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  本文選題：衰老標記蛋白30　切入點：人晶狀體上皮細胞　出處：《廣西醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:觀察氧化應激對人晶狀體上皮細胞(human lens epithelial cells,HLECs)中衰老標記蛋白30(Senescence Marker Protein30,SMP30)表達變化的影響,為今后研究其在人晶狀體上皮細胞及白內障發(fā)生發(fā)展過程中的作用奠定基礎。方法:用不同濃度過氧化氫(0、100、200、300μM)刺激人晶狀體上皮細胞24小時,建立不同濃度急性氧化應激模型,通過光鏡、MTT檢測分析細胞形態(tài)、狀態(tài),Western blot檢測SMP30蛋白的表達情況。用不同濃度過氧化氫(0、25、50、75、100、125、150μM)刺激人晶狀體上皮細胞2周,建立慢性氧化應激模型,誘導細胞衰老,通過光鏡、MTT、β-半乳糖苷酶衰老細胞染色、細胞周期檢測細胞氧化應激情況及評估細胞狀態(tài),用q-PCR、Western blot檢測SMP30基因及蛋白的表達變化。結果:過氧化氫刺激HLECs24小時,隨著過氧化氫濃度的增高,各處理組細胞在光鏡下可見密度逐漸下降,變大變圓,300μM處理組細胞破碎,MTT結果提示細胞生存率逐漸降低,各濃度處理組與對照組相比差異均有統(tǒng)計學意義(P0.05),SMP30在100、200μM處理組的蛋白表達量均較對照組升高(P均0.05)。過氧化氫刺激HLECs14天后25μM處理組細胞形態(tài)與對照組無明顯差異,50-125μM處理組細胞密度逐漸下降,細胞呈衰老狀態(tài),150μM處理組細胞碎裂。MTT結果提示25-50μM處理組較對照組相比生長曲線逐漸下移,75μM處理組僅維持細胞基本代謝活性,100-150μM處理組生長曲線則在早期大幅下降并維持。β-半乳糖苷酶衰老細胞染色顯示隨過氧化氫濃度升高,衰老陽性細胞增多,75-150μM處理組陽性細胞較對照組明顯增多且90%,細胞周期則提示50-100μM處理組細胞增值阻滯于S和G2/M期,PI值逐漸升高。SMP30在25-100μM處理組與對照組相比mRNA表達均下降(P均0.01)。在25-50μM處理組SMP30蛋白的表達量與對照組相比無明顯差異(P=0.695,P=0.126)。在75-100μM處理組SMP30蛋白的表達量均較對照組明顯下降(P均0.01)。結論:SMP30蛋白在處于急性氧化應激狀態(tài)下的人晶狀體上皮細胞中表達上調。而在慢性氧化應激誘導衰老的早期階段SMP30表達無明顯變化,隨著氧化應激增加和衰老加劇,SMP30的表達減少。SMP30參與人晶狀體上皮細胞氧化應激損傷過程,可能與細胞衰老及白內障的發(fā)生發(fā)展有關。
[Abstract]:Objective: to observe the effect of oxidative stress on the expression of senescence Marker protein 30 SMP30 in human lens epithelial cells (HLECs). Methods: human lens epithelial cells were stimulated with different concentrations of hydrogen peroxide for 24 hours and acute oxidative stress models were established. The morphology of human lens epithelial cells was analyzed by light microscopy, and the expression of SMP30 protein was detected by Western blot. Human lens epithelial cells were stimulated with different concentrations of hydrogen peroxide for 2 weeks to establish a chronic oxidative stress model and induce cell senescence. The changes of SMP30 gene and protein expression were detected by light microscope MTT, 尾 -galactosidase staining, cell cycle detection of oxidative stress and evaluation of cell status. Results: hydrogen peroxide stimulated HLECs24 for hours. With the increase of hydrogen peroxide concentration, the density of cells in each treatment group decreased gradually under light microscope, and the MTT results showed that cell survival rate decreased gradually in the treatment group of 300 渭 M. Compared with the control group, the protein expression of P0.05SMP30 in the 100,200 渭 M treatment group was significantly higher than that in the control group (0.05 渭 M). There was no significant difference in cell morphology between the 25 渭 M group treated with hydrogen peroxide and the control group. The cell density decreased gradually in the treatment group. The results of cell fragmentation and MTT showed that the growth curve of 25-50 渭 M treatment group decreased gradually compared with that of the control group. The growth curve of 25-50 渭 M treatment group only maintained the basic metabolic activity of 75 渭 M treatment group was significantly decreased in the early stage. 尾 -galactosidase staining showed that with the increase of hydrogen peroxide concentration, The number of senescent positive cells increased significantly in 75-150 渭 M treatment group compared with the control group, and the cell cycle indicated that the cell proliferation of 50-100 渭 M treatment group was blocked at S and G _ 2 / M phase Pi value gradually increased. The mRNA expression of SMP30 in 25-100 渭 M treatment group was higher than that in the control group. The expression of SMP30 protein in 25-50 渭 M treatment group was not significantly different from that in control group. The expression of SMP30 protein in 75-100 渭 M treatment group was significantly lower than that in control group (P < 0.01). Conclusion the expression of SMP30 protein in the control group is in the state of acute oxidative stress. The expression of SMP30 did not change in the early stage of aging induced by chronic oxidative stress. With the increase of oxidative stress and the decrease of SMP30 expression in lens epithelial cells, SMP30 may be involved in the process of oxidative stress injury in lens epithelial cells, which may be related to cell aging and the development of cataract.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R776.1

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本文編號：1622110