本文選題：非瑟酮　切入點(diǎn)：人晶狀體上皮細(xì)胞　出處：《瀘州醫(yī)學(xué)院》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：研究非瑟酮(fisetin, Fis)在生理狀態(tài)下及氧化應(yīng)激狀態(tài)下對(duì)人晶狀體上皮細(xì)胞(human lens epithelial cells, HLECs)增殖和凋亡的影響。方法：(1)實(shí)驗(yàn)準(zhǔn)備：將凍存的人晶狀體上皮細(xì)胞株(SRA01/04)復(fù)蘇后,置于含10%胎牛血清的低糖DMEM培養(yǎng)基(葡萄糖終濃度5.56mmol/L)中培養(yǎng),傳代,收集對(duì)數(shù)生長(zhǎng)期細(xì)胞進(jìn)行以下實(shí)驗(yàn)。(2)分組及干預(yù)：用0.25%胰酶消化收集同一代貼壁細(xì)胞,制成細(xì)胞懸液計(jì)數(shù)后,隨機(jī)分為空白對(duì)照組(不加干預(yù))；Fis組(分別加入5μg/ml、10μg/ml、20μg/ml濃度的Fis)；H2O2組(加入300μmol/L過氧化氫(Hydrogen peroxide, H2O2)); Fis+H2O2組(先加入5μg/ml、10μg/ml、20μg/mlFis,1小時(shí)后加入300μmol/L H2O2共同培養(yǎng))。(3)形態(tài)學(xué)觀察：培養(yǎng)12h和24h后倒置顯微鏡觀察以上各組細(xì)胞的形態(tài)學(xué)改變,隨機(jī)照相(×100倍,×200倍),對(duì)比各組細(xì)胞形態(tài)學(xué)變化。(4)Fis對(duì)HLECs增殖的觀察：采用四甲基偶氮唑鹽(methyl thiazolyl tetrazolium, MTT)比色法分別檢測(cè)Fis在有或無H2O2作用12小時(shí)和24小時(shí)后對(duì)HLECs增殖的影響。(5)Fis對(duì)H202誘導(dǎo)的HLECs凋亡的觀察：采用異硫氰酸(FITC-Annexin-v, Annexin-V)和碘化丙錠(Propidium Iodide, PI)標(biāo)記的雙染色免疫熒光法,流式細(xì)胞術(shù)(flow cytometric analysis, FCM)檢測(cè)Fis在與H202共同作用24小時(shí)后HLECs凋亡率的變化。結(jié)果：(1)與空白對(duì)照組比較,加入5～20μg/mlFis培養(yǎng)12小時(shí)和24小時(shí),對(duì)HLECs的增殖無明顯影響(P0.05),細(xì)胞形態(tài)無明顯變化。(2)與空白對(duì)照組比較,H2O2組較多細(xì)胞出現(xiàn)細(xì)胞密度降低以及典型的細(xì)胞形態(tài)學(xué)改變,細(xì)胞增殖能力明顯降低,凋亡率明顯增加,差異有顯著統(tǒng)計(jì)學(xué)意義(P0.01)。加入5-20μg/mlFis預(yù)處理組,H2O2誘導(dǎo)的典型形態(tài)改變的細(xì)胞減少,細(xì)胞增殖能力明顯改善,并且隨時(shí)間和Fis濃度的增加作用更明顯(P0.05)；Fis作用24h時(shí),細(xì)胞凋亡率逐漸降低,與H2O2組比較,差異有統(tǒng)計(jì)學(xué)意義(P0.05),并且隨Fis濃度增加作用更明顯(P0.05)。結(jié)論：(1)在生理?xiàng)l件下,在一定濃度和作用時(shí)間范圍內(nèi),非瑟酮對(duì)HLECs的增殖無明顯影響。(2)在氧化應(yīng)激條件下,在一定濃度和作用時(shí)間范圍內(nèi),非瑟酮能明顯改善HLECs的增殖能力,且呈劑量-效應(yīng)與時(shí)間-效應(yīng)關(guān)系。(3)在氧化應(yīng)激條件下,在一定濃度范圍內(nèi),非瑟酮能明顯抑制HLECs凋亡,且呈劑量-效應(yīng)關(guān)系。
[Abstract]:Aim: to study the effects of Fisetin (Fis) on proliferation and apoptosis of human lens epithelial cells (HLECs) in physiological and oxidative stress. Methods: the cryopreserved human lens epithelial cells (LECs) were resuscitated after cryopreserved human lens epithelial cells (HLECs) were resuscitated. Cultured in 10% fetal bovine serum low-glucose DMEM medium (glucose final concentration 5.56 mmol / L), subcultured, collected logarithmic growth phase cells for the following experiment. 2. Intervention: digesting and collecting the same generation of adherent cells with 0.25% trypsin. After making cell suspension count, They were randomly divided into two groups: control group (Fis group without intervention) (adding 5 渭 g / ml 10 渭 g / ml 10 渭 g / ml ~ (20) 渭 g/ml) to H _ 2O _ 2 group (adding #number0# 渭 mol/L hydrogen peroxide, H _ 2O _ 2), Fis H2O2 group (5 渭 g 路ml ~ (10) 渭 g 路ml ~ (10) 渭 g 路ml ~ (20) 渭 g 路ml ~ (-1)) and then adding 300 渭 mol/L H2O2 for 1 hour. Morphological observation: 12 h and 24 h later, the morphology of Fis H2O2 group was observed. The morphological changes of the above groups were observed by inverted microscope. Random radiographs (脳 100 times, 脳 200 times) were used to compare the morphological changes of cells in each group. The proliferation of HLECs was observed by Fis thiazolyl tetrazolium (MTT) colorimetric method. The proliferation of HLECs was detected by Fis after 12 hours and 24 hours of H2O2 treatment, respectively. Observation of HLECs apoptosis induced by H202 by FITC-Annexin-V (Annexin-V) and Propidium Iodide (Pi) labeled by FITC-Annexin-V (Pi). Flow cytometric analysis (FCM) was used to detect the changes of HLECs apoptosis rate in Fis treated with H202 for 24 hours. Results compared with control group, Fis was cultured with 5 渭 g/mlFis for 12 hours and 24 hours. Compared with the control group, more cells in H 2O 2 group had decreased cell density and typical cell morphological changes, and the cell proliferation ability was significantly decreased, and the apoptosis rate was significantly increased compared with the control group. The difference was statistically significant (P 0.01). In the 5-20 渭 g/mlFis pretreatment group, the cells with typical morphological changes induced by H _ 2O _ 2 decreased, the cell proliferation ability was improved significantly, and the effect was more obvious with the increase of the concentration of Fis and the concentration of P0.05Fis for 24 h. The apoptosis rate decreased gradually, and the difference was statistically significant compared with that of H2O2 group, and the effect was more obvious with the increase of Fis concentration. Conclusion: under physiological conditions, in a certain concentration and within a certain time range, the cell apoptosis rate was significantly increased with the increase of Fis concentration. There was no significant effect of fenetherone on the proliferation of HLECs. (2) under oxidative stress, under certain concentration and action time range, fenetherone could obviously improve the proliferative ability of HLECs, and showed a dose-effect and time-effect relationship under oxidative stress. In a certain concentration range, fenetherone could significantly inhibit HLECs apoptosis, and showed a dose-effect relationship.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R776.1

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