發(fā)布時間：2018-05-11 17:51

  本文選題：自發(fā)性高血壓大鼠 + S1P2�。� 參考：《西南醫(yī)科大學》2017年碩士論文

【摘要】：目的:篩選出能顯著抑制原代培養(yǎng)的自發(fā)性高血壓大鼠(Spontaneously hypertensive rats,SHR)陰莖海綿體平滑肌細胞內1-磷酸鞘氨醇受體2(sphingosine-1-phosphate receptor 2,S1P2)基因表達的siRNA慢病毒載體,觀察S1P2基因沉默后對RhoA/Rho激酶信號通路的影響。方法:SHR及SD大鼠各5只采用組織塊貼壁法進行原代培養(yǎng)陰莖海綿體平滑肌細胞并采用差速貼壁法純化,將純化后的細胞隨機分成6組,每組5個樣本,每組每個樣本1.0×105個細胞,分別為:A組、B組、C組(分別攜帶靶向S1P2基因的siRNA-1、2、3號靶點的SHR慢病毒轉染組)、D組(攜帶慢病毒空載體SHR轉染組)、E組(SHR未轉染病毒對照組)、F組(SD大鼠對照組)。將靶向S1P2的siRNA-1、2、3號片段的慢病毒載體及攜帶慢病毒的空載體,以感染復(MOI)=60分別轉染A組、B組、C組、D組SHR陰莖海綿體平滑肌細胞,轉染72 h后于熒光顯微鏡下觀察細胞內熒光表達情況,用Western印跡分析S1P2、ROCK1、ROCK2、eNOS在各組陰莖海綿體平滑肌細胞內的表達變化,RT-PCR檢測各組陰莖海綿體平滑肌細胞中S1P2、ROCK1、ROCK2的mRNA的表達情況。結果:熒光顯微鏡下觀察A組、B組、C組、D組細胞轉染效率均80%;與E組mRNA、蛋白表達[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組[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)]均無明顯差距,A組[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組[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組[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組[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)]均顯著低于E組(P0.05)。而e NOS蛋白表達,A組(0.495±0.156)、B組(0.535±0.098)、C組eNOS(0.536±0.112)、D組(0.551±0.165)與E組(0.518±0.155)相比均無顯著差別,但F組(0.888±0.069)顯著高于E組。結論:SHR的陰莖海綿體平滑肌細胞中S1P2基因表達上調,激活RhoA/Rho激酶信號通路。針對大鼠S1P2基因設計的三組siRNA慢病毒載體轉染至SHR大鼠陰莖海綿體平滑肌細胞后均能顯著抑制SHR陰莖海綿體平滑肌內S1P2的表達,下調ROCK1、ROCK2表達,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. 緇撴灉:鑽у厜鏄懼井闀滀笅瑙傚療A緇，

本文編號：1874959