碘造影剂对重组腺病毒转染大鼠脂肪干细胞转染效率的影响

Effect of iodine contrast medium on transfection efficiency of adipose derived stem cells transfected by recombinant adenovirus

目的初步探讨碘造影剂对腺病毒(Ad)转染大鼠脂肪干细胞(ADSCs)转染效率的影响。方法2015年12月—2016年1月于武汉大学心血管病研究所进行实验。体外培养大鼠ADSCs,用携带有TBX18基因的重组腺病毒载体(Ad-TBX18-GEP)转染ADSCs。根据是否加入碘造影剂将分选的脂肪干细胞分为空白对照组(A)组、腺病毒转染组(B)组、腺病毒转染+造影剂0.1 ml组(C)组、腺病毒转染+造影剂0.25 ml组(D)组、腺病毒转染+造影剂0.5 ml组(E)组。荧光显微镜下观察绿色荧光蛋白(GFP)在靶细胞的表达情况,以每视野内平均阳性表达细胞所占比率判断基因转染效率。结果(1)腺病毒转染ADSCs的最适感染复数(MOI)值为100。(2)除A组外,其余各组在荧光显微镜下均可见绿色荧光,且B组绿色荧光蛋白表达最多,C、D、E组表达明显减少,B组的转染效率明显高于C、D、E组[分别为(77.63±1.74)%、(59.50±1.98)%、(45.10±2.28)%、(25.13±3.20)%],差异有统计学意义(F=1 321.74,P<0.01)。结论碘造影剂明显影响腺病毒对脂肪干细胞的转染效率,且随着造影剂剂量的增加,转染效率逐渐降低。

Objective To investigate the effect of iodine contrast agent on the transfection efficiency of rat adipose derived stem cells（ADSCs） transfected with adenovirus（Ad）.Methods From December 2015 to January 2016 at the Wuhan University Institute of cardiovascular diseases carried on this study.In vitro cultured rat ADSCs,ADSCs gene was transfected with recombinant adenovirus vector（Ad-TBX18-GFP） with TBX18 gene.According to whether the addition of iodine contrast agent will be sorted,fat stem cells were divided into blank control group（group A）,adenovirus transfection group（group b）,transfected with adenovirus＋contrast agent 0.1 ml group（Group C）,adenovirus transfection＋contrast agent0.25 ml（Group D） group,transfected with adenovirus＋contrast agent 0.5 ml group（Group E）.The expression of green fluorescent protein（GFP） in target cells was observed under fluorescence microscope,and the percentage of average positive expression cells in each field of view was judged by the rate of gene transfection efficiency.Results（1） The optimal MOI of adenovirus transfection ADSCs was 100.（2）Except for group A,the rest of the group under the fluorescence microscope with visible green fluorescence and group B with green fluorescent protein expressed the most,C,D,E group significantly reduced the expression,transfection efficiency in group B was significantly higher than that of C,D,E group[respectively（77.63 ±1.74）%,（5.95±1.98）%,（45.10 ±2.28）%,（25.13±3.20）%,the difference were statistically significant（F=1 321.74,P0.01）].Conclusion The effect of adenovirus on the transfection efficiency of the fat stem cells was obviously affected by the iodine contrast agent,and the transfection efficiency was gradually decreased with the increase of the dose of contrast agent.