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目的研究单纯疱疹病毒胸腺激酶(HSV-tk)基因系统对人眼球筋膜囊成纤维细胞(HTFs)增殖的抑制作用及其作用机制。方法构建含HSV-tk基因的逆转录病毒载体pL(tk)SN并转入包装细胞系PT67进行病毒包装,G418筛选抗性克隆并扩大培养收集病毒上清,测定病毒滴度。透射电镜观察鉴定病毒颗粒;聚合酶链反应(PCR)技术鉴定tk基因在病毒基因组中的整合。用病毒感染HTFs细胞,G418筛选并扩大培养,逆转录聚合酶链反应(RT-PCR)技术和免疫印迹法检测tk基因的表达;加不同浓度前药更昔洛韦(GCV)后,以MTT法检测tk基因对HTFs细胞增殖的抑制作用;采用Hoechst33258染色及流式细胞仪检测凋亡细胞;电镜观察细胞形态学改变。结果酶切鉴定结果显示成功构建了逆转录病毒载体pL(tk)SN,电镜下包装细胞产生的病毒颗粒呈椭圆形,直径约100nm;病毒基因组中,PCR扩增出了tk基因;测得病毒滴度为4×107cfu/ml;被转染的HTFs细胞,用RT-PCR及免疫印迹法检测到tk基因在mRNA与蛋白水平均有表达;HSV-tk-GCV系统对HTFs细胞增殖的抑制作用表现为剂量依赖性,5×10-3g/L的GCV作用5d可将细胞全部杀死,IC50为6×10-4g/L,HSV-tk-GCV系统对HTFs细胞的杀伤机制表现为坏死与凋亡,凋亡率随GCV浓度的升高而增加。结论HSV-tk基因系统能有效抑制人眼球筋膜囊成纤维细胞的增殖,作用机制表现为细胞的坏死与凋亡。(中华眼科杂志,2006,42:212-217)
Objective To investigate the inhibitory effect of the herpes simplex virus thymidine kinase (HSV-tk) gene system on the proliferation of human eyeball fascicular fibroblasts (HTFs) and its mechanism. Methods The retroviral vector pL (tk) SN containing HSV-tk gene was constructed and transformed into the packaging cell line PT67 for virus packaging. The resistant clones were screened by G418 and expanded to collect the virus supernatant. The virus titers were determined. Transmission electron microscopy was used to identify the virus particles. Polymerase chain reaction (PCR) was used to identify the integration of tk gene in the viral genome. HT4 cells were infected with virus and screened and expanded by G418. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression of tk gene. After adding different concentrations of ganciclovir (GCV) Method to detect the inhibitory effect of tk gene on the proliferation of HTFs cells. Apoptotic cells were detected by Hoechst 33258 staining and flow cytometry. Morphological changes were observed by electron microscopy. Results The results of restriction enzyme digestion showed that the recombinant retroviral vector pL (tk) SN was successfully constructed. The virus particles produced by packaging cells under electron microscope were oval with a diameter of about 100 nm. The tk gene was amplified by PCR from the virus genome. The titer was 4 × 107cfu / ml. The transfected HTFs cells were detected by RT-PCR and Western blotting at the mRNA and protein levels of tk gene expression; HSV-tk-GCV system HTFs cell proliferation inhibition Showed a dose-dependent manner, 5 days after 5 × 10-3g / L GCV treatment, the cells were completely killed with an IC50 of 6 × 10-4g / L. The killing mechanism of HSV-tk-GCV on HTFs cells was necrosis and Apoptosis and apoptosis rate increased with the increase of GCV concentration. Conclusion The HSV-tk gene system can effectively inhibit the proliferation of human eyeball fibroblasts, the mechanism of which is manifested as cell necrosis and apoptosis. (Chinese Journal of Ophthalmology, 2006,42: 212-217)