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  

Experimental research of hsv1-tk-gfp plasmid transfection into HXO-44 retinal matricyte using SonoVue combined with ultrasonic irradiation

作　　者：贾晓[1] 高璐[2] 杜联芳 伍瑛[1] 李云华[1] 金利芳[1] 李凡[1] 

机构地区：[1]上海交通大学附属上海市第一人民医院超声科,200080 [2]第二军医大学附属长征医院脊柱外科

出　　处：《中华医学超声杂志（电子版）》2013年第5期415-419,共5页Chinese Journal of Medical Ultrasound(Electronic Edition)

基　　金：国家自然科学基金青年科学基金项目(81000617)

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/cm2的条件下,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/cm2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/cm2,1.0 W/cm2,1.5 W/cm2),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 1×105 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/cm2 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/cm2 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.

关 键 词：视网膜母细胞瘤 基因 超声检查 造影剂 

分 类 号：R739.7[医药卫生—肿瘤]