本文關(guān)鍵詞： 缺氧 肺動(dòng)脈平滑肌細(xì)胞 活性氧 缺氧誘導(dǎo)因子 增殖　出處：《華中科技大學(xué)》2007年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 目的 1.觀察在缺氧條件下,大鼠肺動(dòng)脈平滑肌細(xì)胞中活性氧(ROS)的變化趨勢(shì)。 2.探討在缺氧條件下,大鼠肺動(dòng)脈平滑肌細(xì)胞中ROS的變化是否調(diào)控缺氧誘導(dǎo)因子-1apha(HIF-1α)的表達(dá),進(jìn)而影響平滑肌細(xì)胞的增殖。 材料與方法 本實(shí)驗(yàn)采用組織塊貼壁法原代培養(yǎng)大鼠肺動(dòng)脈平滑肌細(xì)胞(PASMCs),用胰蛋白酶消化法傳代細(xì)胞,并將PASMCs隨機(jī)分成3組:常氧組(21%O2,24小時(shí),A組),缺氧組(5%O2,24小時(shí),B組),缺氧+Mn-TBAP組(5%O2,24小時(shí),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(cè)不同組中細(xì)胞的平均熒光強(qiáng)度,從而反映細(xì)胞內(nèi)活性氧的表達(dá)水平;用逆轉(zhuǎn)錄-聚合酶聯(lián)反應(yīng)(RT-PCR)和免疫組織化學(xué)鏈霉菌抗生物素蛋白-過(guò)氧化酶連接(SP)法分別測(cè)定不同組中HIF-1αmRNA和HIF-1α蛋白的表達(dá)水平;用四甲基偶氮唑藍(lán)(MTT)比色法檢測(cè)不同組的細(xì)胞增殖程度。 結(jié)果 1.缺氧組細(xì)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(xì)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(dá)明顯增強(qiáng)(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(xì)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(xì)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(jié)論 缺氧條件下,肺動(dòng)脈平滑肌細(xì)胞中ROS含量是增加的;缺氧可以使肺動(dòng)脈平滑肌細(xì)胞中HIF-1αmRNA和蛋白表達(dá)均明顯升高,同時(shí)促進(jìn)平滑肌細(xì)胞的增殖;當(dāng)減少肺動(dòng)脈平滑肌細(xì)胞中ROS含量時(shí),能抑制上述缺氧對(duì)HIF-1α和平滑肌細(xì)胞增殖的促進(jìn)作用。這些說(shuō)明:1.在肺動(dòng)脈平滑肌細(xì)胞中,ROS可能作為一種信號(hào)分子參與缺氧感受信號(hào)轉(zhuǎn)導(dǎo)途徑;2.在肺動(dòng)脈平滑肌細(xì)胞中,缺氧對(duì)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(xì)胞內(nèi)活性氧、HIF-1αmRNA和蛋白及細(xì)胞增殖的變化趨勢(shì)一致,說(shuō)明活性氧與HIF-1αmRNA和蛋白表達(dá)以及細(xì)胞增殖有關(guān)�；钚匝蹩赡軈⑴c某些信號(hào)轉(zhuǎn)導(dǎo)通路,或者通過(guò)影響特定羥化酶的活性,從而促進(jìn)HIF-1α表達(dá)。同時(shí)ROS通過(guò)調(diào)節(jié)HIF-1α的表達(dá),進(jìn)而影響平滑肌細(xì)胞的增殖。因此,ROS可能在肺動(dòng)脈高壓的發(fā)病機(jī)制和缺氧信號(hào)轉(zhuǎn)導(dǎo)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(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R363

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本文編號(hào)：1467637