2种腺病毒介导的eGFP对人成纤维细胞转染效率的研究

Comparison of Two Different Adenovirus Mediated Transfection of Enhanced Green Fluorescent Protein into Human Skin Fibroblast in vitro

目的:比较2种不同腺病毒(adenovirus,AD)介导的增强型绿色荧光蛋白(enhanced green fluorescent protein,eGFP)对人皮肤成纤维细胞(Human Fibroblast,HFB)的转染效率。方法:原代分离培养人皮肤成纤维细胞并鉴定,按照感染复数MOI为10、25、50、100转染传2代的人成纤维细胞,流式细胞检测AD5-EGFP和AD5/F35-EGFP对HFB的转染效率,荧光显微镜观察转染后的荧光强度。MTT法检测AD5-EGFP和AD5/F35-EGFP对HFB的毒性。结果:两种腺病毒对HFB的转染效率随着MOI增加而增高。AD5-EGFP和AD5/F35-EGFP在MOI为10、25、50、100时转染效率分别为9.44+3.44%、31.96+11.19%、51.7+7.24%、69.99+3.26%和22.82+8.98%、55.08+2.19%、63.37+7.24%、88.63+2.58%。两种病毒在转染过程中,细胞生长正常,MTT显示各转染组与未转染组的吸光度值无统计学差异。结论:AD5/F35载体对HFB嗜性显著高于AD5。在皮肤组织的基因修饰中,AD5/F35比AD5更具优越性。

Objective： To compare the transduction rate of AD5-eGFP and AD5/F35-eGFP to human skin fibroblast （HFB）. Methods： Two recombinant adenoviruses （ADV）encoding enhanced green fluorescent protein （eGFP）were constructed and transfected in vitro to the human fibroblast（ HFB ）at MOI 10,25,50 and 100. In twenty four hours after the infection, the expression rates of EGFP on cultured HFB were assessed by flow cytometry and the transfected cells were observed by fluorescence microscope. The killing effect of virus on infective target cells was assayed by MTT. Results： The results showed that the transfection efficiencies of AD5-EGFP and AD5/F35-EGFP were 9.44＋3.44%, 31.96＋11.19, 51.7＋7.24, 69.99＋3.26% and 22.82＋8.98%, 55.08＋2.19%, 63.37＋7.24%, 88.63＋2.58% at MOI 10, 25, 50 and 100, respectively. Transfection efficiency increased with the increase of MOI. Conclusions： Transfection efficiency for HFB of AD5/F35 is higher than that of AD5. It is shown that AD5/F35 can take place of AD5 in skin gene therapy.