晶状体上皮细胞启动子LEP503调控的自杀基因系统细胞特异性表达研究

The cell-type-specific HSV-tk gene expression of suicide gene therapy system regulated by lensspecific promoter LEP503

目的探讨人晶状体上皮细胞（HLEC）特异性启动子LEP503调控的Cre／LoxP增强型自杀基因（HSV-tk／GCV）系统在眼内主要细胞中的特异性表达及杀伤作用。方法实验研究。利用分子克隆技术，构建HLEC特异性启动子LEP503调控的HSV—tk／GCV系统并进行慢病毒包装。分别在HLEC、视网膜色素上皮细胞（RPEC）、小鼠成纤维细胞（NIH3T3）、293T细胞中转人此系统，3d后观察4组细胞增强型绿色荧光蛋白（EGFP）表达情况，并收集HLEC和RPEC，通过RT-PCR检测HSV—tk的表达差异。20mg／L丙氧鸟苷（GCV）作用后，CCK-8试剂盒和流式细胞仪检测HSV-tk／GCV系统对HLEC和RPEC的杀伤作用。采用单因素方差分析Scheffe两两比较法对各组RPEC活性进行比较。结果HSV-tk／GCV系统在RPEC、NIH3T3、293T细胞中EGFP表达效率分别为62．3％，68．3％，75．8％；在HLEC中EGFP表达效率为17．5％。RT—PCR检测显示HLEC内有较高的HSV—tk表达，相对灰度值为4．01，而RPEC中HSV-tk表达微弱，相对灰度值为0．29。20mg／LGCV作用72h后流式细胞仪检测：HLEC组的细胞凋亡率为76．51％，而RPEC组仅为2．44％。CCK-8试剂盒检测细胞增殖抑制情况显示：20mg／LGCV作用72h后LEP503调控的HSV—tk／GCV转染的RPEC活性0．9198±0．06与正常RPE组0．9672±0．02和阴性对照RPE组0．89274-0．07相比，差异无统计学意义[组间均数差值（MD）分别为MD1=-0．047，P=0．671；MD2=0．027，P=0．912]。结论HLEC特异性启动子LEP503调控的Cre／LoxP增强型自杀基因系统在HLEC中能特异性表达HSV．tk，GCV作用后可特异性抑制HLEC增殖，但对RPEC无明显杀伤作用。

Objective To explore the specific expression of HSV-tk gene and killing effects on ocular leading cells of the enhanced specific HSV-tk/GCV gene therapy system regulated by lens-specific promoter LEPS03. Methods Experimental research. The enhanced specific HSV-tk/GCV gene system of two vectors were constructed （Lenti-LEPS03-HSVtk-Cre and Lenti-HPGK-Loxp-EGFP-pA-Loxp-HSVtk）. The lentiviral vectors were produced by transient transduction of transfering vectors, packaging vectors and enveloping vector into 293T cells. Virus was collected with uhracentrifugation and resuspended with 1 ml phosphate buffered saline and stored at -80 ~C. The HLEC and RPEC, NIH3T3, 293T cells were transduced with the enhanced specific HSV-tk gene system. The specific expressions of EGFP and HSV-tk were detected by fluorescence microscopy, flow cytometry and RT-PCR. The killing effects of HLEC and RPEC at the concentration of 20 mg/L GCV were assayed and compared by flow eytometry and CCK-8 kit. Difference of RPE cell viability among groups was evaluated by analysis of variance （ANOVA）. Results Expression efficiency of EGFP in RPEC group was 62. 3% ,68.3% in NIH3T3 group,75.8% in 293T group, whereas 17.5% in HLEC group. There was higher expression of HSV-tk at mRNA level in HLEC group than that in RPEC group. The relative intensity of HSV-tk mRNA in HLEC group transduced with the enhanced specific HSV-tk gene system was 4.01, whereas 0. 29 in RPEC group. At the concentration of 20 mg/L GCV after 72 hours, the percentage of apoptosis detected by the flow cytometry in HLEC group transduced by the enhanced specific HSY-tk gene system was 76. 51% , and 2.44% in RPEC group. There was no significant difference in the RPE cell viability among the enhanced specific HSV-tk gene combination-RPE group, normal-RPE group and negative-RPE control group at the concentration of 20 mg/L GCV after 72 hours （MD1 = -0.047,P=0.671;MD2 =0.027,P=0.912）. Conclusions The enhanced specific HSV-tk gene system express HSV-tk selectively in HLEC. At the concentration of 20 mg/L GCV,it is effective against the proliferation of HLEC in vitro, but has less kill effect on RPEC.