重组腺相关病毒载体介导的apoA-I和/或LCAT在肌源性细胞中的表达  

Expression of Lecithin Cholesterol Acyltransferase and/or apoA-I Mediated by Recombinant Adeno-associated Virus in Myogenic Cells

作　　者：王立峰[1] 范乐明[1] 陈丙莺[2] 刘宝瑞[3] 王若宁[2] 魏恩会[1] 

机构地区：[1]南京医科大学动脉粥样硬化中心,南京210029 [2]南京医科大学生物化学教研室,南京210029 [3]南京大学医学院附属鼓楼医院肿瘤科,南京210008

出　　处：《生物化学与生物物理学报》2002年第1期33-38,共6页

基　　金：江苏省科委应用基础资助项目;No.BJ980 87~~

摘　　要：卵磷脂胆固醇酰基转移酶 (lecithincholesterolacyltransferase,LCAT)是产生血浆中大部分胆固醇酯 (CE)的主要酶 ,并且是胆固醇逆转运 (reversecholesteroltransfer,RCT)中的一个关键因子。将含有人apoA I和 /或LCATcDNA的rAAVAIL/rAAVL表达载体与 pDG质粒经磷酸钙共沉淀法转导入 2 93T细胞中 ,包装为重组腺辅助病毒 (rAAV) ;以Iodixanol密度梯度离心 ,继以肝素亲和层析等方法分离、纯化及浓缩rAAV ;斑点杂交鉴定制备的rAAV颗粒数达 7× 10 14 个 /L(rAAVAIL)和 1× 10 14 个 /L(rAAVL)。以rAAVAIL/rAAVL转染小鼠肌源性细胞C2C12 ,经ELISA、Western印迹和 [3 H] 胆固醇标记底物的放射化学方法结果分别表明 ,apoA I、LCAT在转染细胞中持续 30天表达 ;分化为多核的肌管细胞后 ,仍保持这种表达能力。PCR结果表明 ,目的基因已整合入细胞基因组。以rAAV为载体 ,能有效介导人LCAT、apoA I基因在肌源性细胞中表达 。Lecithin cholesterol acyltransferase (LCAT) is the major enzyme producing most plasma cholesterol esters(CE)and a key participant in the process of reverse cholesterol transfer (RCT). The aim of this research is to co-express LCAT and it's natural activator apoA-I, with the recombinant adeno-associated virus vectors in the skeletal muscle cells, in order to pave a new way for gene therapy of the primary or secondary LCAT deficiency. 293T cells was cotransfected with pDG and rAAVAIL/rAAVL plasmids to produce infectious rAAV, and non-ionic iodixanol gradient centrifugation, followed by heparin affinity chromatography, were performed for seperation, purification and concentration of rAAV. The particle numbers of rAAV, assayed by dot blot, were 7×10 14/L (rAAVAIL) and 1×10 14/L (rAAVL). These vectors were then transduced into C2C12 myoblasts. The result of ELISA and Western blot for human apoA-I, and [ 3H]-cholesterol-labeled radiochemical methods for LCAT activity, showed that the expression of human apoA-I cDNA and/or human LCAT cDNA in transduced C2C12 cells lasted for 30 days, even after myoblasts were differentiated into myotubes. PCR products for the transgene indicated the long-term persistence of transduced vector sequences. The results indicate that the methods used for production and purification of rAAV is efficient, and rAAV vector mediated the expression and secretion of LCAT and apoA-I gene in C2C12 myoblasts successfully. It suggests that the use of rAAV vectors mediating the high efficiency, long-term expression of human LCAT cDNA and/or apoA-I cDNA in skeletal muscle in vivo can be a safe and fesible strategy for the gene therapy of LCAT deficiency.

关 键 词：卵磷脂胆固醇酰基转移酶 胆固醇逆转运 骨骼肌细胞 重组腺相关病毒 基因转移 表达 

分 类 号：Q786[生物学—分子生物学]