【作者】 刘江；

【导师】 吴延庆；

【作者基本信息】 南昌大学 ， 内科学， 2011， 硕士

【摘要】 目的：研究热休克蛋白22(Heat shock protein 22, HSP22)对缺氧/复氧(hypoxia/ reoxygenation H/R)损伤人脐静脉内皮细胞(HUVECs)的保护作用,并探讨其机制。方法：实验分为正常对照组、脂质体(Lipo)组、空质粒转染对照组(空白质粒组)和目的基因转染组(RNAi组)4组,Lipo组在HUVECs中仅加入Lipo,空白质粒组在HUVECs中加入Lipo和空质粒(pGcsi-U6/Neo/GFp/shRNA), RNAi组在HUVECs中加入Lipo和目的基因(pGcsi-U6/Neo/GFp/shRNA/HSP22)。经G418筛选各组细胞后,分别于第2d,10d,20d,30d在荧光显微镜下观察转染情况。各组HUVECs缺氧24h/复氧Oh后,Western blot印迹检测HSP22蛋白表达情况。再将正常对照组、空白质粒组和RNAi组先予缺氧24h处理后分别复氧Oh、3h、6h、12h,采用MTT检测细胞生长抑制情况,RT-PCR和Western blot印迹检测Bcl-2和Caspase-3基因和蛋白表达情况,RT-PCR检测NF-κB和IK-Bα基因表达情况。结果：1.正常对照组、Lipo组、空白质粒组和RNAi组HUVECs缺氧24h/复氧Oh后,HSP22蛋白表达(0.38±0.05、0.43±0.03、0.45±0.06、0.20±0.02),RNAi组显著低于正常对照组、Lipo组和空白质粒组(P<0.05)；而正常对照组、Lipo组和空白质粒组之间HSP22蛋白表达无显著差异(P>0.05)。2.相同复氧时间点(0,3,6和12h)RNAi组细胞生长抑制率高于空白质粒组和正常对照组细胞抑制率(58.5±2.1% vs.41.6±5.4%,62.7±5.4% vs.45.6±3.7 %,65.4±8.4% vs.48.5±5.2%和68.6±6.7%vs.52.9±3.4%,P<0.051。3.相同复氧时间点(0,3,6和12h)RNAi组Bcl-2基因和蛋白表达均低于空白质粒组和正常对照组(1.85±0.06 vs.2.65±0.15,1.66±0.04 vs.2.35±0.08,1.09±0.05 vs.2.05±0.08,0.69±0.04 vs.1.75±0.09, P<0.05)和(0.54±0.04 vs.1.05±0.05, 0.32±0.05 vs.0.75±0.03,0.29±0.02 vs.0.55±0.03,0.13±0.04 vs.0.45±0.07,P<0.05)。4.相同复氧时间点(0h,3,6和12h)RNAi组Caspase-3基因和蛋白表达均高于空白质粒组和正常对照组(2.75±0.04 vs.1.75±0.07,2.96±0.04 vs.1.95±0.06, 3.59±0.05 vs.2.45±0.05,3.59±0.05 vs.2.77±0.09, P<0.05)和(3.59±0.05 vs.1.75±0.04, 6.18±0.24 vs.2.05±0.11,8.82±0.07 vs.2.65±0.12,9.36±0.11 vs.3.05±0.12,P<0.05)。5.相同复氧时间点(0h,3,6和12h)RNAi组NF-κB基因表达均高于空白质粒组和正常对照组。(2.54±0.03 vs.2.05±0.02,3.26±0.05vs.2.55±0.04,3.85±0.03 vs.3.12±0.05,4.34±0.06 vs.3.87±0.04, P<0.05);相同复氧时间点(0h,3,6和12h)RNAi组Iκ-Bα基因表达均低于空白质粒组和正常对照组(2.95±0.02 vs.3.65±0.04, 2.68±3.17 vs.3.05±0.04,1.75±0.03 vs.2.16±0.02,1.62±0.05 vs.1.89±0.04, P<0.05)。结论：1.H/R损伤诱导下HUVECs可出现HSP22的表达,构建RNAi质粒并转染至HUVECs可显著降低HSP22的表达。2.HSP22在H/R所致HUVECs损伤中发挥保护作用,其机制与上调Bcl-2和下调Capase-3抗凋亡；上调IK-Bα和下调NF-κB抗炎症有关。更多还原

【Abstract】 Objective:Probe to the role and underlying mechanism of Heat shock protein 22 (HSP22) in injury of HUVECs induced by hypoxia/reoxygenation (H/R).Methods:A RNAi plasmid of HSP22 gene (pGcsi-U6/Neo/GFp/shRNA/HSP22) was constructed and transfected into HUVECs to download the expression of HSP22 which could be up-regulated by stress such as heat and H/R. According to the different intervention method, HUVECs were divided into 4 groups, i.e. Control group, Lipo group, Blank control group (i.e.pGcsi-U6/Neo/GFp/shRNA transfection group) and RNAi group(i.e. pGcsi-U6/Neo/GFp/shRNA/HSP22 transfection group). And these cells were subjected to hypoxia for 24h followed by reoxygenation for 0,3, 6,12h. After intervention of H/R, the rate of HUVECs growth inhibition was assayed by MTT and the expression of IK-Ba, NF-κB, Bcl-2 and Caspase-3 were measured by RT-PCR and Western blot.Results:1.The expression of HSP22 protein in RNAi group was obviously lower than others(0.20±0.02 vs.0.33±0.03 vs.0.45±0.06 vs.0.48±0.05 respectively in reoxyenation for Oh,3,6,12h, P<0.05), and there was no obviously difference among the Control, Lipo and Blank control group (P>0.05)2. The rate of HUVECs growth inhibition in RNAi group were obviously higher than Blank control group at the same time point of reoxygenation (58.5±2.1% vs.41.6±5.4%,62.7±5.4% vs.45.6±3.7%,65.4±8.4% vs.48.5±5.2% and 68.6±6.7% vs.52.9±3.4% respectively in reoxyenation for Oh,3,6,12h, P<0.05)3.The expression of bcl-2 mRNA and protein in RNAi group were obviously lower than Blank control group at the same time point of reoxygenation(1.85±0.06 vs.2.65±0.15,1.66±0.04 vs.2.35±0.08,1.09±0.05 vs.2.05±0.08,0.69±0.04 vs.1.75±0.09 respectively in reoxyenation for Oh,3,6,12h, P<0.05) and (0.54±0.04 vs.1.05±0.05,0.32±0.05 vs.0.75±0.03,0.29±0.02 vs.0.55±0.03,0.13±0.04 vs.0.45±0.07 respectively in reoxyenation for Oh,3,6,12h, P<0.05).4. The expression of Caspase-3 mRNA and protein in RNAi group is obviously higher than the Blank control group at the same time point of reoxygenation. (mRNA expression:2.75±0.04 vs.l.75±0.07,2.96±0.04 vs.1.95±0.06, 3.59±0.05 vs.2.45±0.05,3.59±0.05 vs.2.77±0.09 respectively in reoxyenation for Oh, 3,6,12h, P<0.05)and (protein expression:3.59±0.05 vs.1.75±0.04,6.18±0.24 vs.2.05±0.11,8.82±0.07 vs.2.65±0.12,9.36±0.11 vs.3.05±0.12 respectively in reoxyenation for Oh,3,6,12h, P<0.05).5. The expression of NF-κB mRNA in Blank control group were obviously higher than the RNAi group at the same time point of reoxygenation.(2.54±0.03 vs.2.05±0.02,3.26±0.05vs.2.55±0.04,3.85±0.03 vs.3.12±0.05,4.34±0.06 vs.3.87±0.04, P<0.05). And the expression of Iκ-BαmRNA in Blank control group were obviously lower than the RNAi group at the same time point of reoxygenation (2.95±0.02 vs.3.65±0.04,2.68±3.17 vs.3.05±0.04,1.75±0.03 vs.2.16±0.02,1.62±0.05 vs.1.89±0.04 respectively in reoxyenation for Oh,3,6,12h, P<0.05).Conclusion:1. The expression of HSP22 could be induced by H/R intervention in HUVECs and inhibited by transfection of RNAi plasmid of HSP22 gene. The transfection of Lipo and control plasmid did not influent the express of HSP22 in HUVECs.2. HSP22 might play a protective role in the injury of HUVECs induced by H/R.3. The protective mechanism of HSP22 might involve in the up-regulateing of bcl-2 and down-regulateing of Caspase-3 which contribute to protecting HUVECs away from apoptosis induced by H/R.4. The protective mechanism of HSP22 also might involve in inhibition of inflammation which presented as up-regulateing of IK-Ba and down-regulateing of NF-κB.更多还原