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(P<0.05)。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(P<0.05)。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(P<0.05)。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。
Objective To explore the pathogenic mechanism of myoglobin-induced endoplasmic reticulum stress （ERS） and apoptosis in tubular epithelial cells in acute kidney injury （AKI） mouse model of crush syndrome. Methods Eighteen C56BL/6 mice were randomly divided into control group, modeling 8 h group and modeling 24 h group. The AKI model of crush syndrome was established by intramuscular injection of 50 % glycerol saline solution into thigh （8 μL/g）, while equivalent volume of physiological saline was injected in control group. AKI was diagnosed when serum creatinine （sCr） level increased to double value of control group. The mice in the experimental groups were sacrificed at the time points of 8 h and 24 h after injection respectively. Serum Cr was detected and renal tissues was observed under electron microscopy. Apoptosis was detected by TUNEL technique. Marker proteins and mRNA of apoptosis and ERS were detected by immunohistochemistry and real-time PCR. Human kidney proximal tubular cell （HK-2） cells cultured in vitro were randomly divided into control, intervention 6 h and intervention 12 h groups. Control group were incubated in standard cell culture （DMEM/F12） and the two intervention groups were incubated in special DMEM/F12 in which ferrohemoglobin was added. After 6 h and 12 h incubation, the cells were collected and apoptosis was detected by flow cytometry. Results AKI model of crush syndrome was successfully established, which was proved with sCr doubling at the 8 h after the intramuscular injection of glycerol saline. Swelling of endoplasmic reticulum and mitochondria in proximal tubular epithelial cells was more obvious in the two model groups than that in control group. TUNEL staining showed the percentage of positive cells in AKI groups was higher than that in control group （P〈0.05）. Immunohistochemistry and real-time PCR showed the expressions of caspase3, caspasel2 and CHOP in AKI groups were higher than those in control group （P〈0.05）. Flow cytometry showed cell
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2015年第1期22-26,共5页
Journal of Sichuan University(Medical Sciences)
基金
国家自然科学基金面上项目(No.81270818)资助
