HIF-1α小干扰RNA对骨髓间充质干细胞HIF-1α、SDF-1α和VEGF基因表达的影响

发布时间：2018-08-20 09:01

【摘要】： 第一部分MSCs的培养及HIF-1αsiRNA的筛选目的:贴壁法培养骨髓间充质干细胞(MSCs,mesenchymal stem cells), RT-PCR法筛选抑制效果最强的RNA干扰序列。方法:贴壁法培养MSCs,取第3-5代细胞用于实验,倒置显微镜下观察细胞形态,并用流式细胞仪检测其表面标志物CD11b/c、CD34、CD44、CD90。将MSCs分为五组,单纯缺氧组(未经任何干预)、脂质体组(转染空载的脂质体)、siRNA609组(转染siRNA609)、siRNA658组(转染siRNA658)、siRNA2070组(转染siRNA2070)。将以上五组细胞在缺氧条件下培养24h,RT-PCR检测其HIF-1α的基因表达。结果:1.流式细胞仪检测CD11b/c阴性、CD34阴性、CD44阳性、CD90阳性细胞分别为84.2%±0.2%、97.91%±0.7%、99.8%±0.9%、97.7%±0.4%,CD44+/CD34-细胞数为99.4%±0.4%。2.转染siRNA细胞内均能检测到绿色荧光信号,lipsome组和空白对照组未见到绿色荧光信号。3.RT-PCR结果显示,与单纯缺氧组相比siRNA609、siRNA658、siRNA2070组HIF-1α的基因表达明显降低(P 0.05),其中siRNA658组抑制效果最强(P 0.05)。结论:1.培养的细胞表达MSCs相应的表面标记物。2.MSCs可以转染HIF-1αsiRNA序列。3. siRNA658干扰序列抑制效果最强。第二部分HIF-1α小干扰RNA对MSCs的HIF-1α、SDF-1α和VEGF基因表达的影响目的:HIF-1α小干扰RNA对MSCs HIF-1α、基质细胞衍生因子-1α(SDF-1α,stromal derived factor 1α)和血管内皮生长因子(VEGF,Vascular endothelial growth factor)基因表达的影响。方法:将MSCs四组,常氧对照组(无干预因素)、缺氧组(缺氧24h)、脂质体对照组(转染空载脂质体后缺氧24h)、RNA干扰组(转染脂质体介导的RNA干扰序列后缺氧24h)。RT-PCR法检测MSCs的HIF-1α、SDF-1α和VEGF mRNA表达水平, ELISA检测MSCs培养上清HIF-1α、SDF-1α和VEGF蛋白表达水平,CCK8检测MSCs培养上清对大鼠平滑肌细胞增殖的影响。结果:1.RT-PCR显示,同常氧对照组相比缺氧组HIF-1α、SDF-1α、VEGF基因表达增高(P 0.05),同脂质体对照组相比RNA干扰组HIF-1α、SDF-1α、VEGF基因表达降低。2.ELISA检测发现,同常氧对照组相比缺氧组条件培养液中的HIF-1α、SDF-1α、VEGF含量增加(P 0.05),同脂质体对照组相比RNA干扰组HIF-1α、SDF-1α、VEGF含量降低(p0.05)。3.CCK8检测发现,同常氧对照组的相比缺氧组条件培养上清可以刺激平滑肌细胞增殖(P 0.05),同脂质体对照组相比RNA干扰组缺氧培养上清促进增殖的能力减弱(P 0.05)。结论:1.抑制HIF-1α表达可以使MSCs的SDF-1α和VEGF基因表达降低。2.缺氧可以使HIF-1α,SDF-1α和VEGF基因表达增高。3.HIF-1α是MSC调控SDF-1α和VEGF基因表达的主要因素之一。4.RNA干扰可以降低缺氧培养上清对大鼠平滑肌细胞的促增殖作用。
[Abstract]:Part one: culture of MSCs and screening of HIF-1 伪 siRNA objective: to screen the most effective RNA interference sequence of bone marrow mesenchymal stem cells (MSCs / mesenchymal stem cells), RT-PCR) cultured by adherent method. Methods: MSCs were cultured by adherent method. The cells of the 3-5 passage were used in the experiment. The morphology of the cells was observed under inverted microscope, and the surface marker CD11b / cpCD34, CD444 and CD90 were detected by flow cytometry. MSCs was divided into five groups: hypoxia group (without any intervention), liposome group (transfection of empty liposome) siRNA609 group (transfected siRNA609), siRNA658 group (transfected siRNA658) and siRNA2070 group (transfected siRNA2070). The gene expression of HIF-1 伪 was detected by RT-PCR in the above five groups cultured in anoxic condition for 24 h. The result is 1: 1. Flow cytometry showed that the positive cells of CD44 and CD44 were 84.2% 卤0.2% and 97.91% 卤0.710% respectively by flow cytometry. The number of CD44 / CD34- cells was 99.4% 卤0.40.41% 卤0.97% 卤0.97%. The results of RT-PCR showed that the gene expression of HIF-1 伪 in siRNA609 siRNA658siRNA2070 group was significantly lower than that in hypoxic group (P 0.05), and the inhibitory effect of siRNA658 group was the highest (P 0.05). Conclusion 1. The cultured cells expressed the corresponding surface marker of MSCs. 2. MSCs could transfect HIF-1 伪 siRNA sequence. 3. SiRNA658 interference sequence had the strongest inhibitory effect. The second part is the effect of HIF-1 伪 small interfering RNA on the expression of HIF-1 伪 -SDF-1 伪 and VEGF gene in MSCs. Objective to investigate the effects of small interfering RNA on the expression of MSCs HIF-1 伪, SDF-1 伪 -stromal derived factor 1 伪 and vascular endothelial growth factor) in MSCs. Methods: four groups of MSCs, Normoxic control group (no intervention factor), hypoxia group (hypoxia 24 h), liposome control group (hypoxia 24 h after transfection of no-loaded liposome) RNA interference group (hypoxia 24 h after transfection of liposome mediated RNA interference sequence) .RT-PCR method to detect HIF-1 伪 -SDF-1 伪 and VEGF mRNA expression water in MSCs The expression levels of HIF-1 伪 -SDF-1 伪 and VEGF protein in the supernatant of MSCs culture were detected by ELISA and the effect of MSCs supernatant on the proliferation of rat smooth muscle cells was detected by CCK8. Results 1. RT-PCR showed that the expression of HIF-1 伪 -SDF-1 伪 mRNA in hypoxia group was higher than that in normoxic control group (P0. 05), and that in RNA interference group was lower than that in liposome control group. Compared with the normoxic control group, the content of HIF-1 伪 -SDF-1 伪 -VEGF in conditioned medium of hypoxia group was increased (P 0.05), and the content of HIF-1 伪 -SDF-1 伪 -VEGF in RNA interference group was lower than that in liposome control group (p0.05). Compared with the normoxic control group, the conditioned supernatant of hypoxia group could stimulate the proliferation of smooth muscle cells (P0. 05), and the supernatant of hypoxia culture supernatant of RNA interference group was weaker than that of liposome control group (P 0. 05). Conclusion 1. Inhibiting the expression of HIF-1 伪 could decrease the expression of SDF-1 伪 and VEGF genes in MSCs. Hypoxia can increase the expression of HIF-1 伪 -SDF-1 伪 and VEGF genes. 3.HIF-1 伪 is one of the main factors of MSC regulating the expression of SDF-1 伪 and VEGF genes. 4. RNA interference can reduce the proliferation of rat smooth muscle cells induced by hypoxia culture supernatant.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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