摘要
目的:筛选能显著抑制原代培养的自发性高血压大鼠(spontaneously hypertensive rats,SHR)阴茎海绵体平滑肌细胞内1-磷酸鞘氨醇受体2(sphingosine-1-phosphate receptor 2,S1P2)基因表达的siRNA慢病毒载体。方法:SHR及SD大鼠各5只用于原代培养阴茎海绵体平滑肌细胞并分成6组,每组5个样本,每个样本约1.0×105个细胞,分别为携带靶向S1P2基因的siRNA-1~3号靶点的SHR慢病毒转染组(SHR siRNA-1、SHR siRNA-2和SHR siRNA-3组)、携带慢病毒空载体的SHR转染组(SHR GFP组)、SHR未转染对照组(SHR control组)和SD大鼠对照组(SD control组)。将靶向S1P2的siRNA片段的慢病毒载体,以感染复数(MOI)=60转染各组SHR阴茎海绵体平滑肌细胞,于转染72 h后荧光显微镜下观察细胞荧光表达情况,用Western blot分析各组S1P2、ROCK1、ROCK2和eNOS在阴茎海绵体平滑肌细胞内的表达变化,RT-PCR检测各组阴茎海绵体平滑肌细胞S1P2、ROCK1和ROCK2的mRNA的表达。结果:荧光显微镜下观察SHR siRNA-1、SHR siRNA-2、SHR siRNA-3和SHR GFP组细胞转染效率均>80%;SHR GFP组与SHR control组相比,S1P2、ROCK1和ROCK2的mRNA和蛋白表达无明显差异,SHR siRNA-1、SHR siRNA-2、SHR siRNA-3和SD control组均显著低于SHR control组(P<0.05)。而SHR siRNA-1、SHR siRNA-2、SHR siRNA-3和SHR GFP组eNOS蛋白表达与SHR control组相比均无显著差别,但SD control组显著高于SHR control组(P<0.05)。结论:3组针对S1P2的siRNA慢病毒载体均能显著抑制SHR阴茎海绵体平滑肌细胞内S1P2的表达,下调ROCK1和ROCK2表达,其中靶向S1P2的siRNA-1抑制效率最高。
AIM： To screen the lentiviral vector carrying siRNA with higher efficiency of suppressing the sphingosine-1-phosphate receptor 2 （ S 1P 2 ） gene expression in the primarily cultured corpus cavernosum smooth muscle cells of spontaneously hypertensive rats （SHR）. METHODS： SHR and SD rats （n=5 each） were used for primarily cul-turing corpus cavernosum smooth muscle cells. The cells were randomly divided into 6 groups ： SHR siRNA-1, SHR siRNA-2, SHR siRNA-3 , SHR GFP, SHR control （ SHR non-transfection group） , and SD control （ SD rat control group）.Each group had 5 samples with 1.0 x 105 cells of each sample. At 72 h after transfection （ MOI = 60 ） with lentiviral vectors carrying S1P2 siRNA into the SHR corpus cavernosum smooth muscle cells, the expression of GFP was observed under fluorescence microscope. The protein expression of S1P2, ROCK1, ROCK2 and eNOS in the corpus cavernosum smooth muscle cells, and the mRNA expression of S1P2, ROCK1 and ROCK2 were determined by by Western blot and RT-PCR. RESULTS ： The transfection efficiency of the corpus cavernosum smooth muscle cells in SHR siRNA-1, SHR siRNA-2,SHR siRNA-3 and SHR GFP groups were 〉80%. Compared with SHR control group, the mRNA levels and the protein expression of S1P2, ROCK1 and ROCK2 in SHR GFP group showed no remarkable changes, while those in SHR siRNA-1, SHR siRNA-2, SHR siRNA-3 and SD control groups were significantly lower than those in SHR control group （P 〈 0. 05 ） . The protein expression of eNOS in SHR siRNA-1, SHR siRNA-2, SHR siRNA-3 and SHR GFP groups were not significant-ly changed as compared with SHR control group, but that in SD control group was significantly higher than that in SHR con-trol group. CONCLUSION： Three groups of siRNA lentiviral vectors targeting S1P2 inhibit the expression of S1P2 in the corpus cavernosum smooth muscle cells of SHR, and by silencing the S1P2 expression, the expression of ROCK1 and ROCK2 is inhibited. Among them,
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2017年第7期1338-1344,共7页
Chinese Journal of Pathophysiology
基金
四川省科技厅基金资助项目(No.14JC0803)
四川省教育厅基金资助项目(No.15ZA0164)
四川省人力资源和社会保障厅留学回国人员科技项目(川人社办发[2016-64]号)
