声诺维联合超声辐照介导载单纯疱疹病毒Ⅰ型胸苷激酶基因质粒转染 HXO－44视网膜母细胞的实验研究

摘要

目的探讨超声造影剂声诺维（SonoVue）联合超声辐照介导载单纯疱疹病毒Ⅰ型胸苷激酶基因（hsv1－tk－gfp）质粒转染HXO－44视网膜母细胞的可行性。方法在24孔板中设置6组：空白对照组（只有细胞，第1组）；细胞＋质粒（第2组）；细胞＋质粒＋超声辐照（第3组）；细胞＋质粒＋超声辐照＋声诺维（第4组）（辐照声强：0．5、1．0、1．5 W／cm2，辐照时间：40、60、80 s，造影剂浓度：10％、20％）；细胞＋质粒＋超声辐照＋声诺维＋昔洛韦（第5组）（辐照方案同第4组，昔洛韦设置不同的浓度：1 mg／L、10 mg／L、100 mg／L、1 g／L）；细胞＋超声辐照＋声诺维（第6组）（辐照方案同第4组）；细胞数量为1×105个／孔，质粒用量为1μl每孔。转染48 h后流式细胞仪检测各组绿色荧光蛋白转染率，MTT法观察细胞存活情况。结果辐照声强为1．5 W／cm2、辐照时间为60 s、造影剂浓度为20％的条件下绿色荧光蛋白转染率最高。加入1 g／L的昔洛韦后，辐照声强为0．5、1．0、1．5 W／cm件下，HXO－44视网膜母细胞的存活率分别为（95．29±0．94）％、（92．58±1．29）％、（77．26±16．20）％，细胞存活率差异有统计学意义（F＝11．402，P＜0．05）；辐照声强为1．5 W／cm2时细胞存活率较辐照声强为0．5、1．0 W／cm 2时下降，且差异均有统计学意义（t＝12．776、10．856，P＜0．01）。结论超声造影剂联合一定强度的超声辐照可增强载hsv1－tk－gfp质粒转染HXO－44视网膜母细胞率；hsv1－tk－gfp质粒转染率超过10％的肿瘤细胞在加入前体药物昔洛韦（浓度高于1 g／L）可出现明显的凋亡。%Objective To explore the feasibility of SonoVue combined with ultrasonic irradiation in mediating hsv1-tk-gfp plasmid DNA transfection into HXO-44 retinal matricyte.Methods Six groups were designed in this experiment:(1) only cells ( control group );(2) cells +plasmid;(3) cells +plasmid +ultrasonic irradiation;(4)cells+plasmid+ultrasonic irradiation +SonoVue[with different acoustic intensity (0.5 W/cm 2 ,1.0 W/cm 2 ,1.5 W/cm 2 ), radiating time (40 s,60 s,80 s) and micro bubble concentration (10%,20%)];(5)cells+plasmid+ultrasonic irradiation+SonoVue+drug(GCV)[with the same conditions in the fourth group in addition to different drug concentrations (1 mg/L,10 mg/L,100 mg/L,1 g/L)];cells+ultrasonic irradiation+SonoVue(with the same conditions in the fourth group ).There were 1 μl plasmid and 1 ×10 5 cells in each hole.The transfection efficiency was assessed after 48 h using flow cytometry.And the cell apoptosis was detected by MTT test .Results Under the conditions of 1.5 W/cm 2 acoustic intensity,60 s radiating time and 20% micro bubble concentration, the highest transfection rate was reached.Using 1 g/L GCV,the survival rate of HXO-44 retinal matricyte under the acoustic intensity of 0.5 W/cm2 ,1.0 W/cm2 and 1.5 W/cm2 were (95.29 ±0.94)%,(92.58 ±1.29)%,(77.26 ±16.20)%respectively with significant difference (F=11.402,P<0.05).Under 1.5 W/cm 2 irradiation,cells appeared obvious apoptosis ( t =12.776,10.856, P <0.01).Conclusions Sonovue combined with ultrasonic irradiation can enhance the transfection efficiency of hsv1-tk-gfp plasmid into HXO-44 retinal matricyte. Cells appear obvious apoptosis when the hsv1-tk-gfp plasmid transfection rate above 10% with GVC concentration higher than 1 g/L.