本文選題：晶狀體上皮細(xì)胞 + 茶多酚��； 參考：《山東中醫(yī)藥大學(xué)》2010年博士論文

【摘要】：目的：通過體內(nèi)外實驗探討高糖及茶多酚干預(yù)對大鼠晶狀體上皮細(xì)胞的影響,闡明茶多酚對糖尿病性白內(nèi)障的預(yù)計療效及作用機制。 方法：體外培養(yǎng)大鼠晶狀體上皮細(xì)胞,培養(yǎng)基中葡萄糖濃度分別設(shè)定為5mmol/l、30mmol/l兩種濃度,在30mmol/l葡萄糖濃度培養(yǎng)基中分別加入茶多酚配成0.1μmol/l和0.3μmol/l茶多酚濃度的培養(yǎng)基進行細(xì)胞培養(yǎng)。用MMT檢測晶狀體上皮細(xì)胞的活性,流式細(xì)胞儀檢測LEC細(xì)胞死亡率和線粒體膜電位,線粒體活性氧的變化,以及使用免疫細(xì)胞化學(xué)和免疫熒光法檢測內(nèi)質(zhì)網(wǎng)表達量的變化。利用統(tǒng)計學(xué)分析高糖及茶多酚干預(yù)對大鼠晶狀體上皮細(xì)胞的影響。體內(nèi)試驗通過STZ尾靜脈注射誘導(dǎo)建立糖尿病大鼠模型,分為正常組、STZ誘導(dǎo)糖尿病組(STZ-DM)和茶多酚干預(yù)糖尿病組(TP-DM)三組,通過HE染色、電鏡觀察大鼠晶狀體的超微結(jié)構(gòu),生化法測定各組大鼠晶狀體中GSH、GR及CAT生化指標(biāo)的活性,RT-PCR法檢測各組大鼠晶狀體中UCPs、MnSODmRNA的表達量變化,探討茶多酚對STZ誘導(dǎo)糖尿病大鼠晶狀體上皮細(xì)胞干預(yù)的影響。 結(jié)果：在高葡萄糖濃度條件下培養(yǎng)的上皮細(xì)胞逐漸喪失原有特征,形態(tài)表現(xiàn)多樣,漸則細(xì)胞腫脹,出現(xiàn)空泡和顆粒,細(xì)胞數(shù)量明顯減少,傳代后容易老化,死亡率CDR(%)明顯升高,線粒體膜電位MMP(FI)降低,線粒體活性氧增多,內(nèi)質(zhì)網(wǎng)的陽性率明顯降低；而茶多酚干預(yù)組細(xì)胞則較好的保持了上皮細(xì)胞的形態(tài),細(xì)胞之間緊密相靠,排列規(guī)則,較少出現(xiàn)細(xì)胞腫脹、空泡和顆粒等,傳代未見老化,細(xì)胞死亡率降低,線粒體膜電位升高,線粒體活性氧的產(chǎn)生明顯減少,并且內(nèi)質(zhì)網(wǎng)的陽性率明顯升高。低濃度與高濃度的茶多酚干預(yù)效果差別不明顯。體外實驗表明在建模8周時,STZ-DM組76.2%的糖尿病大鼠晶狀體表現(xiàn)為Ⅲ級混濁,而TP-DM組糖尿病大鼠晶狀體混濁發(fā)展不明顯,30%的糖尿病大鼠晶狀體表現(xiàn)為Ⅱ級混濁。第12周時STZ-DM組糖尿病大鼠晶狀體完全混濁,而TP-DM組61.1%糖尿病大鼠晶狀體表現(xiàn)Ⅲ級混濁。掃描電鏡觀察大鼠的晶狀體的超微結(jié)構(gòu),發(fā)現(xiàn)STZ-DM組大鼠的晶狀體纖維嚴(yán)重受損,而TP-DM組受損明顯減輕；通過HE染色觀察大鼠的晶狀體細(xì)胞形態(tài),發(fā)現(xiàn)STZ-DM組的大鼠晶狀體纖維細(xì)胞內(nèi)有大量未被降解的細(xì)胞器,無細(xì)胞器區(qū)域縮小。而TP-DM組晶狀體纖維細(xì)胞未被降解的細(xì)胞器明顯減少,無細(xì)胞器區(qū)域顯著擴大。通過對大鼠晶狀體中CAT的活性和GSH的含量進行測量,發(fā)現(xiàn)STZ-DM大鼠晶狀體中CAT的活性和GSH的含量均明顯下降,而TP-DM組晶狀體中保持了CAT的活性和GSH的含量；應(yīng)用RT-PCR的方法對大鼠晶狀體中UCPs、MnSOD表達的變化進行檢測,發(fā)現(xiàn)STZ-DM大鼠晶狀體中UCP2及MnSOD mRNA表達量逐漸減少,而TP-DM組大鼠晶狀體中的UCP2及MnSOD mRNA表達增加且維持在較高水平。 結(jié)論：大鼠晶狀體上皮細(xì)胞在高糖條件下培養(yǎng)時細(xì)胞凋亡的各項特征明顯加強；而應(yīng)用茶多酚干預(yù)后,以上細(xì)胞凋亡現(xiàn)象對比高糖組均有明顯減輕。表明茶多酚可延緩和阻止晶狀體上皮細(xì)胞受損傷,對高糖條件下晶狀體上皮細(xì)胞的凋亡具有抑制作用,對防治糖尿病性白內(nèi)障有可預(yù)計的積極療效。體內(nèi)實驗表明茶多酚可以顯著減輕糖尿病性白內(nèi)障大鼠晶狀體的超微結(jié)構(gòu)的損傷,維持晶狀體內(nèi)正常的細(xì)胞器降解,可以保持CAT的活性和GSH的含量,維持晶狀體正常的氧化還原狀態(tài),甚至逆轉(zhuǎn)了抗氧化酶類活性的降低；茶多酚可以上調(diào)糖尿病大鼠晶狀體中的UCP2及MnSOD mRNA表達并使其維持在較高水平,進而影響晶狀體中活性氧的清除,阻止或者延緩晶狀體的混濁,顯著延緩糖尿病性白內(nèi)障的發(fā)生。
[Abstract]:Objective : To study the effects of high glucose and tea polyphenols on lens epithelial cells in rats by in vivo and in vivo experiments , and clarify the expected curative effect and mechanism of tea polyphenols on diabetic cataract .


Methods : The rat lens epithelial cells were cultured in vitro . The concentration of glucose in the culture medium was set to 5 mmol / l and 30 mmol / l . The effects of high glucose and tea polyphenols on the rat lens epithelial cells were determined by HE staining and electron microscope .


Results : The epithelial cells cultured under the condition of high glucose concentration gradually lose their original characteristics , the morphology is varied , the cells are swollen , vacuoles and particles appear , the number of cells is obviously reduced , the mortality CDR ( % ) is obviously increased , the mitochondrial membrane potential MMP ( FI ) is decreased , the mitochondrial active oxygen is increased , and the positive rate of the endoplasmic reticulum is obviously reduced ;
The results showed that the lens performance of diabetic rats was not significantly higher than that in TP - DM group .
The activity of CAT and the content of GSH in the lens were measured by HE staining , while the activity of CAT and the content of GSH were observed in the lens of TP - DM group .
The changes of UCP and MnSOD in the lens of rats were detected by RT - PCR , and the expression of UCP2 and MnSOD mRNA in the lens was gradually decreased , while the expression of UCP2 and MnSOD mRNA in the lens of TP - DM rats was increased and maintained at a higher level .


Conclusion : The characteristics of apoptosis in rat lens epithelial cells were significantly enhanced when cultured in high glucose condition .
The results showed that tea polyphenols can delay and prevent lens epithelial cells from being damaged , and can inhibit the apoptosis of lens epithelial cells under the condition of high glucose .
The tea polyphenol can raise the expression of UCP2 and MnSOD mRNA in the lens of diabetic rats and maintain it at a high level , thereby affecting the removal of active oxygen in the lens , preventing or delaying the opacity of the lens , and obviously delaying the occurrence of diabetic cataract .

【學(xué)位授予單位】：山東中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R587.1;R776.1

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