本文選題：自發(fā)性高血壓大鼠　切入點：1-磷酸鞘氨醇受體3　出處：《西南醫(yī)科大學》2017年碩士論文

【摘要】：目的:篩選出能夠高效特異性沉默自發(fā)性高血壓大鼠(spontaneously hypertensive rats,SHR)陰莖海綿體平滑肌細胞1-磷酸鞘氨醇受體3(sphingosine-1-phosphate receptor 3,S1PR3)基因表達的siRNA慢病毒載體,并觀察其對SHR陰莖海綿體平滑肌細胞ROCK1、ROCK2、eNOS表達的影響[1]。方法:以大鼠S1PR3基因mRNA序列作為干擾靶點,設(shè)計并合成3對靶向S1PR3的siRNA序列(siRNA1、siRNA2、siRNA3)及1對陰性對照序列,構(gòu)建并包裝成慢病毒載體[1]。體外培養(yǎng)SHR陰莖海綿體平滑肌細胞及魏-凱二氏大鼠(Wistar-Kyoto rats,WKY)陰莖海綿體平滑肌細胞,隨機分為A組(SHR對照組)、B組(攜帶陰性對照病毒的SHR陰莖海綿體平滑肌細胞轉(zhuǎn)染組)、C~E組(分別攜帶靶向S1PR3基因siRNA1~3號靶點慢病毒的SHR陰莖海綿體平滑肌細胞轉(zhuǎn)染組)、F組(WKY對照組),以感染復(fù)數(shù)(MOI)=60轉(zhuǎn)染SHR陰莖海綿體平滑肌細胞,轉(zhuǎn)染72h后觀察細胞綠色熒光蛋白(green fluorescent protein,GFP)的表達情況,并用RT-PCR和Western blotting檢測轉(zhuǎn)染后細胞中S1PR3、ROCK1、ROCK2、eNOS mRNA及蛋白的表達情況[1]。結(jié)果:經(jīng)基因測序證明合成的攜帶S1PR3基因siRNA 1~3號靶點的寡核苷酸鏈序列插入正確。熒光顯微鏡下觀察發(fā)現(xiàn)攜帶靶向S1PR3的siRNA慢病毒載體及陰性對照病毒載體均能有效轉(zhuǎn)染SHRCCSM細胞,且轉(zhuǎn)染效率均80%[1]。C、D、E、F組的S1PR3、ROCK1、ROCK2 mRNA及蛋白的表達較A組下降(p0.05),其中E組抑制作用最為明顯,使S1PR3基因mRNA及蛋白表達的抑制效率分別為(34.2±2.9)%、(77.7±4.7)%;ROCK1基因mRNA及蛋白表達的抑制效率分別為(33.3±1.4)%、(51.1±7.3)%;ROCK2基因mRNA及蛋白表達的抑制效率分別為(30.8±3.6)%、(58.32±5.5)%。S1PR3、ROCK1、ROCK2 mRNA及蛋白的表達在A組與B組之間均無明顯改變,A組eNOS基因mRNA及蛋白表達分別與B、C、D、E比較,無明顯差別,但較F組顯著降低(p0.01);與F組比較,E組S1PR3、ROCK1、ROCK2 mRNA及蛋白的表達均無明顯差別,A、B、C、D組的S1PR3、ROCK1、ROCK2基因mRNA及蛋白表達高于F組(p0.05),A、B、C、D、E組eNOS基因mRNA及蛋白表達較F組顯著降低(p0.01)[1]。結(jié)論:本研究構(gòu)建的3條攜帶不同位點的S1PR3基因的siRNA慢病毒載體均能夠顯著抑制SHR陰莖海綿體平滑肌細胞S1PR3基因的表達,并能有效的抑制SHR陰莖海綿體平滑肌細胞中上調(diào)的RhoA/Rho激酶信號通路,其中攜帶siRNA3慢病毒載體的抑制效率最高[1]。
[Abstract]:Objective: to screen out the efficient silencing of spontaneously hypertensive rats (spontaneously hypertensive, rats, SHR) corpus cavernosum smooth muscle cells of sphingosine 1-phosphate receptor (sphingosine-1-phosphate 1- 3 receptor 3, S1PR3) of the lentiviral vector of siRNA gene expression, and to observe the ROCK2 of SHR ROCK1, corpus cavernosum smooth muscle cells, the expression of eNOS: [1]. method. The rat S1PR3 gene mRNA sequence as target, designed and synthesized 3 pairs of siRNA targeting sequence of S1PR3 (siRNA1, siRNA2, siRNA3) and 1 negative control sequences, constructed and packaged into lentivirus vector [1]. in vitro SHR penile corpus cavernosum smooth muscle cells and Wei - Kay's rats (two Wistar-Kyoto rats. WKY) corpus cavernosum smooth muscle cells, were randomly divided into A group (SHR group), B group (SHR corpus cavernosum smooth muscle cells in transfection group carrying negative control virus group (C~E), respectively, articles SHR corpus cavernosum smooth muscle cells in transfection group with S1PR3 gene targeted siRNA1~3 targeting lentivirus), F group (WKY group), with the multiplicity of infection (MOI) in =60 transfected SHR cells was observed in corpus cavernosum smooth muscle cells transfected with green fluorescent protein 72h (green fluorescent protein, GFP) expression, and RT-PCR and Western blotting detection of transfected cells in S1PR3, ROCK1, ROCK2, eNOS, mRNA and the expression of [1]. protein by gene sequencing proved that the synthesis of oligonucleotides carrying S1PR3 gene sequence of siRNA 1~3 target. Insert the correct found with targeting siRNA lentiviral vector and negative S1PR3 virus vector can effectively control transfection of SHRCCSM cells under the fluorescence microscope, and the transfection efficiency were 80%[1].C, D, E, F and S1PR3 in group ROCK1, the expression of ROCK2 protein and mRNA decreased compared with the A group (P0.05), the inhibition of E group is the most obvious. S1PR3鍩哄洜mRNA鍙婅泲鐧借〃杈劇殑鎶戝埗鏁堢巼鍒嗗埆涓，

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