论文部分内容阅读
目的:采用短发夹 RNA(shRNA)特异性沉默人鼻黏膜上皮细胞(HNEC)缺氧诱导因子-1α(HIF-1α)基因,观察对其表达血管内皮生长因子(VEGF)、转化生长因子(TGF)β1、碱性成纤维细胞生长因子(bFGF)的影响。方法体外培养原代鼻黏膜细胞,设计合成以腺病毒为载体GFP标记的shRNA ,将HNEC分为空白对照组、阴性对照组(ad-HIF shRNA-neg )、ad-HIF shRNA干扰组。采用RT-PCR和Western blotting技术检测转染后相应因子蛋白和mRNA的表达情况。结果成功合成ad-HIF shRNA并转染入 HNEC 中。转染ad-HIF shRNA后, HIF-1α蛋白及mRNA表达分别下降40%和39%(q =31.469,16.590);同时 VEGF、TGF-β1、bFGF 的蛋白及mRNA表达也随之下降,差异均有统计学意义(P<0.05)。结论 HIF-1αmRNA的shRNA能有效地使HNEC的HIF-1α基因沉默,进而有效地抑制VEGF、TGF-β1和bFGF的表达,提示HNEC中HIF-1α可能通过直接或间接途径调控着VEGF、bFGF和TGF-β1的表达。“,”Objective To investigate the effects of silencing hypoxia inducible factor-1α(HIF-1α)by transfecting short hairpin RNA(shRNA)into cultured human nasal epithelial cells (HNECs)and its further regulations on vascular endo-thelial growth factor (VEGF),transforming growth factor (TGF)β1 and basic fibroblast growth factor (bFGF). Methods HNECs were isolated from 10 inferior turbinate-cutting patients’mucosa.Two HIF-shRNA fragments were built and the best silencing shRNA sequence was used for transfection.We separated cells into three groups:the blank group,the negative control group and the ad-HIFshRNA interference group.The protein and gene expressions were detected by Western blotting and PCR.Results HNECs were transfected with GFP marketed adenovirus which carry the best silencing HIF-1αshRNA.The protein and mRNA expressions of HIF-1αwere significantly reduced 40% and 39%(q=31.469,16.590),and following,the protein and mRNA expressions of VEGF,TGF-β1 and bFGF were reduced (P<0.05).Conclusion Transfection of ad-HIFshRNA can effectively silence the expression of HIF-1αgene and protein and furthermore inhibit the expressions of VEGF,TGF-β1 and FGF-2,which suggests HIF-1αregulates the expression of VEGF,bFGF and TGF-β1 through direct or indirect ways.Thus silencing HIF-1αmay provide a new way towards allergic rhiniti’s treatment.