转化生长因子β3重组腺病毒载体转染退变髓核细胞后的增殖活性  

Proliferative activity of degenerated nucleus pulposus cells transfected by recombinant adenovirus vectors containing transforming growth factor beta 3

作　　者：宋希元[1] 彭圣智[1] 

机构地区：[1]济宁医学院基础医学与法医学院,山东省济宁市272067

出　　处：《中国组织工程研究》2012年第24期4408-4412,共5页Chinese Journal of Tissue Engineering Research

基　　金：济宁医学院青年基金项目~~

摘　　要：背景:通过基因干扰促进某些基因的表达,调控细胞生物学活性刺激有利于椎间盘组织再生的基质合成来达到治疗目的。目的:构建含有转化生长因子β3的腺病毒表达载体,观察其对退变髓核细胞增殖的影响。方法:制作椎间盘突变模型,从而获取原代退变椎间盘髓核细胞,提取总RNA,调取转化生长因子β3cDNA克隆至穿梭质粒pAdTrack-CMV,采用pAdEasy系统进行细菌内同源重组得到含目的基因的腺病毒载体,脂质体转染293A细胞进行包装并扩增病毒。结果与结论:成功构建有较强感染能力的含转化生长因子β3基因重组腺病毒表达载体,滴度达1010pfu/L,MTT法检测表明Ad-TGF-β3可改善退变髓核细胞生物学特性,为研究转化生长因子β3的作用机制及将其用于椎间盘退变的基因治疗提供实验和理论依据。BACKGROUND： Gene interference can promote certain gene expressions, change the cell matrix synthesis condition by regulating the cell biological activity and stimulate the matrix synthesis which is benefit for the regeneration of the intervertebrai discs to achieve therapeutic purposes. OBJECTIVE： To construct a recombinant adenovirus vector containing transforming growth factor-β3 gene and to detect its effects on the proliferation of degenerated nucleus pulposus cells. METHODS： Intervertebral disc degenerated model was constructed to gain the primary degenerated nucleus pulposus cells. Total RNA was extracted. Then the cDNA of the transforming growth factor-β3 was cloned into the shuttle plasmid pAdTrack-CMV. The recombinant adenovirus vector containing transforming growth factor-β3 was constructed by homogenous recombination in bacteria using PAdEasy system. The correct recombinant was packaged by liposome and transfected into 293A cells to amplify. RESULTS AND CONCLUSION： The adenovirus vector containing transforming growth factor-β3 was successfully constructed with high infection; the titer was 10^10 pfu/L. MTT assay showed that the adenovirus vector containing transforming growth factor-β3 significantly enhanced the activity of degenerated nucleus pulposus cells; it provides experimental and theoretical basis for research on the mechanism of transforming growth factor-β3 and its application on the gene therapy for intervertebral disc degeneration.

关 键 词：转化生长因子Β3 腺病毒 转染 髓核细胞 椎间盘退变 

分 类 号：R318[医药卫生—生物医学工程]