微泡造影剂结合超声介导一氧化氮合成酶基因转染血管平滑肌细胞

摘要

目的 探讨微泡造影剂及超声辐照介导内皮型一氧化氮合成酶(eNOS)基因转染体外培养大鼠血管平滑肌细胞(VSMCs)的可行性.方法 实验分为A、B、C、D、E5组,分别为空白对照组、单纯质粒浸泡组、质粒+微泡造影剂组、质粒+超声辐照组、质粒+超声辐照+造影剂组.用重组真核表达载体pcDNA3.1- eNOS经微泡造影剂及超声辐照介导转染体外培养的大鼠VSMCs,辐照条件为超声探头频率为10 MHz,机械指数为1.9,辐照时间为10 min,重组真核细胞表达载体pcDNA3.1-eNOS 5μg/mL.辐照48 h后,采用RT-PCR、Western blot及免疫组化检测VSMCs内eNOSmRNA和蛋白的表达.结果 RT -PCR检测E组eNOS mRNA的表达最显著,积分吸光度(IA)比值为(91.11±3.41)％,B、C、D组也有少量表达,IA比值分别为(26.10±1.32)％、(31.42±2.43)％、(35.05±2.25)％,与E组相比均P＜0.05.蛋白印迹分析eNOS蛋白表达,A组有极少量表达,B、C、D组也有少量表达,IA比值分别为(22.12±1.33)％、(25.42±2.41)％、(33.11±3.11)％,而E组表达最明显,IA比值为(84.22±9.22)％,与各组相比均P＜0.05.结论 超声辐照结合微泡造影剂能显著提高eNOS基因在VSMCs的转染效率,提高细胞内一氧化氮合成酶的表达.%Objective To investigate the feasibility and efficacy of ultrasound and microbubble contrast agent-mediated transfection of endothelial nitric oxide synthase into vascular smooth muscle cells(VSMCs)in rats. Methods Five groups were designed:blank control group(A) .simple plasmid immersion group(B) ,plasmid+microbubble contrast agent group(C) ,plasmid + ultrasonic irradiation group(D) ,plasmid+ultrasonic irradiation+microbubble contrast agent group(E). The rat VSMCs were exposed to the diagnostic ultrasonography for 10 min in vitro in the presence or absence of ultrasound microbubble contrast a-gent,and the ultrasonic probe frequency was 10 MHz,mechanical index was 1. 9,and the plasmid of pcDNA3. 1-eNOS concentration in the solution was 5 μg/mL. RT-PCR and Western blot were used to detect the eNOS mRNA and protein expression in VSMCs after 48 h of the exposure. Results The strongest expression of eNOS mRNA was detected in group E with the integral absorbance(IA)ratio being(91. 11 ± 3. 41) %. There was weak expression of eNOS mRNA in groups B,C and D with IA ratio being(26. 10 ± 1. 32) %, (31. 42 ± 2. 43)%, and (35. 05 ± 2. 25)% respectively. There was significant difference in IA ratio between group E and groups B,C,D(P<0. 05). There was very little expression of eNOS protein expression in group A,weak expression in groups B,C and D with IA ratio being(22. 12±1. 33) %, (25. 42 ± 2. 41) % and(33. 11 ± 3. 11)%, and strong expression in group E with IA ratio being(84. 22 ± 9. 22) %, P<0. 05. Conclusion The ultrasound combined with microbubble contrast agent can significantly improve the eNOS transfection efficiency in VSMCs, and increase the expression of eNOS in VSMCs.