心肌细胞内质网应激介导APPswe/PS1dE9转基因成年小鼠发生心肌肥厚的研究

摘要

目的:探讨内质网应激在APPswe/PS1dE9转基因成年小鼠发生心肌肥厚中的作用.方法:选择APPswe/PS1dE9转基因成年小鼠及野生型(WT)成年小鼠各10只,分别为转基因实验组及对照组、野生型实验组及对照组,每组各5只,实验组背部皮下注射低剂量异丙肾上腺素(ISO)(2 mg/kg),每日1次,连续4周;对照组注射同等体积的生理盐水,4周后麻醉各组小鼠,描记体衷心电图,计算心率,测量体重后处死,取出心脏,测量心脏质量及左室质量,计算左心室重量指数(LV厂/BW)及全心重量比(HW/BW).用于HE染色法测定心肌细胞病理形态学变化,应用Western Blot方法及免疫组织化学方法检测内质网应激相关蛋白即GRP78、JNK、P-JNK及CaMKII的表达.结果:APPswe/PSldE9转基因小鼠在低剂量的ISO诱导后较WT小鼠心脏明显肥大,APPswe/PS1dE9转基因实验组较转基因对照组及WT小鼠实验组及对照组HW/BW及LVW/BW显著增加(P＜0.05);HE染色可见APPswe/PS1dE9转基因实验组较转基因对照组及WT实验组、对照组心肌细胞直径明显变大,细胞密度变小,毛细血管密度减少,细胞间质增多,细胞间距变大.Western blot结果显示APPswe/PS1 dE9转基因实验组GRP78、p-JNK及CaMKII的表达较转基因对照组及WT两组明显增加(P＜0.05,P＜0.01),而转基因对照组与WT两组无明显差异.免疫组化结果显示APPswe/PS1dE9转基因实验组GRP78及CaMKII蛋白在心肌细胞胞浆中表达阳性率80％,明显高于野生型实验组(40％)(P＜0.05),两组对照组表达均为阴性;转基因实验组p-JNK表达为90％,JNK表达为30％,其它三组均为阴性.结论:APPswe/PSl dE9转基因成年小鼠在较低剂量的ISO诱导后较WT实验组更容易出现心肌肥厚,内质网应激参与了APPswe/PS1 dlE9转基因成年小鼠心肌肥厚的发生.%Objective:The present study was designed to explore the role of ER stress in cardiac hypertrophy of adult APPswe /PS1dE9 transgenic mice.Methods:10 adult APPswe/PS1dE9 transgenic mice and 10 C57BL/6 wild type (WT) mice were divided into the transgenic experimental and control group,WT experimental and control group,respectively,with 5 mice in each group.Experimental groups received a low dose ofIsoproterenol (ISO) (2 mg/kg) once a day for 4 weeks to induce cardiac hypertrophy,while control groups received the same volume of normal saline.After 4 weeks,the mice were anesthetized,followed by,electrocardiogram (ECG) recording and the measurement of the heart rate and body weight before being sacrificed.The heart was dissected out,and the masses of heart and the left ventricle were measured,the left ventricule mass index (LVW/BW) and the whole heart weight ratio (HW/BW) were calculated.HE staining was used to observe the pathological and morphological changes of cardiomyocytes,and Western Blot and immunohistochemistry were used to detect the expressions of ER stress relevant proteins-GRP78,JNK,P-JNK and CaMKII.Results:Compared with WT experimental mice,the ventricular wall in the APPswe/PS1dE9 transgenic experimental mice was apparently hypertrophic after the induction by low doses of ISO,and the HW/BW and the LVW/BW were also significantly increased in the APPswe/PS1dE9 transgenic experimental mice than those in the transgenic control mice,WT experimental and control mice (P＜0.05).HE staining showed that compared with the transgenic control mice,WT experimental and WT control mice,in the adult APPswe/PS1dE9 transgenic experimental mice,the cardiomyocyte diameter was obviously increased,cell density was decreased,the capillary density was decreased,the intercellular substance was increased,and the intercellular space was increased.Western blot showed that the expression of GRP78,p-JNK and CaMKII in the experimental group of adult APPswe / PS1dE9 transgenic mice were significantly higher than those in the transgenic control mice and WT mice (P＜0.05,P＜0.01).There were no significant difference among the control group oftransgenic mice and the two groups of WT mice.Immunohistochemistry showed that the positive rates of GRP78 and CaMKII in the cytoplasm of cardiomyocytes of the APPswe/PS1 dE9 transgenic experimental mice were significantly higher than those of experimental WT mice (80％&40 ％)(P＜0.05),and the expressions in the two control groups were negative.The positive rates of p-JNK and JNK in the APPswe/PS1dE9 transgenic experimental mice were 90％ and 40％ respectively,and the expressions were negative in other three groups.Conclusions:The adult APPswe/PSldE9 transgenic mice are more prone to cardiac hypertrophy than WT mice after the induction with a low dose of ISO.ER stress is involved in the formation of cardiac hypertrophy in adult APPswe/PS1dE9 transgenic mice.