c-FLIP蛋白在小鼠脓毒症急性肾损伤中的表达及意义  

Expression and significance of c-FLIP protein in sepsis mice with acute kidney injury

作　　者：樊恒[1] 乐健伟[1] 陈国栋[1] 孙敏[1] 叶继辉[1] 朱永定[1] 朱建华[1] Fan Heng;Le Jianwei;Chen Guodong;Sun Min;Ye Jihui;Zhu Yongding;Zhu Jianhua(Department of lntensive Care Unit,Ningbo First Hospital,Ningbo 315010,Zhefiang,China)

机构地区：[1]宁波市第一医院重症医学科,浙江宁波315010

出　　处：《中华危重病急救医学》2018年第12期1132-1136,共5页Chinese Critical Care Medicine

基　　金：浙江省宁波市自然科学基金(2017A610188);浙江省自然科学基金(LQ18H150001);浙江省医药卫生科技计划项目(2017KY134,2018KY669).

摘　　要：目的观察细胞型Fas相关死亡域样白细胞介素-1β转换酶抑制蛋白(c-FLIP)在小鼠脓毒症急性肾损伤(SAKI)中的表达并探讨其意义。方法按随机数字表法将30只雄性ICR小鼠分为正常对照组(Normal组)、假手术组(Sham组)和SAKI组,每组10只。采用盲肠结扎穿孔术(CLP)制备小鼠SAKI模型;Sham组不结扎、穿刺盲肠,其余手术步骤同SAKI组;Normal组不做任何处理。于CLP术后24h取小鼠眼眶血和肾脏组织,采用酶联免疫吸附试验(ELISA)检测血肌酐(SCr)和尿素氮(BUN)水平;取肾组织行苏木素-伊红染色,于光镜下观察肾组织病理学改变并评估损伤严重程度;用免疫组化法检测肾组织中c-FLIP表达;用蛋白质免疫印迹试验(Western Blot)检测肾组织中c-FLIP、Bax和天冬氨酸特异性半胱氨酸蛋白酶3(caspase-3)蛋白表达。采用Pearson法分析SAKI时肾组织中c-FLIP与Bax和caspase-3蛋白表达的相关性。结果Normal组和Sham组肾小管上皮细胞结构规则、完整,间质未见炎性细胞浸润,肾损伤评分均为(1.30±0.48)分;免疫组化显示肾小管上皮细胞可见大量c-FLIP阳性表达(IA:120.20±3.87、116.70±3.46);WesternBlot显示肾组织中c-FLIP呈高表达(c-FLIP/GAPDH:0.99±0.01、0.98±0.02),Bax和caspase-3呈低表达(Bax/GAPDH:0.16±0.04、0.19±0.03,caspase-3/GAPDH:0.24±0.04、0.23±0.05)。与Sham组相比,SAKI组肾小管上皮细胞变性、坏死,间质有大量炎性细胞浸润,肾损伤评分显著升高(分:4.60±0.52比1.30±0.48,P<0.01),血清SCr和BUN水平均显著升高[SCr(μmol/L):193.90±13.54比24.50±3.78,BUN(mmol/L):81.60±7.26比5.20±0.92,均P<0.01],肾组织中c-FLIP阳性细胞明显减少(IA:17.11±0.82比116.70±3.46,P<0.01),c-FLIP蛋白表达水平明显下降(c-FLIP/GAPDH:0.29±0.03比0.98±0.02,P<0.01),而Bax和caspase-3蛋白表达水平均明显升高(Bax/GAPDH:0.87±0.06比0.19±0.03,caspase-3/GAPDH:0.88±0.07比0.23±0.05,均P<0.01)。相关性分析显示,SAKI小鼠肾组织中c-FLIP蛋白与Bax蛋白Objective To observe the expression of cellular Fas-associated death domain-like interleukin-1β converting enzyme inhibit protein(c-FLIP)in sepsis mice with acute kidney injury(SAKI)and explore its significance.Methods Thirty male ICR mice were divided into the normal control group(Normal group),sham operation group(Sham group)and SAKI group by random number table method,with 10 mice in each group.The SAKI model of mice was established by cecal ligation and puncture(CLP);the Sham group was not ligated and the cecum was not punctured,and other surgical procedures were the same as the SAKI group;the Normal group did not experience any treatment.The serum and renal tissues of mice in each group were harvested 24 hours after CLP model establishment.The levels of serum creatinine(SCr)and blood urea nitrogen(BUN)were detected by enzyme linked immunosorbent assay(ELISA).The renal tissues were stained with hematoxylin-eosin(HE),and the pathological changes of renal tissues were observed under light microscope and the severity of injury was determined.The expression of c-FLIP in renal tissues was detected by immunohistochemistry.The expression of c-FLIP,Bax and caspase-3 protein in renal tissue was detected by Western Blot.The correlation between c-FLIP expression and Bax,caspase-3 protein expressions in renal tissues were analyzed by Pearson test.Results In the Normal group and the Sham group,the renal tubular epithelial cells were regular and intact,and no interstitial inflammatory cell infiltration was observed;the renal injury score was both 1.30±0.48;immunohistochemistry showed a large amount of c-FLIP positive expression in renal tubular epithelial cells(IA:120.20±3.87,116.70±3.46);Western Blot showed high expression of c-FLIP in renal tissues(c-FLIP/GAPDH:0.99±0.01,0.98±0.02),and low expressions of Bax and caspase-3(Bax/GAPDH:0.16±0.04,0.19±0.03,caspase-3/GAPDH:0.24±0.04,0.23±0.05).Compared with the Sham group,in the SAKI group,renal tubular epithelial cells were degenerated and necrosis,and a large numbe

关 键 词：C-FLIP 急性肾损伤 ICR小鼠 抑制蛋白 CASPASE-3蛋白表达 脓毒症 WESTERNBLOT 肾小管上皮细胞 

分 类 号：R459.7[医药卫生—急诊医学] R692[医药卫生—治疗学]