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

Transfection of Nitric Oxide Synthase Gene into Vascular Smooth Muscle Cells Mediated by Ultrasound Combined with Microbubble Contrast Agent

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、E 5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10MHz,机械指数为1.9,辐照时间为10min,重组真核细胞表达载体pcDNA3.1-eNOS 5μg/mL。辐照48h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 agent,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（P0.05）.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）%,P0.05.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.