本文關(guān)鍵詞： 白內(nèi)障 �；撬嵝苊撗跄懰� 內(nèi)質(zhì)網(wǎng)應(yīng)激 細(xì)胞凋亡 葡萄糖　出處：《山東大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的 內(nèi)質(zhì)網(wǎng)是細(xì)胞內(nèi)蛋白質(zhì)修飾和折疊的場所,未折疊或錯誤折疊蛋白質(zhì)在內(nèi)質(zhì)網(wǎng)腔中蓄積以及細(xì)胞內(nèi)環(huán)境的破壞將導(dǎo)致內(nèi)質(zhì)網(wǎng)應(yīng)激(endoplasmic reticulum stress, ERS)。適宜的應(yīng)激有利于細(xì)胞內(nèi)環(huán)境的恢復(fù),嚴(yán)重而持久的內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)則可誘導(dǎo)細(xì)胞發(fā)生凋亡。已有研究證實(shí),白內(nèi)障的發(fā)生也與內(nèi)質(zhì)網(wǎng)應(yīng)激有關(guān),在葡萄糖缺乏、高血糖、同型半胱氨酸、衣霉素等內(nèi)質(zhì)網(wǎng)應(yīng)激刺激因子作用下,均可誘導(dǎo)晶狀體上皮細(xì)胞發(fā)生內(nèi)質(zhì)網(wǎng)應(yīng)激,導(dǎo)致細(xì)胞凋亡。近年發(fā)現(xiàn),�；撬嵝苊撗跄懰�(tauroursodeoxycholic acid,TUDCA)可通過抑制內(nèi)質(zhì)網(wǎng)應(yīng)激途徑介導(dǎo)的細(xì)胞凋亡,有效減輕疏水性膽汁酸誘導(dǎo)的肝細(xì)胞凋亡,提高細(xì)胞生存能力。本研究采用高濃度葡萄糖誘導(dǎo)建立晶狀體上皮細(xì)胞凋亡模型,旨在觀察TUDCA對晶狀體上皮細(xì)胞凋亡的影響以及內(nèi)質(zhì)網(wǎng)凋亡途徑相關(guān)蛋白的變化,探討TUDCA對高濃度葡萄糖環(huán)境下晶狀體上皮細(xì)胞的保護(hù)作用及其機(jī)制。方法 人晶狀體上皮細(xì)胞(human lens epithelial cells, HLECs)株SRA01/04用含10%胎牛血清的DMEM完全培養(yǎng)基培養(yǎng),取對數(shù)生長期細(xì)胞在不同濃度葡萄糖培養(yǎng)液中培養(yǎng)24小時,誘導(dǎo)建立晶狀體上皮細(xì)胞凋亡模型,并采用不同濃度TUDCA(0.2、0.5、1.0、2.0mmol／L)進(jìn)行干預(yù)。應(yīng)用MTT法檢測不同濃度藥物刺激后的細(xì)胞增殖抑制率；應(yīng)用Hoechst 33258熒光染色法觀察凋亡細(xì)胞核形態(tài)學(xué)改變；Annexin V-FITC/PI雙染后流式細(xì)胞儀檢測細(xì)胞凋亡率；Western blot技術(shù)檢測細(xì)胞GRP78的表達(dá)。 結(jié)果 1.不同濃度葡萄糖對HLECs活性的影響 不同濃度葡萄糖孵育細(xì)胞24小時后,各濃度葡萄糖組增殖抑制率均高于對照組,且隨著葡萄糖濃度的增加,細(xì)胞增殖抑制率亦升高(P0.01)。取葡萄糖作用24小時后細(xì)胞增殖抑制率接近50%的250mmol／L葡萄糖濃度為HLECs凋亡誘導(dǎo)濃度,制作晶狀體上皮細(xì)胞凋亡模型。后續(xù)實(shí)驗(yàn)顯示,在此濃度葡萄糖培養(yǎng)液中培養(yǎng)細(xì)胞24小時,多數(shù)細(xì)胞出現(xiàn)典型凋亡細(xì)胞核形態(tài)改變,24小時細(xì)胞凋亡率為(57.94±1.29)%。 2. TUDCA干預(yù)對高濃度葡萄糖環(huán)境下HLECs活性的影響 與對照組相比,模型組細(xì)胞增殖抑制率明顯升高(P0.01)；與模型組相比,TUDCA干預(yù)各組細(xì)胞增殖抑制率明顯降低(P均0.01)。 3. TUDCA干預(yù)對高濃度葡萄糖環(huán)境下HLECs細(xì)胞形態(tài)學(xué)的影響 Hoechst33258染色可見,對照組細(xì)胞無明顯凋亡發(fā)生,細(xì)胞呈彌散均勻熒光。以250mmol／L葡萄糖培養(yǎng)細(xì)胞24小時后可見典型的凋亡細(xì)胞形態(tài)改變,凋亡細(xì)胞體積變小,核致密、固縮,核或細(xì)胞質(zhì)內(nèi)可見致密的顆粒狀強(qiáng)熒光,加入2.0mmol／LTUDCA共同培養(yǎng)后,凋亡細(xì)胞明顯減少。 4. TUDCA干預(yù)對高濃度葡萄糖環(huán)境下HLECs細(xì)胞凋亡率的影響 流式細(xì)胞術(shù)檢測各組細(xì)胞凋亡率,與對照組相比,模型組(250mmol／L葡萄糖)細(xì)胞凋亡率顯著增高,加入TUDCA干預(yù)后細(xì)胞凋亡率明顯降低(P均0.01)。 5. TUDCA干預(yù)對高濃度葡萄糖環(huán)境下HLECs細(xì)胞GRP78蛋白表達(dá)的影響 Western blot檢測顯示,與對照組相比,250mmol／L葡萄糖作用細(xì)胞24小時后GRP78蛋白表達(dá)顯著增加,加入TUDCA干預(yù)后GRP78表達(dá)明顯受到抑制(P0.05)。 結(jié)論 高濃度葡萄糖可以刺激晶狀體上皮細(xì)胞發(fā)生內(nèi)質(zhì)網(wǎng)應(yīng)激,誘導(dǎo)細(xì)胞凋亡。TUDCA可通過內(nèi)質(zhì)網(wǎng)應(yīng)激途徑抑制高濃度葡萄糖誘導(dǎo)的細(xì)胞凋亡,對晶狀體上皮細(xì)胞產(chǎn)生保護(hù)作用。
[Abstract]:objective
The endoplasmic reticulum is a place protein modification and folding in cells, unfolded or misfolded proteins accumulate in the endoplasmic reticulum cavity and intracellular environmental damage will lead to endoplasmic reticulum stress (endoplasmic reticulum, stress, ERS). The suitable stress is conducive to the intracellular environment recovery, severe endoplasmic reticulum stress response is lasting can induce the cell apoptosis. Studies have confirmed that the occurrence of cataract is associated with endoplasmic reticulum stress, in the absence of glucose, high blood glucose, homocysteine, tunicamycin and endoplasmic reticulum stress stimulation factor, can induce endoplasmic reticulum stress in lens epithelial cells, leading to cell apoptosis. In recent years, tauroursodeoxycholic deoxidation cholic acid (tauroursodeoxycholic acid, TUDCA) through inhibition of cell apoptosis mediated by endoplasmic reticulum stress pathway, reduce the apoptosis of liver cells induced by hydrophobic bile acid, improve the fine Cell viability. This study uses high glucose induced apoptosis of lens epithelial cell model, to observe the effect of TUDCA on apoptosis of lens epithelial cells and changes of endoplasmic reticulum apoptosis related proteins, to investigate the protective effect of TUDCA on lens epithelial cells in high glucose environment and its mechanism.
Human lens epithelial cells (human lens epithelial cells, HLECs) strain SRA01/04 containing 10% fetal bovine serum DMEM medium, the logarithmic growth phase were cultured for 24 hours in different concentrations of glucose, induced apoptosis of lens epithelial cell model, and using different concentrations of TUDCA (0.2,0.5,1.0,2.0mmol / L) to intervene. Detection of different concentrations of drug stimulation by MTT method after the cell proliferation inhibition rate; to observe the changes of cell apoptosis morphology by Hoechst 33258 fluorescence staining; apoptosis rate of flow cytometry after staining with Annexin V-FITC/PI; GRP78 Western blot to detect the expression of cell technology.
Result
The effect of 1. different concentrations of glucose on the activity of HLECs
The cells were incubated in different concentrations of glucose after 24 hours, the inhibition rate of glucose group were higher than the control group, and with the increase of glucose concentration, the inhibition of cell proliferation was increased (P0.01). The effect of glucose after 24 hours, cell proliferation inhibition rate of 250mmol / L glucose concentration is close to 50% for the HLECs apoptosis inducing concentration production apoptosis of lens epithelial cell model. Subsequent experiments showed that this concentration of glucose in cultured cells for 24 hours, typical changes of apoptotic nuclear morphology appears in most cells, the cell apoptosis rate was 24 hours (57.94 + 1.29)%.
Effect of 2. TUDCA intervention on HLECs activity in high concentration glucose environment
Compared with the control group, the cell proliferation inhibition rate of the model group increased significantly (P0.01), and the proliferation inhibition rate of TUDCA intervention group was significantly lower than that of the control group (P = 0.01).
The effect of 3. TUDCA intervention on the morphology of HLECs cells in high concentration glucose environment
Hoechst33258 staining showed that the control group had no obvious cell apoptosis, cells were well dispersed in 250mmol / L. The fluorescent glucose change visible typical morphology of apoptosis cells after 24 hours, the apoptosis of smaller cell volume, nuclear pyknosis, dense granular cytoplasm or nucleus strong fluorescence dense, adding 2.0mmol / LTUDCA after incubation, apoptotic cells decreased significantly.
Effect of 4. TUDCA intervention on the apoptosis rate of HLECs cells in high concentration glucose environment
The apoptotic rate of each group was detected by flow cytometry. Compared with the control group, the apoptotic rate of the model group (250mmol / L glucose) increased significantly, and the apoptotic rate decreased significantly after the intervention of TUDCA (P 0.01).
Effect of 5. TUDCA intervention on the expression of GRP78 protein in HLECs cells with high concentration of glucose
Western blot analysis showed that compared with the control group, the expression of GRP78 protein increased significantly after 24 hours of 250mmol / L glucose treatment, and GRP78 expression was significantly inhibited after TUDCA intervention (P0.05).
conclusion
High glucose can stimulate the endoplasmic reticulum stress and induce apoptosis of lens epithelial cells..TUDCA can inhibit the apoptosis of high glucose induced by endoplasmic reticulum stress pathway, and protect lens epithelial cells.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R776.1

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