重组腺相关病毒介导的HSV—tk／GCV体系对兔晶状体上皮细胞的抑制作用

Inhibitory effects of HSV-tk/GCV system mediated by recombinant adeno-associated virus 2 on rabbit lens epithelial cells

目的探讨2型重组腺相关病毒（rAAV2）载体介导的单纯疱疹病毒胸苷激酶基因／丙氧鸟苷体系（HSV—tk／GCV）对体外培养兔晶状体上皮细胞（N／N1003A）的抑制作用，以及晶状体上皮细胞死亡的机制。方法实验研究。rAAV2载体介导增强型绿色荧光蛋白基因（EGFP）转染体外培养N／N1003A细胞，倒置荧光显微镜观察细胞中EGFP的表达，流式细胞仪检测病毒的转染效率。重组病毒rAAV2／HSV—tk转染体外培养的N／N1003A细胞为实验组，以没有转染重组病毒的细胞作为对照组，MTT法检测HSV—tk／GCV体系对细胞作用的浓度依赖性、时间依赖性和旁观者效应；相差显微镜、透射电镜、Hoechst33258染色观察细胞凋亡、坏死改变，流式细胞仪检测细胞凋亡率和细胞周期的变化。不同浓度GCV对两组细胞的作用结果采用两因素析因设计的方差分析；两组细胞周期和凋亡的比较采用两样本t检验。结果rAAV2载体能介导EGFP基因稳定高效转染N／N1003A细胞。GCV对两组细胞的杀伤作用有剂量-效应依赖关系（F=13076．239，P〈0．001）；实验组N／N1003A—tk细胞的存活率低于对照组，差异有统计学意义（F=53947．119，P〈0．001）。实验组GCV的IC50为2mg／L，而对照组IC50为524mg／L。GCV的杀伤效应随时间延长而增强，且存在明显的旁观者效应。N／N1003A-tk细胞在GCV作用下出现明显的细胞凋亡和坏死，细胞的凋亡率7．18％±2．04％，与对照组（3．50％±0．56％）比较差异有统计学意义（t=3．83，P〈0．01）；S期细胞比例明显增加，G0／G1期细胞比例明显减少，与对照组比较差异有统计学意义（S期细胞比例：f=3．55，P〈0．01；G0／G1期细胞比例：t=4．29，P〈0．01）。结论GCV可有效杀伤重组腺相关病毒rAAV2／HSV-tk转染的兔晶状体上皮细胞，并具有较强的旁观者效应。

Objective To study the inhibitory effects of recombinant adeno-associated virus 2 （rAAV2）-mediated herpes simplex virus thymidine kinase/ganciclovir （HSV-tk/GCV） system on the rabbit lens epithelial cells （N/N1003A） in vitro and to investigate the mechanism of cell death. Methods After N/N1003A cells had been transfected with rAAV2-EGFP, expression of enhanced green fluorescent protein （EGFP） were observed by inverted fluorescent microscope and the transfection efficiency was detected by flow cytometry. N/N1003A cells were infected by recombinant virus rAAV2/HSV-tk as the treated group, and the uninfected N/N1003A cells were used as the controls. The dose- and time-dependent efficiency and bystander effect of HSV-tk/GCV system on the cells were studied by MTT assay. Apoptosis and necrosis were observed by phase contrast microscope, electron microscope and Hoechst33258 stain. Apoptotic cell rate and cell cycle were detected by flow cytometry. Results rAAV2 vector encoding EGFP gene could be transfected into N/N1003A cells stably and efficiently. The effects of GCV on these two groups were dose- dependent （F = 13 076. 239 ,P 〈0. 001 ）. The difference of percentages of survival cells between the study group and the control group at various doses of GCV was statistically significant （ F = 53 947. 119, P 〈 0. 001 ）. The 50% of the inhibitory concentration （ IC50 ） of GCV in the study group was 2 mg/L and was 524 mg/L in the control group. The killing efficiency of GCV increased with the prolongation of time and showed significant bystander effect. Cell apoptosis and necrosis were observed in N/N1003A-tk cells transfected by GCV, and the percentage of apoptotic cells was significantly higher than that of the control group （t = 3.83, P 〈 0.01 ）. The percentages of N/N1003A-tk cells in the S phase of the cell cycle was significantly higher than that of the control group （t = 3.55 ,P 〈 0.01 ）. Whereas the percentages of the Go/ G~ phase in GCV treated cells was significantly lower than that of the control group （t = 4.29, P 〈 0.01 ）. Conclusions GCV can kill efficiently the N/N1003A cells infected by recombinant virus rAAV2/HSV-tk, and there is strong bystander effect. Recombinant adeno-associated virus-mediated HSV-tk/GCV suicide gene system may provide an effective approach for the treatment of lens posterior capsular opacification.