高同型半胱氨酸血症通过内质网应激抑制ApoE敲除鼠肾脏CFTR的表达  

Hyperhomocysteinemia Inhibit Cystic Fibrosis Transmembrane Conductance Regulator Expression of Apolipoprotein E-knockout Mice Renal via Endoplasmic Reticulum Stress

作　　者：马文斌[1] 张辉[1] 赵丽[1] 周龙霞[2] 陈久凯 王艳华[1] 杨晓玲[2] 田珏[2] 张鸣号[2] 徐华[2] 姜怡邓[2,3] 

机构地区：[1]宁夏医科大学检验学院,宁夏银川市750004 [2]宁夏医科大学基础医学院,宁夏银川市750004 [3]宁夏医科大学总医院,宁夏银川市750004

出　　处：《中国动脉硬化杂志》2015年第3期237-242,共6页Chinese Journal of Arteriosclerosis

基　　金：国家自然科学基金资助项目(81160044;81360073);宁夏教育厅项目(NGY2012063;NGY2012056)

摘　　要：目的探讨高同型半胱氨酸血症通过内质网应激抑制载脂蛋白E基因敲除(Apo E-/-)鼠肾脏囊性纤维化跨膜转导调节因子(CFTR)的表达。方法实验分组:正常对照组(n=6):选择健康5周龄雄鼠(SPF级C57BL/6J),饲以正常饮食。另选5周龄雄性纯合子Apo E-/-鼠(SPF级近交系c57BL/6)18只,随机分为3组,每组6只:Apo E-/-对照组、高蛋氨酸组、干预组,分别给予正常饮食、高蛋氨酸饮食、高蛋氨酸饮食加0.006%叶酸及0.0004%维生素B12。喂养14周后眼球取血,分离血清,用酶联免疫吸附法检测血清同型半胱氨酸水平;实时定量荧光PCR检测小鼠肾脏CFTR、葡萄糖调节蛋白78(GRP78)、活化转录因子6(ATF6)、蛋白激酶样内质网激酶(PERK)、C/EPB同源蛋白(CHOP)mRNA的表达以及免疫组织化学法检测小鼠肾脏CFTR蛋白的表达。结果Apo E-/-对照组、高蛋氨酸组及干预组中血清同型半胱氨酸水平均增加,其中以高蛋氨酸组增加最显著(P<0.01)。实时定量荧光PCR结果显示,高蛋氨酸组CFTR mRNA的表达较正常对照组和Apo E-/-对照组显著降低(P<0.01,P<0.05),干预组CFTR mRNA的表达较高蛋氨酸组显著增加(P<0.01);免疫组织化学法检测CFTR蛋白的表达与其mRNA的表达变化一致。高蛋氨酸组GRP78、ATF6、PERK、CHOP mRNA的表达较正常对照组和Apo E-/-对照组显著增加(P<0.01),干预组GRP78、ATF6、PERK、CHOP mRNA的表达较高蛋氨酸组显著降低(P<0.01)。小鼠肾脏GRP78、ATF6、PERK、CHOP mRNA表达与CFTR mRNA表达的相关系数分别为:r=-0.7192,P<0.01;r=-0.5501,P<0.01;r=-0.7772,P<0.01;r=-0.6785,P<0.01。结论高同型半胱氨酸血症可能通过内质网应激抑制Apo E敲除鼠肾脏CFTR的表达。Aim To explore the hyperhomocysteinemia( HHcy) inhibit cystic fibrosis transmembrane conductance regulator( CFTR) expression of apolipoprotein E-knocked-out( Apo E- /-) mice renal via endoplasmic reticulum stress( ERS). Methods 18 male Apo E- /-mice were randomly divided into 3 groups( n = 6 for each group) :Apo E- /-control group,high methionine group and intervention group. They were respectively fed with nomal diet and high methionine diet and high methionine diet plus 0. 006% folate and 0. 0004% vitamin B12 for 14 weeks. After 14 weeks,the blood of the mice was gotten using a unilateral enucleation method,the serum homocysteine( Hcy) was detected by enzyme-linked immunosorbent assay( ELISA). The protein and mRNA expressions of CFTR in the renal of mice were detected by immunohistochemical assay and real time polymerase chain reaction( RT-PCR); the mRNA expressions of glucose regulated protein 78( GRP78),activating transcription factor 6( ATF6),protein kinase RNA-like endoplasmic reticulum kinase( PERK),C / EPB homologous protein( CHOP) in the renal of mice were detected by RT-PCR. Results The result showed that the content of Hcy was significantly higher in Apo E- /-control group,high methionine group and intervention group; an remarkable increase was observed in the high methionine group( P < 0. 01). The results of RT-PCR and immunohistochemical assay exposed a remarkable down-expressed CFTR mRNA and protein in high methionine group( P < 0. 01). The results of RT-PCR exposed a remarkable up-expressed GRP78,ATF6,PERK,CHOP mRNA in high methionine group,while intervention group decreased GRP78,ATF6,PERK,CHOP levels compared with high methionine group. There were negative correlations between GRP78,ATF6,PERK,CHOP mRNA and CFTR mRNA( r=- 0. 7192,P < 0. 01; r =- 0. 5501,P < 0. 01; r =- 0. 7772,P < 0. 01; r =- 0. 6785,P < 0. 01). Conclusion HHcy inhibits CFTR expression of Apo E-knockout mice renal might via endoplasmic reticulum stress.

关 键 词：高同型半胱氨酸血症 内质网应激 囊性纤维化跨膜转导调节因子 

分 类 号：R285.5[医药卫生—中药学]