靶向S1P2基因的siRNA有效片段的慢病毒载体筛选

发布时间：2018-05-11 17:51

  本文选题：自发性高血压大鼠 + S1P2　； 参考：《西南医科大学》2017年硕士论文

【摘要】：目的:筛选出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,观察S1P2基因沉默后对RhoA/Rho激酶信号通路的影响。方法:SHR及SD大鼠各5只采用组织块贴壁法进行原代培养阴茎海绵体平滑肌细胞并采用差速贴壁法纯化,将纯化后的细胞随机分成6组,每组5个样本,每组每个样本1.0×105个细胞,分别为:A组、B组、C组(分别携带靶向S1P2基因的siRNA-1、2、3号靶点的SHR慢病毒转染组)、D组(携带慢病毒空载体SHR转染组)、E组(SHR未转染病毒对照组)、F组(SD大鼠对照组)。将靶向S1P2的siRNA-1、2、3号片段的慢病毒载体及携带慢病毒的空载体,以感染复(MOI)=60分别转染A组、B组、C组、D组SHR阴茎海绵体平滑肌细胞,转染72 h后于荧光显微镜下观察细胞内荧光表达情况,用Western印迹分析S1P2、ROCK1、ROCK2、eNOS在各组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中S1P2、ROCK1、ROCK2的mRNA的表达情况。结果:荧光显微镜下观察A组、B组、C组、D组细胞转染效率均80%;与E组mRNA、蛋白表达[S1P2(0.874±0.047)、(0.690±0.122);ROCK1(0.819±0.055)、(0.731±0.134);ROCK2(0.854±0.098)、(0.852±0.128)]相比D组[S1P2(0.905±0.091)、(0.701±0.155);ROCK1(0.871±0.073)、(0.774±0.135);ROCK2(0.857±0.131)、(0.825±0.115)]均无明显差距,A组[S1P2(0.560±0.169)、(0.238±0.027);ROCK1(0.630±0.059)、(0.421±0.160);ROCK2(0.614±0.127)、(0.486±0.189)]、B组[S1P2(0.731±0.126)、(0.501±0.056)、ROCK1(0.726±0.057)、(0.567±0.134)、ROCK2(0.724±0.146)、(0.652±0.119)]、C组[S1P2(0.624±0.008)、(0.431±0.083);ROCK1(0.702±0.051)、(0.568±0.117);ROCK2(0.649±0.166)、(0.600±0.102)]、F组[S1P2(0.540±0.167)、(0.147±0.046);ROCK1(0.511±0.152)、(0.291±0.107);ROCK2(0.568±0.068)、(0.474±0.207)]均显著低于E组(P0.05)。而e NOS蛋白表达,A组(0.495±0.156)、B组(0.535±0.098)、C组eNOS(0.536±0.112)、D组(0.551±0.165)与E组(0.518±0.155)相比均无显著差别,但F组(0.888±0.069)显著高于E组。结论:SHR的阴茎海绵体平滑肌细胞中S1P2基因表达上调,激活RhoA/Rho激酶信号通路。针对大鼠S1P2基因设计的三组siRNA慢病毒载体转染至SHR大鼠阴茎海绵体平滑肌细胞后均能显著抑制SHR阴茎海绵体平滑肌内S1P2的表达,下调ROCK1、ROCK2表达,其中靶向S1P2基因的siRNA-1的抑制效率最高。
[Abstract]:Objective: to screen out the lentivirus vector of siRNA that can significantly inhibit the expression of 2(sphingosine-1-phosphate receptor 2P S1P2gene in the smooth muscle cells of the cavernous body of primary cultured spontaneously hypertensive rats. To observe the effect of S1P2 gene silencing on RhoA/Rho kinase signaling pathway. Methods the primary cultured smooth muscle cells (SMC) of cavernous corpus cavernosum were cultured by tissue mass adherence method and purified by differential adherent method. The purified cells were randomly divided into 6 groups, 5 samples in each group, 1.0 脳 105 cells in each group. They were divided into two groups: group C (siRNA-1m2with specific S1P2 gene) and group D (group D with SHR lentivirus transfected with lentivirus empty vector SHR). The lentivirus vector targeting siRNA-1m2and 3 fragment of S1P2 and the empty vector carrying lentivirus were transfected into SHR cavernous smooth muscle cells of SHR in group A and C respectively. After 72 hours of transfection, the intracellular fluorescence expression was observed under fluorescence microscope, and the expression of S1P _ 2-rock _ (1) rock _ (2) and Enos in the smooth muscle cells of the cavernous body of the penis was analyzed by Western blot. The expression of S1P _ (2) ~ (2) ~ (2) ROCK1 ~ (1) roCK2 mRNA was detected by RT-PCR in the smooth muscle cells of the cavernous body of the penis in each group. 缁撴灉:鑽у厜鏄惧井闀滀笅瑙傚療A缁，

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