高糖、高同型半胱氨酸环境对足细胞内质网应激相关蛋白GRP78及CHOP表达的影响

发布时间：2018-03-17 17:23

本文选题：葡萄糖　切入点：同型半胱氨酸　出处：《山西医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：观察体外高糖和/或高同型半胱氨酸(high homocysteine, hHcy)环境下，小鼠肾小球足细胞内质网应激相关蛋白：葡萄糖调节蛋白78(glucose regulatedprotein78, GRP78/BiP)、生长阻滞和DNA损伤诱导基因153(growth arrest andDNAdamage inducible gene153, CHOP/GADD153)的表达，初步探讨高糖和/或hHcy诱导内质网应激(endoplasmic reticulum stress, ERS)导致足细胞凋亡的相关机制，观察抗氧化剂白藜芦醇(resveratrol, RES)对足细胞的保护作用。方法：分化后的条件永生化小鼠足细胞分6组：正常对照组、高渗对照组(葡萄糖5mmol/L+甘露醇15mmol/L)、高糖组(葡萄糖20mmol/L)、hHcy组(hHcy200umol/L)、hHcy+高糖组(hHcy200umol/L+葡萄糖20mmol/L)、混合+RES组(RES20umol/L预先干预半小时+hHcy200umol/L+葡萄糖20mmol/L)，作用24小时后结束干预。采用流式细胞术AnnexinV-FITC/PI法测定各组细胞凋亡率；Real-time PCR技术分析各组细胞GRP78、CHOP mRNA的表达情况；Western blot技术评价各组细胞GRP78、CHOP蛋白的表达情况。结果：高糖组的凋亡率、GRP78、CHOP mRNA及蛋白表达明显高于正常对照组及高渗对照组(P 0.05)；hHcy组的凋亡率、GRP78、CHOP mRNA及蛋白表达显著高于正常对照组(P 0.05)；hHcy+高糖组的凋亡率、GRP78、CHOPmRNA及蛋白表达分别与高糖组、hHcy组比较均显著升高(P 0.05)；混合+RES组较hHcy+高糖组，凋亡率、GRP78、CHOP mRNA及蛋白表达显著下降(P 0.05)，上述各组差异均有统计学意义；高渗对照组较正常对照组,凋亡率、GRP78、CHOP mRNA及蛋白表达水平无显著变化(P0.05)，差异无统计学意义。结论：体外高糖、hHcy环境能诱导小鼠足细胞凋亡，hHcy和高糖共同环境下对足细胞损伤及致凋亡作用更明显，提示hHcy、高糖两者有协同作用；内质网应激参与其凋亡过程，，抗氧化剂RES能抵抗高糖和hHcy共同环境下诱导的足细胞凋亡，阻断内质网应激CHOP通路为其机制之一。
[Abstract]:Aim: to investigate the expression of high homocysteine (hHcyine) stress associated proteins in glomerular podocyte endoplasmic reticulum (ER): glucose regulated protein 78 (GRP 78 / BiPN), growth arrest and DNA damage inducible gene 153 arrest andDNAdamage inducible genetics 153, CHOP- GADD153) in high glucose and / or hyperhomocysteine (hHcyine) environment in vitro. To investigate the mechanism of endoplasmic reticulum stress induced by high glucose and / or hHcy in podocyte apoptosis, and to observe the protective effect of antioxidant resveratrol on podocyte. Methods: the conditioned immortalized mouse podocytes after differentiation were divided into 6 groups: normal control group. The hypertonic control group (glucose 5 mmol / L mannitol 15 mmol / L), the high glucose 20 mmol / L Hcy group (20 mmol / L hHcy group), the hHcy200 mol / L glucose 20 mmol / L group, the mixed RES group Res 20 mol / L pretreated with hHcy200umol/L 20 mmol / L L / L, and the flow cytometry AnnexinV-FITC/PI / L were used. Methods the expression of GRP78 chop mRNA was analyzed by real-time PCR and Western blot was used to evaluate the expression of GRP78 chop protein. Results: the expression of GRP78 / CHOPmRNA and protein in high glucose group was significantly higher than that in normal control group and hyperosmotic control group. The expression of GRP78 / CHOPmRNA and protein in hyperosmotic control group was significantly higher than that in normal control group (P 0.05 Hcy group) and the expression of GRP78 CHOPmRNA and protein in hyperosmotic control group was significantly higher than that in control group (P 0.05 Hcy high glucose group). Compared with high glucose group, Hcy group significantly increased P 0.05, and mixed RES group was higher than hHcy high glucose group. The expression of GRP78 chop mRNA and protein decreased significantly (P 0.05), and there was no significant difference between the hypertonic control group and the normal control group in the expression of GRP78 chop mRNA and protein (P 0.05), but there was no significant difference in the expression of GRP78 chop mRNA and protein between the hypertonic control group and the normal control group (P 0.05). Conclusion: Hcy in vitro can induce apoptosis of podocyte in mice. Hcy can induce apoptosis and damage of podocyte in vitro, suggesting that hHcyand high glucose have synergistic effect, endoplasmic reticulum stress is involved in the apoptosis process of podocyte. The antioxidant RES can resist the apoptosis of podocyte induced by high glucose and hHcy. Blocking endoplasmic reticulum stress CHOP pathway is one of its mechanisms.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.2;R692

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