草酸钙结晶诱导肾小管上皮细胞内质网应激的研究

发布时间：2018-05-17 19:17

本文选题：草酸钙 + 人肾小管上皮细胞　；参考：《沈阳医学院》2017年硕士论文

【摘要】：目的内质网是蛋自翻译后修饰的场所,内质网应激被视为诱导细胞发生凋亡的第二条信号通路,是诱发多种慢性、代谢性疾病发病的重要途径。本项目研究肾结石形成与内质网应激的关系。方法本研究采用100μg/mL的草酸钙结晶刺激HK-2人肾小管上皮细胞3h、6h、12h和24h,Oh为对照组,用Real-time PCR法检测内质网应激标志基因BIP/GRP78,同时检测内质网应激相关信号通路中CHOP、PERK、JNK、P38和CasPase-3等基因mRNA的表达情况,用Western blot法检测内质网应激相关蛋白BIP/GRP78、CHOP、P-PERK、P-JNK、P-P38、XBP1、Cleaved-CasPase-3 和CasPase-12的表达情况,用TUNEL法检测草酸钙结晶刺激24h时细胞凋亡的情况。结果用100μg/mL的草酸钙结晶刺激HK-2细胞后,内质网应激标志基因BIP/GRP78和CHOP基因的mRNA表达升高,内质网应激信号通路中的PERK、JNK、P38基因的mRNA表达均升高。当用草酸钙结晶刺激HK-2细胞6h,BIP/GRP78蛋白表达升高,暴露12h,CHOP蛋白表达升高与对照组Oh相比具有统计学差异,P-PERK、P-JNK、P-P38和XBP1蛋白分别在草酸钙结晶刺激3h、3h、6h和3h时蛋白表达与对照组相比升高,Western blot检测结果与Real-time RCR结果一致。TUNEL检测发现24小时结晶暴露后细胞发生凋亡,凋亡率约达25%。Western blot显示内质网应激特异性蛋白CasPase-12和Cleaved-CasPase-3的表达升高。结论我们研究发现草酸钙结晶诱导肾小管上皮细胞发生了内质网应激,结晶诱导的肾小管细胞凋亡是通过线粒体凋亡通路和内质网凋亡通路共同引起的。这些发现提示内质网应激可能是肾结石形成过程的重要发病机制。
[Abstract]:Objective the endoplasmic reticulum (ER) is a place where eggs are modified after translation. Endoplasmic reticulum stress is regarded as the second signal pathway to induce apoptosis, and it is an important way to induce many chronic and metabolic diseases. The purpose of this study was to study the relationship between renal stone formation and endoplasmic reticulum stress. Methods in this study, 100 渭 g/mL calcium oxalate crystals were used to stimulate HK-2 human renal tubular epithelial cells for 12 h and 24 h respectively as control group. The expression of the endoplasmic reticulum stress marker gene BIP-GRP78, the expression of CHOPPERKP38 and CasPase-3 genes in the endoplasmic reticulum stress-related signaling pathway and the expression of BIPP / GRP78 CHOPP-PERKP-JNKP-P38 and XBP1Cleaved-CasPase-3 and CasPase-12 were detected by Real-time PCR method, and the expression of BIP-PERKP-JNKP-38-XBP1Cleaved-Caspase-3 and CasPase-12 were detected by Western blot method. The apoptosis of calcium oxalate after 24 h was detected by TUNEL method. Results after 100 渭 g/mL calcium oxalate was used to stimulate HK-2 cells, the mRNA expression of endoplasmic reticulum stress marker gene BIP/GRP78 and CHOP gene was increased, and the mRNA expression of PERKN JNKN P38 gene in endoplasmic reticulum stress signaling pathway was increased. The expression of BIPP / GRP78 protein was increased in HK-2 cells stimulated by calcium oxalate crystallization for 6 h. There were significant differences in the expression of chop protein between the control group and the control group at 12 h after exposure. The protein expression of P-PERKU P-JNKP-P38 and XBP1 was increased at 3h and 3h after the crystallization of calcium oxalate, respectively. The results of Western blot and Real-time RCR were consistent with those of the control. It was found that cell apoptosis occurred after 24 hours of crystallization exposure. Apoptosis rate up to 25%.Western blot showed increased expression of ER stress-specific proteins CasPase-12 and Cleaved-CasPase-3. Conclusion We found that calcium oxalate crystallization induced endoplasmic reticulum stress in renal tubular epithelial cells. The apoptosis of renal tubular cells induced by calcium oxalate was induced by mitochondrial apoptosis pathway and endoplasmic reticulum apoptosis pathway. These findings suggest that endoplasmic reticulum stress may play an important role in the formation of renal calculi.
【学位授予单位】：沈阳医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692.4

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