CHOP在糖尿病大鼠膀胱内质网应激中的作用研究

发布时间：2018-12-23 10:46

【摘要】：目的:通过观察CHOP在糖尿病大鼠DSM细胞内质网应激中的表达变化,探讨内质网应激与糖尿病膀胱的关系。通过研究CHOP在糖尿病膀胱发生发展中的变化及作用,为DCP的病因学研究提供依据和其治疗提供新的靶点。方法:健康成年雌性Sprague-Dawley(SD)大鼠45只,随机分为一组正常对照组和四组实验组,实验组通过腹腔注射链脲菌素50mg/kg诱导1型糖尿病大鼠模型,注射后72小时尾静脉采血,血糖浓度300mg/dl为成膜标准,正常对照组腹腔注射同等剂量生理盐水,于不同的时点(造模成功后4周,8周,12周,16周),切除膀胱组织,电镜下观察内质网超微结构,应用TUNEL技术检测糖尿病大鼠膀胱逼尿肌细胞的凋亡水平,RT-PCR技术测定DSM细胞内质网应激标志性因子CHOPmRNA的表达水平,western-blotting技术测定DSM细胞内质网应激标志性因子CHOP蛋白的表达水平。结果:电镜下,正常对照组内质网排列整齐,实验组由4周至16周内质网排列逐渐紊乱,肿胀程度不断加重,并出现融合,呈空泡样结构,附着核糖小体增多,逼尿肌细胞核扭曲甚至塌陷。正常对照组,凋亡DSM细胞少见,细胞核染色浅,实验组由4周至16周凋亡DSM细胞明显增多,细胞核染色逐渐加深,逼尿肌细胞凋亡率不断升高,并且都高于正常对照组。RT-PCR结果显示,4W组、8W组、12W组、16W组CHOP mRNA表达水平逐渐增加,并且都高于正常对照组。western-blotting结果显示,实验组由4W组至16W组CHOP蛋白表达水平明显高于正常对照组,并呈递增趋势。结论:内质网应激参与了糖尿病大鼠DSM细胞凋亡过程,CHOP与糖尿病膀胱病程进展呈正相关。ERS在DCP发生发展中如何发生作用以及CHOP通过哪些途径影响逼尿肌细胞功能仍不清楚,今后我们将围绕时间序列做进一步的研究。
[Abstract]:Aim: to investigate the relationship between endoplasmic reticulum stress and diabetic bladder by observing the expression of CHOP in DSM cells of diabetic rats. By studying the changes and roles of CHOP in the development of diabetic bladder, we can provide a new target for the etiological study and therapy of DCP. Methods: Forty-five healthy adult female Sprague-Dawley (SD) rats were randomly divided into a normal control group and four experimental groups. Type 1 diabetic rats were induced by intraperitoneal injection of streptozotocin (50mg/kg) in the experimental group. 72 hours after injection, blood samples were collected from the tail vein, the blood glucose concentration (300mg/dl) was taken as the membrane forming standard, and the normal control group received intraperitoneal injection of the same dose of normal saline at different time points (4 weeks, 8 weeks, 12 weeks, 16 weeks after successful modeling) to remove bladder tissue. The ultrastructure of endoplasmic reticulum (ER) was observed under electron microscope, the apoptosis level of bladder detrusor muscle cells in diabetic rats was detected by TUNEL technique, and the expression of endoplasmic reticulum stress marker factor (CHOPmRNA) in DSM cells was detected by RT-PCR technique. Western-blotting technique was used to detect the expression of CHOP protein, the iconic factor of endoplasmic reticulum stress in DSM cells. Results: under electron microscope, the endoplasmic reticulum was arranged neatly in the normal control group, and the endoplasmic reticulum in the experimental group was gradually disordered from 4 weeks to 16 weeks, the swelling degree was getting worse, and the swelling degree was increasing. The endoplasmic reticulum appeared vacuolar structure, and the number of attached ribose bodies increased. The nucleus of the detrusor muscle distorts or even collapses. In the normal control group, apoptotic DSM cells were rare and the nuclear staining was shallow. In the experimental group, the apoptotic DSM cells increased significantly from 4 weeks to 16 weeks, the nuclear staining gradually deepened, and the apoptosis rate of detrusor muscle cells increased continuously. RT-PCR results showed that the expression of CHOP mRNA in 4W, 8W, 12W and 16W groups increased gradually, and were higher than those in normal control group. Western-blotting results showed that the expression of CHOP mRNA in 4W group, 8W group, 12W group and 16W group were higher than that in normal control group. The expression of CHOP protein in the experimental group from 4 W to 16 W was significantly higher than that in the normal control group and showed an increasing trend. Conclusion: endoplasmic reticulum stress is involved in the apoptosis of DSM cells in diabetic rats, and CHOP is positively correlated with the progression of diabetic bladder. It is not clear how ERS plays a role in the development of DCP and how CHOP affects the function of detrusor cells. In the future, we will do further research around time series.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R587.2

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