牛磺酸熊脱氧胆酸对高糖环境下晶状体上皮细胞保护作用的实验研究

发布时间：2018-02-13 15:14

本文关键词： 白内障牛磺酸熊脱氧胆酸内质网应激细胞凋亡葡萄糖　出处：《山东大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的内质网是细胞内蛋白质修饰和折叠的场所,未折叠或错误折叠蛋白质在内质网腔中蓄积以及细胞内环境的破坏将导致内质网应激(endoplasmic reticulum stress, ERS)。适宜的应激有利于细胞内环境的恢复,严重而持久的内质网应激反应则可诱导细胞发生凋亡。已有研究证实,白内障的发生也与内质网应激有关,在葡萄糖缺乏、高血糖、同型半胱氨酸、衣霉素等内质网应激刺激因子作用下,均可诱导晶状体上皮细胞发生内质网应激,导致细胞凋亡。近年发现,牛磺酸熊脱氧胆酸(tauroursodeoxycholic acid,TUDCA)可通过抑制内质网应激途径介导的细胞凋亡,有效减轻疏水性胆汁酸诱导的肝细胞凋亡,提高细胞生存能力。本研究采用高浓度葡萄糖诱导建立晶状体上皮细胞凋亡模型,旨在观察TUDCA对晶状体上皮细胞凋亡的影响以及内质网凋亡途径相关蛋白的变化,探讨TUDCA对高浓度葡萄糖环境下晶状体上皮细胞的保护作用及其机制。方法人晶状体上皮细胞(human lens epithelial cells, HLECs)株SRA01/04用含10%胎牛血清的DMEM完全培养基培养,取对数生长期细胞在不同浓度葡萄糖培养液中培养24小时,诱导建立晶状体上皮细胞凋亡模型,并采用不同浓度TUDCA(0.2、0.5、1.0、2.0mmol／L)进行干预。应用MTT法检测不同浓度药物刺激后的细胞增殖抑制率；应用Hoechst 33258荧光染色法观察凋亡细胞核形态学改变；Annexin V-FITC/PI双染后流式细胞仪检测细胞凋亡率；Western blot技术检测细胞GRP78的表达。结果 1.不同浓度葡萄糖对HLECs活性的影响不同浓度葡萄糖孵育细胞24小时后,各浓度葡萄糖组增殖抑制率均高于对照组,且随着葡萄糖浓度的增加,细胞增殖抑制率亦升高(P0.01)。取葡萄糖作用24小时后细胞增殖抑制率接近50%的250mmol／L葡萄糖浓度为HLECs凋亡诱导浓度,制作晶状体上皮细胞凋亡模型。后续实验显示,在此浓度葡萄糖培养液中培养细胞24小时,多数细胞出现典型凋亡细胞核形态改变,24小时细胞凋亡率为(57.94±1.29)%。 2. TUDCA干预对高浓度葡萄糖环境下HLECs活性的影响与对照组相比,模型组细胞增殖抑制率明显升高(P0.01)；与模型组相比,TUDCA干预各组细胞增殖抑制率明显降低(P均0.01)。 3. TUDCA干预对高浓度葡萄糖环境下HLECs细胞形态学的影响 Hoechst33258染色可见,对照组细胞无明显凋亡发生,细胞呈弥散均匀荧光。以250mmol／L葡萄糖培养细胞24小时后可见典型的凋亡细胞形态改变,凋亡细胞体积变小,核致密、固缩,核或细胞质内可见致密的颗粒状强荧光,加入2.0mmol／LTUDCA共同培养后,凋亡细胞明显减少。 4. TUDCA干预对高浓度葡萄糖环境下HLECs细胞凋亡率的影响流式细胞术检测各组细胞凋亡率,与对照组相比,模型组(250mmol／L葡萄糖)细胞凋亡率显著增高,加入TUDCA干预后细胞凋亡率明显降低(P均0.01)。 5. TUDCA干预对高浓度葡萄糖环境下HLECs细胞GRP78蛋白表达的影响 Western blot检测显示,与对照组相比,250mmol／L葡萄糖作用细胞24小时后GRP78蛋白表达显著增加,加入TUDCA干预后GRP78表达明显受到抑制(P0.05)。结论高浓度葡萄糖可以刺激晶状体上皮细胞发生内质网应激,诱导细胞凋亡。TUDCA可通过内质网应激途径抑制高浓度葡萄糖诱导的细胞凋亡,对晶状体上皮细胞产生保护作用。
[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.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R776.1

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