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  

Expression of recombinant adeno-associated virus/human coagulant factor IX gene in human/rat colon carcinoma cells

作　　者：马泳泳[1] 陈方平[2] 杜建伟[2] 陈焱[2] 彭建强[3] 吴小兵[3] 解勤之[2] 

机构地区：[1]温州医学院第一附属医院血液内科,浙江温州325000 [2]中南大学湘雅医院血液科,湖南长沙410008 [3]中国疾病控制中心病毒预防控制所病毒基因工程国家重点实验室,北京100052

出　　处：《温州医学院学报》2008年第2期100-105,共6页Journal of Wenzhou Medical College

基　　金：国家863基金资助项目(2001AA217181)

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。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.rAAY-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·10^6cell^-1·24h^-1 and （78.0±5.1） ng·10^6cell^-1·24h^-1 of the C26 cells while the negative control was （2.9±0.1） ng·10^6cell^-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 cell safter transduction are capable of producing hFⅨ with an elevated level of hFⅨ antigen and biological activity, rAAY-2/GFP can effectively transducte to the SW480 and C26 cells.

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 

分 类 号：R554.1[医药卫生—血液循环系统疾病]