缺氧条件下大鼠肺动脉平滑肌细胞中活性氧对缺氧诱导因子-1α和细胞增殖作用的研究

发布时间：2018-01-27 05:01

本文关键词： 缺氧肺动脉平滑肌细胞活性氧缺氧诱导因子增殖　出处：《华中科技大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的 1.观察在缺氧条件下,大鼠肺动脉平滑肌细胞中活性氧(ROS)的变化趋势。 2.探讨在缺氧条件下,大鼠肺动脉平滑肌细胞中ROS的变化是否调控缺氧诱导因子-1apha(HIF-1α)的表达,进而影响平滑肌细胞的增殖。材料与方法本实验采用组织块贴壁法原代培养大鼠肺动脉平滑肌细胞(PASMCs),用胰蛋白酶消化法传代细胞,并将PASMCs随机分成3组:常氧组(21%O2,24小时,A组),缺氧组(5%O2,24小时,B组),缺氧+Mn-TBAP组(5%O2,24小时,C组, Mn-TBAP是一种ROS的清除剂)。用激光共聚焦显微镜荧光染色法检测不同组中细胞的平均荧光强度,从而反映细胞内活性氧的表达水平;用逆转录-聚合酶联反应(RT-PCR)和免疫组织化学链霉菌抗生物素蛋白-过氧化酶连接(SP)法分别测定不同组中HIF-1αmRNA和HIF-1α蛋白的表达水平;用四甲基偶氮唑蓝(MTT)比色法检测不同组的细胞增殖程度。结果 1.缺氧组细胞中活性氧(69.59±3.50)比常氧组(27.39±2.25)明显增加(n=6,P0.05);缺氧+Mn-TBAP组细胞内活性氧(41.09±2.47)低于缺氧组(n=6,P0.05),但高于常氧组(n=6,P0.05)。 2.与常氧组(HIF-1αmRNA: 0.16±0.03,蛋白:186.46±5.13)比较,缺氧组HIF-1αmRNA(0.43±0.02)和蛋白(146.93±14.48)的表达明显增强(n=6,P0.05);而缺氧+Mn-TBAP组的HIF-1αmRNA(0.25±0.03)和蛋白(160.07±7.21)均比缺氧组减弱(n=6,P0.05) ,但仍比常氧组升高(n=6,P0.05) 3.缺氧组的平滑肌细胞增殖程度(0.20±0.01)比常氧组(0.12±0.01)显著升高(n=6, P0.05);缺氧+Mn-TBAP组的平滑肌细胞增殖程度(0.16±0.01)比缺氧组有显著的降低(n=6, P0.05),但高于常氧组(n=6, P0.05)。结论缺氧条件下,肺动脉平滑肌细胞中ROS含量是增加的;缺氧可以使肺动脉平滑肌细胞中HIF-1αmRNA和蛋白表达均明显升高,同时促进平滑肌细胞的增殖;当减少肺动脉平滑肌细胞中ROS含量时,能抑制上述缺氧对HIF-1α和平滑肌细胞增殖的促进作用。这些说明:1.在肺动脉平滑肌细胞中,ROS可能作为一种信号分子参与缺氧感受信号转导途径;2.在肺动脉平滑肌细胞中,缺氧对HIF-1α的调节可以发生于转录,翻译和翻译后水平;3.平滑肌细胞内活性氧、HIF-1αmRNA和蛋白及细胞增殖的变化趋势一致,说明活性氧与HIF-1αmRNA和蛋白表达以及细胞增殖有关。活性氧可能参与某些信号转导通路,或者通过影响特定羟化酶的活性,从而促进HIF-1α表达。同时ROS通过调节HIF-1α的表达,进而影响平滑肌细胞的增殖。因此,ROS可能在肺动脉高压的发病机制和缺氧信号转导中具有重要的作用。
[Abstract]:objective
1. the changes of active oxygen (ROS) in the pulmonary artery smooth muscle cells of rats were observed under the condition of hypoxia.
2. to investigate whether the change of ROS in rat pulmonary artery smooth muscle cells can regulate the expression of hypoxia inducible factor -1apha (HIF-1 alpha) and influence the proliferation of smooth muscle cells under anoxic condition.
Materials and methods
This experiment adopts the explant primary culture of rat pulmonary artery smooth muscle cells (PASMCs), using cell trypsin digestion method, and the PASMCs were randomly divided into 3 groups: normoxia group (21%O2,24 h, A group), hypoxia group (5%O2,24 h, B group), hypoxia group (+Mn-TBAP 5%O2,24 hours, C group, Mn-TBAP is a ROS scavenger). The mean fluorescence intensity of cells staining in each group was detected by laser confocal fluorescence microscope, which reflect the expression level of intracellular reactive oxygen species; using reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical streptavidin peroxidase conjugated (SP) method were used to determine the expression level of HIF-1 alpha mRNA and HIF-1 protein in different groups; four methyl thiazolyl tetrazolium (MTT) assay to detect cell proliferation than the degree of different groups.
Result
1., reactive oxygen species (69.59 + 3.50) in hypoxia group increased significantly compared with normoxia group (27.39 + 2.25) (n=6, P0.05); reactive oxygen species in hypoxia +Mn-TBAP group (41.09 + 2.47) were lower than those in hypoxia group (n=6, P0.05), but higher than those in normoxia group (n=6, P0.05).
2. with normal oxygen group (HIF-1 mRNA: 0.16 + alpha 0.03, protein: 186.46 + 5.13), hypoxia group HIF-1 alpha mRNA (0.43 + 0.02) and protein (146.93 + 14.48) was significantly higher (n=6, P0.05); and hypoxia +Mn-TBAP group HIF-1 alpha mRNA (0.25 + 0.03) and protein (160.07 + 7.21) than the hypoxia group decreased (n=6, P0.05), but still higher than the normal oxygen group increased (n=6, P0.05)
3., the proliferation of smooth muscle cells in hypoxia group (0.20 + 0.01) was significantly higher than that in normoxia group (0.12 + 0.01) (n=6, P0.05). The proliferation of smooth muscle cells in hypoxia +Mn-TBAP group (0.16 + 0.01) was significantly lower than that in hypoxia group (n=6, P0.05), but higher than that in normoxic group (n= 6, P0.05).
conclusion
Under hypoxic conditions, the content of ROS in pulmonary artery smooth muscle cells is increased; hypoxia can make the increased expression of HIF-1 protein and mRNA in pulmonary artery smooth muscle cells, and promote the proliferation of smooth muscle cells; when reduce the content of ROS in pulmonary artery smooth muscle cells, can inhibit the proliferation of hypoxic HIF-1 alpha smooth muscle cells to promote note: 1.. The role in pulmonary artery smooth muscle cells, ROS may act as a signaling molecule involved in hypoxia signal transduction pathway; 2. in pulmonary artery smooth muscle cells in the hypoxic regulation of HIF-1 alpha can occur in transcription, translation and post translation level; 3. of active oxygen in the smooth muscle cells, the same trend of proliferation of HIF-1 alpha mRNA and protein and cells, suggesting that reactive oxygen associated with the expression of mRNA and HIF-1 alpha protein and cell proliferation. ROS may be involved in some signal transduction pathways, or by affecting The activity of specific hydroxylase can promote the expression of HIF-1. Meanwhile, ROS can affect the proliferation of smooth muscle cells by regulating the expression of HIF-1. Therefore, ROS may play an important role in the pathogenesis and hypoxia signal transduction of pulmonary hypertension.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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