重组腺相关病毒介导的人凝血因子IX基因在人-鼠结肠癌上皮细胞中的表达.DOCVIP

临床医学论文-重组腺相关病毒介导的人凝血因子IX基因在人/鼠结肠癌上皮细胞中的表达 ????????????????? 作者：马泳泳，陈方平，杜建伟，陈焱，彭建强，吴小兵，解勤之 【摘要】? 目的:探讨重组腺相关病毒/人凝血因子IX 基因（rAAV-2/hFIX）在人/鼠结肠癌上皮细胞(SW480/C26细胞株)中的表达。方法:重组腺相关病毒/绿色荧光蛋白（rAAV-2/GFP）感染SW480/C26细胞，在荧光显微镜下观察GFP的表达，计算转染率。rAAV-2/hFIX感染SW480/C26细胞，检测hFIX基因、FIXAg量及hFIX活性的表达。结果:SW480细胞株/C26细胞株GFP48hr阳性率分别为（57.0±8.5）%和（48.0±5.7）%；转导组14 d和21 d均可见外源hFIX的cDNA片断。SW480细胞转导了rAAV-2/hFIX后在第2天和第21天ELISA法测hFIX抗原量分别为（98.0±4.3）和（30.9±6.5）ng·106cell-1·24h-1。未转导组为（2.7±0.1）ng·106cell-1·24h-1。C26细胞转导了rAAV-2/hFIX后在第2天测hFIX抗原量为（78±5.12）ng·106cell-1·24h-1。未转导组为（2.9±0.1）ng·106cell-1·24h-1。hFIX活性与hFIX抗原浓度明显相关。较对照组亦显著增高。结论:rAAV-2/GFP能有效地转导SW480/C26细胞。rAAV-2/hFIX能有效地转导SW480/C26细胞并表达具有凝血活性的功能蛋白hFIX。 【关键词】? 血友病B; 基因治疗法; 重组腺相关病毒; 人凝血因子IX基因; 结肠上皮细胞 ??? Abstract:? Objective: To study the expression of recombinant adeno-associated virus/human coagulant factor. Methods: rAAV-2/GFP was transferred to human/rat gut epithelial cells, the expression of GFP was detect and the transduction efficiency was measured.rAAV-2/hFIX was transducted to human/rat gut epithelial cells. The expressions of hFIX were studied in the parameters of DNA, protein and biological activity. Results: （57.0±8.5）% SW480 cells expressed the GFP gene on the 2nd day. （48.0±5.7）% C26 cells expressed the GFP gene on the 2nd day.The hFIX cDNA expressions were found in DNA extracted from both the SW480 and C26 cells after transduction. Elevated level of hFIX antigen and biological activities were detected and persisted for 21 days with（98.0±4.3）ng·106cell-1·24h-1 and（78.0±5.1）ng·106cell-1·24h-1 of the C26 cells while the negative control was（2.9±0.1）ng·106cell-1·24h-1.The expression of hFIX antigen was in accordance with coagulant activity. Both the SW480 and C26 cells after transduction showed remarkable higher coagulant activities than that of the negative control. Conclusion:The results indicate that the SW480/C26 cells after transduction are capable of producing hFIX with an elevated level of hFIX antigen and biological activ