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  本文關(guān)鍵詞： 抵抗素 大鼠主動脈內(nèi)皮細(xì)胞 一氧化氮 一氧化氮合酶　出處：《安徽醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的: 抵抗素是新近發(fā)現(xiàn)的一種脂肪因子,在肥胖和超重兒童中血清濃度有不同程度升高,與心腦血管疾病等密切相關(guān)。通過原代培養(yǎng)幼鼠主動脈內(nèi)皮細(xì)胞建立體外模型,以不同濃度抵抗素(resistin)干預(yù),模擬正常和肥胖狀態(tài)下抵抗素水平,從分子、蛋白、酶的活性、基因的表達(dá)及轉(zhuǎn)錄、翻譯后調(diào)節(jié)等多個水平研究幼鼠主動脈內(nèi)皮細(xì)胞一氧化氮(NO)系統(tǒng)的調(diào)控機(jī)制。 方法: 取幼鼠胸主動脈,用植環(huán)貼壁法原代培養(yǎng)大鼠主動脈內(nèi)皮細(xì)胞,消化、傳代,觀察細(xì)胞形態(tài),VIII因子相關(guān)抗原免疫細(xì)胞化學(xué)染色進(jìn)行細(xì)胞鑒定,建立體外細(xì)胞模型。 取傳至3-5代的大鼠主動脈內(nèi)皮細(xì)胞,以不同濃度鼠抵抗素(10-100 ng/ml)干預(yù)24小時。MTT法檢測內(nèi)皮細(xì)胞活性,化學(xué)比色法檢測NO及NOS酶活性,蛋白免疫印跡法檢測細(xì)胞內(nèi)eNOS(內(nèi)皮型一氧化氮合酶)蛋白和P-eNOS(磷酸化一氧化氮合酶)蛋白的表達(dá),RT-PCR檢測eNOSmRNA表達(dá)的變化。 結(jié)果: 幼年大鼠主動脈內(nèi)皮細(xì)胞經(jīng)不同濃度鼠重組抵抗素處理24小時后: 1.光鏡下觀察細(xì)胞形態(tài)均未發(fā)生明顯改變。 2.與對照組和10 ng/ml相比,50 ng/ml和100 ng/ml組細(xì)胞活性下降。 3.各組NO產(chǎn)物及eNOS活性均未發(fā)生改變。 4.與對照組相比,50 ng/ml和100 ng/ml組細(xì)胞eNOS蛋白和mRNA表達(dá)減少。 5.各組P-eNOS表達(dá)未見改變。 結(jié)論: 抵抗素可減低幼年大鼠主動脈內(nèi)皮細(xì)胞的細(xì)胞活性,調(diào)控NO系統(tǒng),使eNOS蛋白和基因表達(dá)水平降低,其但未見通過調(diào)控磷酸化作用影響酶的活性從而降低NO產(chǎn)物的合成。
[Abstract]:Objective: Resistin (resistin) is a newly found fatty factor, and serum levels in obese and overweight children have increased to varying degrees. In vitro model was established by primary culture of aortic endothelial cells of young rats and treated with different concentrations of resistin. Mimic normal and obese levels of resistin from molecular, protein, enzyme activity, gene expression and transcription. The regulation mechanism of nitric oxide (no) system in aortic endothelial cells of young rats was studied at several levels such as posttranslational regulation. Methods: The primary cultured rat aortic endothelial cells were isolated from the thoracic aorta of young rats. The endothelial cells of rat aorta were digested and subcultured. The morphology of the cells was observed and the immunocytochemistry staining of factor VIII related antigen was carried out for cell identification. To establish cell model in vitro. The endothelial cells of rat aorta were collected for 3 to 5 passage. The endothelial cell activity was detected by MTT assay with different concentrations of resistin 10-100 ng / ml for 24 hours. The activity of no and NOS was detected by chemical colorimetry, and the expression of Enos (endothelial nitric oxide synthase) protein and P-eNOS- (phosphorylated nitric oxide synthase) protein were detected by Western blot. The expression of eNOSmRNA was detected by RT-PCR. Results: Young rat aortic endothelial cells were treated with different concentrations of recombinant resistin for 24 hours. 1. There was no obvious change in cell morphology under light microscope. 2.Compared with the control group and 10 ng/ml group, the cell activity of 50 ng/ml and 100 ng/ml groups decreased. 3. No products and eNOS activity did not change in each group. 4. Compared with the control group, the expression of eNOS protein and mRNA was decreased in 50 ng/ml and 100 ng/ml groups. 5. There was no change in the expression of P-Enos in each group. Conclusion: Resistin can reduce the cellular activity of aortic endothelial cells, regulate the no system, and decrease the expression of eNOS protein and gene. However, it was not found that the activity of the enzyme was affected by phosphorylation and the synthesis of no products was decreased.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R363

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本文編號：1490955