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目的观察慢病毒载体(lentiviral vectors,LVs)用于兔角膜上皮细胞基因转染的有效性。方法兔角膜上皮细胞的原代及传代培养并做细胞鉴定;慢病毒载体介导的增强型绿色荧光蛋白(lenti-EGFP)以不同的感染复数(multiplicity of infection,MOI=0、1、10、50、100、500)转染实验细胞,寻找最佳转染剂量;于转染后24、48、72、96h运用倒置荧光显微镜观察不同MOI下增强型绿色荧光蛋白(enhanced green fluorescence protein,EGFP)的表达并计算细胞转染率;RT-PCR方法检测EGFP基因的表达情况。结果 EGFP于转染48h即开始有表达,随着转染时间的延长其表达增强。MOI=1、10、50、100时,角膜上皮细胞转染率随着感染复数的增加而增加,分别为(4.5±0.6)%、(30.4±0.9)%、(51.9±1.4)%、(75.4±0.7)%,各组间差异显著(P<0.05);MOI在100与500时,转染率差异不显著(P>0.05),即最适感染复数为100。RT-PCR结果提示转染细胞组EGFP基因有表达。结论慢病毒载体能够有效转染离体兔角膜上皮细胞。
Objective To observe the effectiveness of lentiviral vectors (LVs) for gene transfection in rabbit corneal epithelial cells. METHODS: Primary and subculture cultures of rabbit corneal epithelial cells were made and identified. Lentivirus vector-mediated lenti-EGFP was expressed in different multiplicity of infection (MOI = 0, 1, 10, 50, 100, 500) transfected cells to find the best transfection dose; At 24, 48, 72, 96 h after transfection, the expression of enhanced green fluorescence protein (EGFP) The expression of EGFP gene was detected by RT-PCR. Results EGFP began to be expressed 48h after transfection and its expression was enhanced with the extension of transfection time. The transfection rate of corneal epithelial cells increased with the increase of the multiplicity of infection at the MOI of 1, 10, 50 and 100, respectively (4.5 ± 0.6), (30.4 ± 0.9)% and (51.9 ± 1.4)%, respectively 75.4 ± 0.7)%, and there was significant difference among the groups (P <0.05). When the MOI was between 100 and 500, the transfection rate was not significantly different (P> 0.05). The results of RT-PCR indicated that EGFP gene was expressed in transfected cells. Conclusion Lentiviral vectors can efficiently transfect rabbit corneal epithelial cells.