本文關鍵詞： 缺氧 肺動脈平滑肌細胞 活性氧 缺氧誘導因子 增殖　出處：《華中科技大學》2007年碩士論文　論文類型：學位論文

【摘要】： 目的 1.觀察在缺氧條件下,大鼠肺動脈平滑肌細胞中活性氧(ROS)的變化趨勢。 2.探討在缺氧條件下,大鼠肺動脈平滑肌細胞中ROS的變化是否調(diào)控缺氧誘導因子-1apha(HIF-1α)的表達,進而影響平滑肌細胞的增殖。 材料與方法 本實驗采用組織塊貼壁法原代培養(yǎng)大鼠肺動脈平滑肌細胞(PASMCs),用胰蛋白酶消化法傳代細胞,并將PASMCs隨機分成3組:常氧組(21%O2,24小時,A組),缺氧組(5%O2,24小時,B組),缺氧+Mn-TBAP組(5%O2,24小時,C組, Mn-TBAP是一種ROS的清除劑)。用激光共聚焦顯微鏡熒光染色法檢測不同組中細胞的平均熒光強度,從而反映細胞內(nèi)活性氧的表達水平;用逆轉(zhuǎn)錄-聚合酶聯(lián)反應(RT-PCR)和免疫組織化學鏈霉菌抗生物素蛋白-過氧化酶連接(SP)法分別測定不同組中HIF-1αmRNA和HIF-1α蛋白的表達水平;用四甲基偶氮唑藍(MTT)比色法檢測不同組的細胞增殖程度。 結果 1.缺氧組細胞中活性氧(69.59±3.50)比常氧組(27.39±2.25)明顯增加(n=6,P0.05);缺氧+Mn-TBAP組細胞內(nèi)活性氧(41.09±2.47)低于缺氧組(n=6,P0.05),但高于常氧組(n=6,P0.05)。 2.與常氧組(HIF-1αmRNA: 0.16±0.03,蛋白:186.46±5.13)比較,缺氧組HIF-1αmRNA(0.43±0.02)和蛋白(146.93±14.48)的表達明顯增強(n=6,P0.05);而缺氧+Mn-TBAP組的HIF-1αmRNA(0.25±0.03)和蛋白(160.07±7.21)均比缺氧組減弱(n=6,P0.05) ,但仍比常氧組升高(n=6,P0.05) 3.缺氧組的平滑肌細胞增殖程度(0.20±0.01)比常氧組(0.12±0.01)顯著升高(n=6, P0.05);缺氧+Mn-TBAP組的平滑肌細胞增殖程度(0.16±0.01)比缺氧組有顯著的降低(n=6, P0.05),但高于常氧組(n=6, P0.05)。 結論 缺氧條件下,肺動脈平滑肌細胞中ROS含量是增加的;缺氧可以使肺動脈平滑肌細胞中HIF-1αmRNA和蛋白表達均明顯升高,同時促進平滑肌細胞的增殖;當減少肺動脈平滑肌細胞中ROS含量時,能抑制上述缺氧對HIF-1α和平滑肌細胞增殖的促進作用。這些說明:1.在肺動脈平滑肌細胞中,ROS可能作為一種信號分子參與缺氧感受信號轉(zhuǎn)導途徑;2.在肺動脈平滑肌細胞中,缺氧對HIF-1α的調(diào)節(jié)可以發(fā)生于轉(zhuǎn)錄,翻譯和翻譯后水平;3.平滑肌細胞內(nèi)活性氧、HIF-1αmRNA和蛋白及細胞增殖的變化趨勢一致,說明活性氧與HIF-1αmRNA和蛋白表達以及細胞增殖有關�；钚匝蹩赡軈⑴c某些信號轉(zhuǎn)導通路,或者通過影響特定羥化酶的活性,從而促進HIF-1α表達。同時ROS通過調(diào)節(jié)HIF-1α的表達,進而影響平滑肌細胞的增殖。因此,ROS可能在肺動脈高壓的發(fā)病機制和缺氧信號轉(zhuǎn)導中具有重要的作用。
[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.

【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R363

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相關期刊論文 前1條

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本文編號：1467637