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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 investigate the expression of recombinant adeno-associated virus / human coagulation factor IX gene (rAAV-2 / hFIX) in human / mouse colon epithelial cells (SW480 / C26 cell line). Methods: SW480 / C26 cells were infected with recombinant adeno-associated virus / green fluorescent protein (rAAV-2 / GFP). The expression of GFP was observed under a fluorescence microscope and the transfection efficiency was calculated. The SW480 / C26 cells were infected with rAAV-2 / hFIX, and the expression of hFIX gene, FIXAg and hFIX activity were detected. Results: The positive rates of GFP48hr in SW480 / C26 cell lines were (57.0 ± 8.5)% and (48.0 ± 5.7)%, respectively. The cDNA fragments of exogenous hFIX were found on the 14th and 21st days of transduction. After transduced with rAAV-2 / hFIX in SW480 cells, the amounts of hFIX antigen in ELISA assay were (98.0 ± 4.3) and (30.9 ± 6.5) ng.106cell-1.24h-1 on day 2 and 21, respectively. Non-transduced group was (2.7 ± 0.1) ng.106cell-1.24h-1. After transduction of rAAV-2 / hFIX with C26 cells, the amount of hFIX antigen measured on day 2 was (78 ± 5.12) ng.106cell-1.24h-1. The non-transduced group was (2.9 ± 0.1) ng.106cell-1.24h-1. hFIX activity was significantly associated with hFIX antigen concentration. Compared with the control group was also significantly higher. Conclusion: rAAV-2 / GFP can effectively transduce SW480 / C26 cells. rAAV-2 / hFIX effectively transduces SW480 / C26 cells and expresses clotting-active functional protein hFIX.