TRADD慢病毒载体构建及其对增生性瘢痕成纤维细胞增殖的影响 袁 月,康秀峰,牟 东,薛世祥,代剑华,彭贵勇 (400038 重庆,第三军医大学西南医院全军消化病研究所) [摘要] 目的 构建抗食管支架术后再狭窄携人TRADD基因慢病毒表达载体,并检测其对增生性瘢痕成纤维细胞增殖的影响。方法 以购买的人TRADD基因为模板,采用PCR法扩增目的基因片段,PCR产物经回收纯化后与线性化的慢病毒表达载体pLVX-EGFP-3FLAG-Puro重组。重组质粒转化DH5a感受态细胞。菌落PCR鉴定转化子,阳性克隆测序无误后,质粒共转染293FT细胞制备慢病毒。Real-time 定量PCR法检测病毒滴度。Western blot检测TRADD-GFP-FLag融合蛋白的表达。MTT法检测TRADD基因慢病毒对增生性瘢痕成纤维细胞增殖的影响。结果 菌落PCR扩增产物经凝胶电泳,阳性克隆得到1200bp片断,基因测序显示重组质粒中TRADD序列与GenBank 中序列一致。包装产生的慢病毒转染293 FT 细胞48 h 后,荧光显微镜下可见大量绿色荧光,×108 IU /ml, Western blot检测到融合蛋白TRADD-GFP-Flag在293FT细胞中获得有效表达,MTT法证实融合蛋白的生物活性,能有效抑制瘢痕成纤维细胞增殖。结论 成功构建并包装了pLVX-TRADD-EGFP-3FLAG-Puro慢病毒表达载体,其可明显抑制增生性瘢痕成纤维细胞的增殖。 [关键词] TRADD;慢病毒表达载体;食管再狭窄;基因治疗 [中图法分类号] [文献标志码] A Construction of lentiviral expression vector containing human TRADD gene for preventing esophageal restenosis Yuan Yue, Kang Xiufeng, Mu Dong, Xue Shixiang, Dai Jianhua, Peng Guiyong(Institute of Gastroenterology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China) [Abstract] Objective To construct and identify a lentiviral expression vector containing the human TRADD gene and to explore the effect of the recombinant lentiviruses on the proliferation of fibroblasts derived from hypertrophic scars. Methods The TRADD specific fragment was amplified by PCR technique and cloned into the EcoRⅠsite of the lentivir