CAJ | 학술논문

目的：高糖高脂饮食加小剂量链脲佐菌素（streptozocin ，STZ）诱导大鼠糖尿病模型，尾静脉注射人重组腺相关病毒介导的截断突变型腺苷酸活化蛋白激酶基因AMPK‐CA（AMPKα1312，T172 D），观察AMPK基因治疗对糖尿病大鼠肾脏的保护作用并进行相关机制研究。方法24只雄性Wistar大鼠随机分为糖尿病组（n＝16）和普通饮食组（n＝8），糖尿病组以高糖高脂饮食加小剂量STZ诱导糖尿病模型，8周后再将其随机分为两组，分别经尾静脉导入表达持续激活型AMPK‐CA的重组腺相关病毒rAAV2‐AMPK‐CA和rAAV2‐GFP作为对照，观察12周后处死实验动物，留取肾脏组织。PAS染色观察比较各组大鼠肾脏的病理改变，Real‐time PCR法检测各组细胞外基质的 mRNA 水平变化， Western blot法检测Ⅳ型胶原蛋白α1（Col4α1）、腺苷酸活化蛋白激酶（AMPK）及磷酸化AMPK、磷酸化的乙酰辅酶A羧化酶（p‐ACC）、雷帕霉素靶蛋白（mTOR）、磷酸化的mTOR及其下游分子磷酸化水平改变。结果与正常对照组（C组）相比，转染rAAV2‐GFP组（GFP组）糖尿病大鼠肾脏内p‐AMPK和p‐ACC蛋白水平显著降低（均 P＜0.05）；与rAAV2‐GFP组相比，转染 rAAV2‐AMPK‐CA的糖尿病大鼠（CA组）体内p‐ACC表达活性显著升高，肾重/体重、肾小球体积、肾小球系膜基质增生等指标有明显改善，细胞外基质纤维连接蛋白（FN）、Col4α1、Col4α5 mRNA表达水平，以及Col4α1、信号分子p‐mTOR、磷酸化的真核延伸因子激酶2（p‐eEF2K）及磷酸化的起始因子4E结合蛋白1（p‐4EBP1）蛋白表达水平显著降低（均 P＜0.05）。结论 rAAV2介导的AMPK‐CA基因治疗可能通过增加糖尿病大鼠肾脏AMPK活性，抑制mTOR信号通路，缓解基质沉积，对糖尿病大鼠肾脏起保护作用。
목적：고당고지음식가소제량련뇨좌균소（streptozocin ，STZ）유도대서당뇨병모형，미정맥주사인중조선상관병독개도적절단돌변형선감산활화단백격매기인AMPK‐CA（AMPKα1312，T172 D），관찰AMPK기인치료대당뇨병대서신장적보호작용병진행상관궤제연구。방법24지웅성Wistar대서수궤분위당뇨병조（n＝16）화보통음식조（n＝8），당뇨병조이고당고지음식가소제량STZ유도당뇨병모형，8주후재장기수궤분위량조，분별경미정맥도입표체지속격활형AMPK‐CA적중조선상관병독rAAV2‐AMPK‐CA화rAAV2‐GFP작위대조，관찰12주후처사실험동물，류취신장조직。PAS염색관찰비교각조대서신장적병리개변，Real‐time PCR법검측각조세포외기질적 mRNA 수평변화， Western blot법검측Ⅳ형효원단백α1（Col4α1）、선감산활화단백격매（AMPK）급린산화AMPK、린산화적을선보매A최화매（p‐ACC）、뢰파매소파단백（mTOR）、린산화적mTOR급기하유분자린산화수평개변。결과여정상대조조（C조）상비，전염rAAV2‐GFP조（GFP조）당뇨병대서신장내p‐AMPK화p‐ACC단백수평현저강저（균 P＜0.05）；여rAAV2‐GFP조상비，전염 rAAV2‐AMPK‐CA적당뇨병대서（CA조）체내p‐ACC표체활성현저승고，신중/체중、신소구체적、신소구계막기질증생등지표유명현개선，세포외기질섬유련접단백（FN）、Col4α1、Col4α5 mRNA표체수평，이급Col4α1、신호분자p‐mTOR、린산화적진핵연신인자격매2（p‐eEF2K）급린산화적기시인자4E결합단백1（p‐4EBP1）단백표체수평현저강저（균 P＜0.05）。결론 rAAV2개도적AMPK‐CA기인치료가능통과증가당뇨병대서신장AMPK활성，억제mTOR신호통로，완해기질침적，대당뇨병대서신장기보호작용。
To investigate the protective effect of adenosine 5’‐monophosphate(AMP)‐activated protein kinase (AMPK)on the kidneys of diabetic rats and the involved mechanism after recombinant adeno‐associated viruses(rAAVs)encoding constitutively active AMPK‐CA(AMPKα1312 ,T172 D) were injected into the diabetic rat models (via tail veins)which were established by using streptozocin(STZ)as well as diets enriched in both glucose and fat.Methods A total of 24 male Wistar rats were randomly divided into normal control(n= 8)and experimental groups(n= 16).Rats in the experimental group were fed on high‐glucose and high‐fat diets and were injected with STZ to induce diabetes mellitus (DM). They were allocated to two subgroups randomly after 8 weeks.Diabetic rats in the two subgroups were injected with rAAV2 expressing AMPK‐CA gene(CA subgroup)or GFP gene(GFP subgroup ,as controls)through tail veins.Rats were killed and the kidneys removed after another 12 weeks. PAS staining was performed to evaluate the renal pathological changes. The expression of extracellular matrix mRNA was detected by Real‐time PCR and protein levels of collagen Ⅳ α1(Col4α1) ,AMPK ,p‐AMPK ,phospho‐acetyl‐CoA carboxylase (p‐ACC) ,mammalian target of rapamycin (mTOR) ,p‐mTOR and its downstream targets were measured by Western blot.Results The expressions of p‐AMPK and p‐ACC proteins were significantly declined in GFP subgroup compared with normal control group. rAAV2‐AMPK‐CA treatment could elevate p‐ACC expression and improve the pathological changes of diabetic nephropathy(kidney weight/body weight ,mean glomerular volume ,mesangial matrix area)in comparison with GFP subgroup. Furthermore ,rAAV2‐AMPK‐CA reversed the mRNA expressions of FN ,Col4α1 and Col4α5 and protein levels of Col4α1 ,p‐mTOR ,p‐eEF2K and p‐4EBP1 which were increased in the GFP group.Conclusion rAAV2‐AMPK‐CA gene treatment could protect the kidney in diabetic rats by increasing the activity of AMPK in the kidney of diabetic rats ,inhibiting the activation of mTOR signaling pathway and alleviating the extracellular matrix deposition.