Expression of Lecithin: Cholesterol Acyltransferaseand/or apoA-I Mediated by Recombinant Adeno-as-sociated Virus in Myogenic Cell
王立峰1
范乐明1
陈丙莺2
刘宝瑞2
王若宁3
魏恩会3
1.Atherosclerosis Research Center, NJMU Nanjing 2100292.Department of Biochemistry, NJMU Nanjing 2100293.Departmentof Oncology, Drum Tower Hospital, School of Medicine, Nanjing University, Nanjing 210008,P. R. China
摘要：Objective Lecithia: cholesterol acyltrmsfer ase (LCAT) is the major enzyme producing most plasma cholesterol esters( CE )and a key partiipant in the process of reverse cholesterol traansfer ( RCT). The aim of the study was to co-express LCAT and its nature activator apoA- I medi ated by recombinant adeno-associated virus vectors in the skeletal muscle cells, and open a new avenue of gene therapy touard the primary or secondary LCAT deficiency. Methods 293T cells were cotrans fected with pDG and rAAVAIL/rAAVL plasmid to produce infectious rAAV, and non-iouic iodixanol gradients centri f ngation followed by heparin affinity chromatography was per formed f or separation . pu rification and concentration of rAAV. The particle numbers of rAAV were assayed by dot-blot, then these vectors transduced C2C12 myoblasts. ELISA and Western Blot asasayed for human apoA- I and 3H-cholesterol labeled radiochemical methods for LCAT activity. Genomic DNA was extracted from transduced C2C12 and analyzed fo the presence of vector sequence by PCR amplifiations. Results The particle mumbers of rAAV were 7× 1014/L (rAAAIL) and 1 × 1014/L (rAAVL). The expres sion of human apoA- I cDNA and/or human LCAT cDNA in transduced C2C12 cells lasted for 3 0 d, even after myoblasts were differentiated into myotubes. PCR products for transgene indiated the long-term persistence of transduced vector sequences. Conclusion The result indicated that the meth ods used for production and purification of rAAV is an effiient and rAAV vector mediate the expres sion and secretion of LCAT and apoA- I gene in C2C12 myoblasts successfully. It suggested 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 might be a safe and fessible strategy to the gene therapy of LCAT deficiency.
机标关键词：
分类号：R3(基础医学)
资助基金：基础研究项目(BJ98087)
论文发表日期：2002-07-02
在线出版日期：2025-08-15（本平台首次上网日期，不代表文献的发表时间）
页数：7( 91-96,126 )
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