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目的:探讨慢病毒载体介导绿色荧光蛋白基因体外转染卵巢颗粒细胞的有效性及在体转染大鼠卵巢组织的最佳剂量及时效性。方法:原代培养大鼠卵巢颗粒细胞,慢病毒载体介导绿色荧光蛋白基因(lenti-GFP)(MOI=20)体外转染颗粒细胞,荧光显微镜下观察绿色荧光蛋白的表达。通过显微注射方法将不同转染剂量低剂量(2×106 TU病毒颗粒,A组;高剂量:10×106 TU病毒颗粒,B组;空白病毒载体,C组;空白对照组,D组)的lenti-GFP在体转染大鼠卵巢组织。于转染第5日观察各剂量组卵巢组织切片的GFP表达情况,确定最佳转染剂量后,分别于转染第5日、第15日、第30日、第45日、第60日、第75日观察大鼠卵巢组织和全身其他组织器官GFP的表达。结果:体外转染实验中,在转染第5日可以观察到颗粒细胞内有GFP表达,并随着转染时间延长其表达量增多。体内转染大鼠卵巢第5日,不同剂量组大鼠卵巢均有明显GFP表达;A组与B组卵巢组织切片荧光强度分别为0.231±0.020及0.231±0.020;组间差异无统计学意义(P=0.976)。以最佳转染剂量(2×106 TU病毒颗粒)转染,随转染时间的延长,GFP的表达量增加,于转染第30日时表达量达到峰值,并可持续高效表达至第75日(转染第5日、第15日、第30日、第45日、第60日、第75日卵巢组织荧光强度值分别为0.231±0.020、0.312±0.021、0.346±0.020、0.357±0.013、0.350±0.013及0.351±0.017)。同时,大鼠全身其它组织器官均有GFP的高效持续表达。结论:lenti-GFP不仅可以在体外有效转染颗粒细胞,并且可以通过活体转染大鼠卵巢后,在卵巢及其他组织器官均高效持续地表达。
OBJECTIVE: To investigate the effectiveness of lentiviral vector-mediated green fluorescent protein gene transfection in ovarian granulosa cells in vitro and the optimal dose and timeliness of transfection of ovarian tissue in vivo. METHODS: Primary cultured rat ovarian granulosa cells were transfected with lenti-GFP (MOI = 20) in vitro and granulosa cells were transfected with lentiviral vector. The expression of green fluorescent protein (GFP) was observed under a fluorescence microscope. Different transfection doses of low dose (2 × 106 TU virus particles, group A; high dose: 10 × 106 TU virus particles, group B; blank virus carrier, group C; blank control group, group D) The lenti-GFP was transfected into rat ovarian tissue in vivo. On the fifth day after transfection, the expression of GFP in ovarian tissue sections of each dose group was observed. After the optimal transfection dose was determined, On the 75th day, the expression of GFP in ovary tissue and other tissues and organs of rats was observed. Results: In vitro transfection experiments, GFP expression in granulosa cells was observed on the 5th day after transfection, and its expression increased with the time of transfection. On the 5th day after transfection in vivo, the expression of GFP in ovaries of rats of different dose groups was significant. The fluorescence intensities of ovarian tissue sections in group A and group B were 0.231 ± 0.020 and 0.231 ± 0.020, respectively. There was no significant difference between the two groups P = 0.976). At the optimal transfection dose (2 × 106 TU), the expression of GFP increased with the prolonging of the transfection time and reached its peak on the 30th day after transfection. Day (day 5, day 15, day 30, day 45, day 60, day 75), the fluorescence intensity values of ovarian tissue were 0.231 ± 0.020,0.312 ± 0.021,0.346 ± 0.020,0.357 ± 0.013, 0.350 ± 0.013 and 0.351 ± 0.017). At the same time, other tissues and organs of rats all have high and sustained expression of GFP. Conclusion: lenti-GFP not only can effectively transfect granulosa cells in vitro, but also can be efficiently and consistently expressed in ovary and other tissues and organs after in vivo transfection of rat ovary.