siRNA沉默过度内质网应激下JNK基因在缺血/再灌注肺损伤中的作用  

Effect of siRNA silencing the role of JNK gene in excessive endoplasmic reticulum stress on lung ischemia/reperfusion injury

作　　者：郝卯林[1] 赵珊[1] 陈海娥[1] 陈丹[1] 宋冬[1] 何金波[1] 汪洋[1] 王万铁[1] 

机构地区：[1]温州医科大学缺血/再灌注损伤研究所,浙江温州325035

出　　处：《中国应用生理学杂志》2014年第1期48-53,共6页Chinese Journal of Applied Physiology

基　　金：浙江省公益技术应用研究项目(2011C37092);浙江省中医药重点研究计划项目(2013ZZ011);浙江省医药卫生科技计划项目(2010KYA132);浙江省中医药科技计划项目(2010ZB088)

摘　　要：目的:探讨siRNA沉默过度内质网应激(ERS)下C-Jun氨基末端激酶(JNK)通路在缺血/再灌注肺凋亡中的作用。方法:采用C57BL/6J小鼠左侧肺原位缺血/再灌注(I/R)损伤模型。实验随机分为7组(n=10),包括假手术组(Sham组),缺血/再灌注组(I/R组),PBS+Lipofectamine2000TM转染试剂组(I/R+PBS+Lipo组),阴性对照组(I/R+SCR组),JNK-siRNA组(I/R+siRNAJNK1组、I/R+siRNAJNK2组、I/R+siRNAJNK3组)。各组实验结束后处死小鼠,留取左肺,分别检测肺湿干重比(W/D)和总肺水含量(TLW);光镜观察肺组织形态结构变化,并进行肺组织损伤评估(IQA);RT-PCR、检测JNK和葡萄糖调节蛋白78(GRP78)的基因表达;Western blot检测磷酸化JNK(p-JNK)和GRP78的蛋白表达;原位缺口末端标记法(TUNEL法)检测肺细胞凋亡指数(AI)。结果:与Sham组相比,I/R+PBS+Lipo组和SCR组各组W/D、TLW、IQA、JNK与GRP78 mRNA和p-JNK、GRP78蛋白质表达量及AI均明显升高(P<0.01),光镜显示肺组织结构出现明显损伤性变化,且各组两两比较,上述各指标均无统计学差异;与I/R+PBS+Lipo组和SCR组相比,JNK-siRNA各组GRP78表达水平无明显变化,余各指标均降低(P<0.05,P<0.01)且肺组织损伤减轻。结论:I/R引起肺组织细胞发生过度的ERS,并通过JNK通路引起细胞凋亡,损伤肺组织。Objective： To investigate the effect of siRNA silencing the role of C-Jun N-terminal Kinase （JNK） gene in excessive endo- plasmic reticulum stress on lung ischemia./reperfusion injury. Methods： Mouse model of pulmonary ischemia reperfusion injury（PIRI） in situ was established with unilateral lung /n vivo. Seventy experimental mice were randomly allocated into seven groups（ n = 10）： Sham group （Sham group）, ischemia reperfusion group （I/R）, PBS ＋ Lipofectamine2（X）ffI＇M transfection reagent group （I/R ＋ PBS ＋ Lipo group）, nega- tive control group （I/R ＋ SCR group）, JNK-siRNA group （I/R ＋ siRNAj^r~I, siRNAjNK2, siRNAj^3）.Mice were euthanized after experimental time out, and left lung tissue was extracted. Wet/dry lung weight ratio （W/D） and total lung water content （TLW） were tested. Light micro- scope, alveolar damage quantitative evaluation index （IQA） and electron microscope were observed. The expression levels of JNK and glucose regulatex protein（GRP78） were detected by RT-PCR and Western blot. Apeptosis of lung tissue was determined by TUNEL. Results： Com- pared with Sham group, all indicators above of I/R ＋ PBS ＋ Lipo group and I/R ＋ SCR group were significantly increased （ P 〈 0.01 ）, and compared with I/R group, those indicators of the three groups all had no notable difference; those indicators were not statistically different be- tween I/R ＋ PBS ＋ Lipe group and I/R ＋ SCR goup, and compared to the three groups, the above indicators in JNK-siRNA group were lower （ P 〈 0.05 , P 〈 0.01） except that the expression levels of GRP78 was not statistically different. Condusion： I/R induces excessive ERS in lung tissue, in which JNK pathway participates in apoptosis, leading to lung tissue injury.

关 键 词：内质网应激 JNK 缺血再灌注损伤 细胞凋亡 

分 类 号：R363[医药卫生—病理学]