重组人心肌营养素对体外培养受损心肌细胞的保护作用  

Protective Effect of Recombinant Human Cardiotrophin-1 on Damaged Cardiomyocyte in vitro

作　　者：刘冠群[1] 孔祥平[2] 佟明华[2] 李茹冰[2] 陈文吟[2] 游松[1] 

机构地区：[1]沈阳药科大学制药工程学院,沈阳110016 [2]中国人民解放军第四五八医院全军肝病中心,广州510602

出　　处：《中国生物化学与分子生物学报》2007年第5期394-399,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金："九五"国家重点科技攻关项目(No.969020104);国家高科技研究发展计划(863计划)项目(No.2002AA2Z3119)~~

摘　　要：心肌营养素是一种具有心肌保护功能的细胞因子.为鉴定人心肌营养素在体外具有促进受损大鼠心肌细胞存活的生物学活性,构建pQE-huCT-1表达载体,在大肠杆菌中表达重组人心肌营养素(rhCT-1);利用含有还原型及氧化型谷胱甘肽的复性体系,恢复变性蛋白的天然构象;通过MTT法鉴定rhCT-1的促进受损大鼠心肌细胞存活的生物学活性.pQE-huCT-1在M15菌中得到高效表达,但95%的目的蛋白以包涵体形式存在;通过变性、复性及Ni-NTA纯化,得到纯度为90%的可溶性重组蛋白.利用无血清培养基培养新生大鼠心肌细胞,造成缺血损伤;利用低浓度阿霉素损伤大鼠心肌细胞,构建阿霉素损伤模型;通过加入rhCT-1治疗,发现rhCT-1具有明显的促进受损心肌细胞存活的作用;心肌细胞琥珀酸脱氢酶(SDH)活性随rhCT-1浓度的升高而增强,且呈显著的剂量依赖性关系;通过测定预防组和治疗组心肌细胞SDH和乳酸脱氢酶(LDH)活性,发现预防组较治疗组在促进细胞线粒体产生SDH和降低培养液上清LDH活性方面效果更加显著,说明rhCT-1可能是在心肌细胞损伤早期通过抑制细胞凋亡而达到心肌保护作用的.本研究通过复性获得了可溶性rhCT-1,它在体外具有显著的促进受损心肌细胞存活的生物学活性.Cardiotrophin-1 has been considered as a cardioprotective cytokine. To identify the activity of human cardiotrophin-1 protecting rat cadiomyocyte from ischaemic stress and adriamycin damage, pQE-huCT vector expressing the recombinant human cardiotrophin-1 （rhCT-1） was constructed and transfected into E. coli. Natural conformation of rhCT-1 was restored by using a glutathione redox system （GSSG/GSH） and its biological activity was determined by MTr test. The results showed that rhCT-1, most （95%） existed in the form of inclusion bodies, was efficiently expressed in E. coli M15. The purity of dissoluble rhCT-1 was increased to 90% by Ni-NTA affinity column after denaturation and renaturation, lschemic damage of cadiocytes from neonatal rat was produced by culture in a serum-free media. Meanwhile the model of myocardial damage was established by treated with low-dose adriamycin. It was found that the survivable time of damaged cadiocytes would be significantly prolonged after treated with rhCT-1. The activity of succinodehvdrogenase （SDH） in cadiocvtes was increased by the treatment with rhCT-1 in a dose-dependent manner. It was evident that rhCT-1 could promote in the release of SDH from mitochondrial and decreased the activity of lactic dehydrogenase （LDH） in supernatant in pre-treatment group compared with those in treatment group. Therefore, we conclude that rhCT-1 may play a role in the protection of cardiocytes by inhibiting apoptosis in initial stage of myocardial damage. To compare the activity of SDH and lactate dehydrogenase （LDH） of pre-treated group with treated group, it was found that the effect of pre-treatment was more significant. It implied that CT-1 might play a role in cardioprotection by inhibiting apoptosis in initial stage of myocardial damage. The dissoluble rhCT-1 with biological activity was obtained. It underlay the base. of application and research.

关 键 词：心肌营养素-1 心肌保护 琥珀酸脱氢酶 阿霉素 

分 类 号：Q71[生物学—分子生物学]