肌红蛋白诱导的内质网应激及细胞凋亡在挤压综合征中的机制初探

发布时间：2018-02-27 06:23

本文关键词： 挤压综合征肌红蛋白内质网应激细胞凋亡　出处：《四川大学学报(医学版)》2015年01期 　论文类型：期刊论文

【摘要】：目的建立挤压综合征急性肾损伤(AKI)小鼠模型,探究肌红蛋白诱导的肾小管上皮细胞内质网应激及细胞凋亡在挤压综合征中的致病机制。方法体内实验:将C56BL/6小鼠随机分为对照组、模型组8h及模型组24h,每组6只,模型组于小鼠大腿外侧肌注50%甘油生理盐水溶液(8μL/g)建立挤压综合征模型,对照组注入等量生理盐水。分别于注射后8、24h麻醉处死动物,检测血清肌酐(sCr),获取完整肾脏标本进行电镜及TUNEL染色观察凋亡,采用免疫组化、实时荧光定量PCR等检测凋亡及内质网应激相关蛋白表达。体外实验:将人近端肾小管上皮细胞随机分为对照组、干预组6h及干预组12h,对照组加入标准细胞培养液,干预组加入等量含马肌红蛋白的细胞培养液,分别于6、12h后收取细胞进行流式分析检测细胞凋亡。结果注射甘油8h后sCr明显升高,挤压综合征模型建立成功。电镜示与对照组相比,模型组肾小管上皮细胞内质网、线粒体等细胞器肿胀较明显。TUNEL染色提示模型组肾组织凋亡细胞百分比高于对照(P0.05)。免疫组化染色和实时荧光定量PCR示模型组肾组织凋亡标志蛋白半胱氨酸天冬氨酸蛋白酶(caspase)3及内质网应激标志蛋白CCAAT增强子结合蛋白同源蛋白(CHOP)、caspase12表达高于对照组(P0.05)。细胞流式分析提示马肌红蛋白干预组细胞凋亡比例较对照组升高(P0.05)。结论内质网应激及凋亡参与了挤压综合征导致AKI的发病机制,肌红蛋白可能是诱导细胞凋亡的重要因素。
[Abstract]:Objective to establish a mouse model of acute renal injury induced by squeeze syndrome. To explore the mechanism of endoplasmic reticulum stress and apoptosis of renal tubular epithelial cells induced by myoglobin in squeeze syndrome. Methods: C56BL / 6 mice were randomly divided into control group (8 h) and model group (24 h) with 6 mice in each group. The model group was injected with 50% 渭 L / g glycerol saline solution on the lateral thigh of mice to establish squeeze syndrome model, and the control group was injected with the same amount of saline. The animals were anesthetized and killed at 824 h after injection. The serum creatinine was detected, the intact kidney samples were obtained, the apoptosis was observed by electron microscope and TUNEL staining, and the apoptosis was observed by immunohistochemistry. In vitro, the human proximal tubular epithelial cells were randomly divided into control group, intervention group for 6 hours and intervention group for 12 hours, the control group was supplemented with standard cell culture medium. In the intervention group, the cell culture medium containing equine myoglobin was added, and the cell apoptosis was detected by flow cytometry after 61h. Results the sCr increased significantly after 8 h of glycerol injection. The model of squeeze syndrome was established successfully. Compared with the control group, the model group showed the endoplasmic reticulum of renal tubular epithelial cells. Tunel staining showed that the percentage of apoptotic cells in renal tissue in model group was higher than that in control group P0.05.The immunohistochemical staining and real-time fluorescence quantitative PCR showed the apoptotic marker protein cysteine aspartate in renal tissue of model group. The expression of protease Caspase-3 and endoplasmic reticulum stress marker protein CCAAT enhancer protein homologue protein (CHOPN) caspase12 was higher than that of control group (P 0.05). Cell flow analysis showed that the percentage of apoptosis in equine myoglobin intervention group was higher than that in control group. Conclusion the endoplasmic reticulum should be increased. Shock and apoptosis are involved in the pathogenesis of AKI induced by squeeze syndrome. Myoglobin may be an important factor in inducing apoptosis.
【作者单位】：四川大学华西医院肾脏内科;
【基金】：国家自然科学基金面上项目(No.81270818)资助
【分类号】：R685

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