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目的:探讨新型阳离子磷酸胆碱聚合物MPC30-DEA70(PC)载MiRNA-195反义寡核苷酸(AMOMiRNA-195)转染心肌细胞及其对心肌细胞肥厚的影响。方法:应用琼脂糖凝胶电泳表征具有不同比值的MPC30-DEA70与AMO-MiRNA-195的复合物,将不同比例的MPC30-DEA70/AMO-MiR-195基因复合物(PC复合物)转染至体外培养的乳鼠心肌细胞。应用倒置荧光显微镜(FMI)及激光共聚焦扫描显微镜(CLSM)观察MPC30-DEA70/AMO-MiR-195(FAM标记)在细胞内的分布及定位;流式细胞仪(FCM)检测转染效率及荧光强度;RT-PCR检测心肌细胞MiRNA-195的表达;RT-PCR法检测心肌肥厚标志物心房钠尿肽(ANP)、α-肌球蛋白重链(α-MHC)、β-肌球蛋白重链(β-MHC)编码基因Nppa、Myh6、Myh7基因的表达。结果:1不同比值的PC复合物在电泳中可见不同程度的电泳迟滞现象,随电性增强而显著;2FMI及CLSM观察到MPC30-DEA70/AMO-MiR-195(FAM标记)分布在细胞核周围,少量进入细胞核。3流式细胞术显示随N/P比值的增加,转染效率明显增大,荧光强度也随之增强;4RT-PCR法显示PC复合物可使MiRNA-195及心肌肥厚标志性基因Nppa、Myh7基因表达降低,且随着N/P比值的增大,MiRNA-195、Nppa及Myh7基因表达明显下降(P<0.05),Myh6基因表达无明显变化。结论:MPC30-DEA70可作为AMO-MiRNA-195的载体转染至体外培养的乳鼠心肌细胞,并下调MiRNA-195及心肌肥厚标志性基因Nppa、Myh7基因表达。
OBJECTIVE: To investigate the effect of AMOMiRNA-195 transfected cardiomyocytes loaded with novel cationic phosphocholine MPC30-DEA70 (PC) on cardiomyocyte hypertrophy. METHODS: Complexes of different ratios of MPC30-DEA70 and AMO-MiRNA-195 were characterized by agarose gel electrophoresis and different ratios of MPC30-DEA70 / AMO-MiR-195 gene complex (PC complex) In vitro cultured neonatal rat cardiomyocytes. The distribution and localization of MPC30-DEA70 / AMO-MiR-195 (FAM marker) in cells were observed by inverted fluorescence microscope (FMI) and confocal laser scanning microscopy (CLSM). The transfection efficiency and The expression of MiRNA-195 in cardiomyocytes was detected by RT-PCR. The levels of atrial natriuretic peptide (ANP), α-MHC, β-myosin Heavy chain (β-MHC) gene encoding Nppa, Myh6, Myh7 gene expression. Results: 1 Different ratios of PC complexes showed different degree of electrophoretic hysteresis in electrophoresis, which was significant with the increase of electrical conductivity. MPC30-DEA70 / AMO-MiR-195 (FAM marker) A small amount into the nucleus. 3 Flow cytometry showed that with the increase of N / P ratio, the transfection efficiency was significantly increased, and the fluorescence intensity was also enhanced. 4RT-PCR method showed that PC complex can make MiRNA-195 and cardiac hypertrophy markers Nppa, Myh7 The mRNA expression of MiRNA-195, Nppa and Myh7 decreased significantly (P <0.05), while the expression of Myh6 gene did not change significantly with the increase of N / P ratio. CONCLUSION: MPC30-DEA70 can be transfected into neonatal rat cardiomyocytes as a vector of AMO-MiRNA-195 and down-regulate gene expression of MiRNA-195 and myocardial marker gene Nppa and Myh7.