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

【摘要】：目的:觀察氧化應(yīng)激對人晶狀體上皮細(xì)胞(human lens epithelial cells,HLECs)中衰老標(biāo)記蛋白30(Senescence Marker Protein30,SMP30)表達(dá)變化的影響,為今后研究其在人晶狀體上皮細(xì)胞及白內(nèi)障發(fā)生發(fā)展過程中的作用奠定基礎(chǔ)。方法:用不同濃度過氧化氫(0、100、200、300μM)刺激人晶狀體上皮細(xì)胞24小時,建立不同濃度急性氧化應(yīng)激模型,通過光鏡、MTT檢測分析細(xì)胞形態(tài)、狀態(tài),Western blot檢測SMP30蛋白的表達(dá)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(xì)胞2周,建立慢性氧化應(yīng)激模型,誘導(dǎo)細(xì)胞衰老,通過光鏡、MTT、β-半乳糖苷酶衰老細(xì)胞染色、細(xì)胞周期檢測細(xì)胞氧化應(yīng)激情況及評估細(xì)胞狀態(tài),用q-PCR、Western blot檢測SMP30基因及蛋白的表達(dá)變化。結(jié)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(xì)胞在光鏡下可見密度逐漸下降,變大變圓,300μM處理組細(xì)胞破碎,MTT結(jié)果提示細(xì)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(xué)意義(P0.05),SMP30在100、200μM處理組的蛋白表達(dá)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(xì)胞形態(tài)與對照組無明顯差異,50-125μM處理組細(xì)胞密度逐漸下降,細(xì)胞呈衰老狀態(tài),150μM處理組細(xì)胞碎裂。MTT結(jié)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(xì)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(xì)胞染色顯示隨過氧化氫濃度升高,衰老陽性細(xì)胞增多,75-150μM處理組陽性細(xì)胞較對照組明顯增多且90%,細(xì)胞周期則提示50-100μM處理組細(xì)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(dá)均下降(P均0.01)。在25-50μM處理組SMP30蛋白的表達(dá)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(dá)量均較對照組明顯下降(P均0.01)。結(jié)論:SMP30蛋白在處于急性氧化應(yīng)激狀態(tài)下的人晶狀體上皮細(xì)胞中表達(dá)上調(diào)。而在慢性氧化應(yīng)激誘導(dǎo)衰老的早期階段SMP30表達(dá)無明顯變化,隨著氧化應(yīng)激增加和衰老加劇,SMP30的表達(dá)減少。SMP30參與人晶狀體上皮細(xì)胞氧化應(yīng)激損傷過程,可能與細(xì)胞衰老及白內(nèi)障的發(fā)生發(fā)展有關(guān)。
[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(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R776.1

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本文編號：1622110