【摘要】：目的探討應(yīng)用siRNA技術(shù)沉默EPAS1(HIF-2α)基因表達(dá)對(duì)低氧條件下培養(yǎng)的大鼠肺動(dòng)脈平滑肌細(xì)胞(PASMCs)增殖的影響。方法原代培養(yǎng)大鼠PASMCs共采用免疫熒光法進(jìn)行鑒定。構(gòu)建特異性EPAS1 siRNA脂質(zhì)體,轉(zhuǎn)染PASMCs,從3個(gè)干擾靶點(diǎn)中選取效果最好的靶點(diǎn)進(jìn)行干擾;在常氧(37℃、5%CO_2、20%O_2)和低氧(37℃、5%CO_2、2%O_2)條件下分別培養(yǎng)24、48、72h,采用Western blotting檢測(cè)PASMCs中EPAS1、VEGF蛋白的表達(dá)水平,CCK-8法檢測(cè)細(xì)胞增殖情況。結(jié)果成功分離培養(yǎng)原代大鼠PASMCs并經(jīng)免疫熒光鑒定證實(shí)。將特異性的EPAS1 siRNA脂質(zhì)體轉(zhuǎn)染到PASMCs,選擇干擾效果最好的靶點(diǎn)2進(jìn)行干擾。與常氧條件比較,低氧條件下PASMCs中VEGF蛋白的表達(dá)及細(xì)胞增殖水平均明顯升高;沉默EPAS1后,無(wú)論低氧還是常氧條件下,PASMCs中VEGF蛋白的表達(dá)及細(xì)胞增殖水平均明顯降低。結(jié)論 EPAS1基因參與了低氧條件下大鼠PASMCs增殖的調(diào)控,其調(diào)節(jié)可能是通過(guò)VEGF介導(dǎo)完成的。
[Abstract]:Objective to investigate the effect of silencing EPAS1 (HIF-2 偽) gene expression by siRNA technique on the proliferation of rat pulmonary artery smooth muscle cells (PASMC) cultured in hypoxia. Methods the primary cultured rat PASMCs was identified by immunofluorescence assay. The specific EPAS1 siRNA liposomes were constructed and transfected into PASMCs, to select the most effective target for interference. The expression level of EPAS1,VEGF protein in PASMCs was detected by Western blotting and the proliferation of cells was detected by CCK-8 method in normal oxygen (37 鈩，

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