两型腺相关病毒对培养人牙髓细胞转染效率的研究

Transfection efficiency of two kinds of recombinant adeno-associated virus in human dental pulp cells in vitro

目的探讨两种不同腺相关病毒(AAV)介导的增强型绿色荧光蛋白rAAV2-EGFP和rAAV2/1-EGFP对人牙髓细胞的转染效率及对人牙髓细胞的毒性,为AAV携带目的基因治疗提供理论依据与实验基础。方法采用组织块加酶消化法培养人牙髓细胞并鉴定组织来源。按照感染复数(MOI)为104、105、5×105转染第2代人牙髓细胞,并选择最适MOI值;流式细胞仪检测转染后7d,rAAV2-EGFP和rAAV2/1-EGFP对人牙髓细胞的转染效率;MTT法检测AAV2-EGFP,AAV2/1-EGFP转染后4、6、8、10、12d人牙髓细胞的增殖活性。结果荧光显微镜下观察rAAV-EGFP对人牙髓细胞的转染效率及荧光蛋白强度,随着MOI值的增加而增高,其中105为转染最适MOI值,7d时,流式细胞仪检测AAV2-EGFP、AAV2/1-EGFP转染率分别为35.8%、76.9%;病毒转染细胞后,细胞生长正常,MTT显示转染后各转染组与未转染组的吸光度值差异无统计学意义。结论rAAV2-EGFP对体外培养的人牙髓细胞转染效率高于rAAV2/1-EGFP,是腺相关病毒中转染人牙髓细胞比较适合的载体。

Objective To compare the transfection rate of rAAV2-EGFP and rAAV2/1-EGFP to human pulp cells. Methods Two recombinant adeno-associated viruses （AAV） encoding enhanced gene fluorescent protein （EGFP） were constructed and transfected to human dental pulp cells at varying multiple of infection （MOI） of 10^4, 10^5 and 5 × 10^5. Twenty four hours after the infection, the expression rates of EGFP on cultured human dental pulp cells were assessed by flow cytometry and the transfected cells were observed under fluorescence microscope. The proliferation rates of the infected cells were assayed by MTT. Results The transfection efficiency of rAAV2-EGFP was increased along with MOI. When MOI was 10^5, the transfection efficiency of rAAV2-EGFP was 76.9%, while the transfection efficiency of rAAV2/1-EGFP was 35.8%. Furthermore, the transfection efficiency of rAAV2-EGFP and rAAV2/1-EGFP showed no significant influence on the cell activity. Conclusion rAAV2-EGFP is more efficient than rAAV2/1-EGFP in transfecting human dental pulp cells.