计学意义(F=231.348,P＜0.05).结论 AAV介导shRNA干扰抑制Claudin-3的表达可下调VEGF、Bcl-2及上调Caspase-3的表达,促进RGC凋亡,抑制RGC轴突生长.

abstractsObjective To study the effect of down-regulation of Claudin-3 mediated by adenoassociated virus (AAV) of shRNA on the cultured retinal ganglion cells (RGCs) in vitro.Methods RGCs isolated from mouse eyes were divided into normal control group,AAV-shScramble group,and AAVshClaudin-3 group.The RGCs in AAV-shScramble group and AAV-shClaudin3 group were treated with AAV-shScramble and AAV-shClaudin-3 respectively 24 hours after cell seeding.Dynamic live cell fluorescence microscopy was used to observe the transfection efficiency 96 hours after transfection.Immunofluorescent staining of β-tubulin was used to measure the length of RGCs' axon.4',6-diamidino-2-phenylindole staining was used to observe the nuclei of apoptotic cells.The mRNA level of Claudin-3 and VEGF was measured by real-time polymerase chain reaction.The protein levels of Claudin-3,vascular endothelial growth factor (VEGF),Bcl-2 and Caspase-3 was determined by Western blot.Results The positive transfection rate was more than 50％ in both AAV-shScramble group and AAV-shClaudin-3 group.The length of RGCs' axon in AAV-shClaudin-3 group was shorter than that in normal control group and AAVshScramble group (F=22 363.274,P＜0.05).Down-regulation of Claudin-3 accelerated RGCs' apoptosis with nuclei shrinkage,tapering,and nucleolus formation of apoptotic bodies.The mRNA levels of Claudin-3 and VEGF in AAV-shClaudin-3 group were lower than those in normal control group and AAV-shScramble group (F=257.408,160.533;P＜0.05).The protein levels of Claudin-3,VEGF and Bcl-2 in AAV-shClaudin-3 group were lower than those in normal control group and AAV-shScramble group (F=129.671,420.552,62.669;P＜0.05),while the protein level of Caspase-3 in AAV-shClaudin-3 group was higher than that in normal control group and AAV-shScramble group (F=231.348,P＜0.05).Conclusion Down-regulation of Claudin-3 increases the expression of Caspase-3,reduces the expression of VEGF and Bcl-2,accelerates RGCs' apoptosis and inhibit the RGCs' axon growth.