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目的以原代培养的小鼠卵巢颗粒细胞为模型,观察人SET基因重组腺病毒载体在卵巢颗粒细胞的感染效率和基因表达效果。方法分离并培养小鼠颗粒细胞。扩增人SET基因重组腺病毒载体AdCMV-SET和对照重组腺病毒载体AdCMV,感染原代培养的小鼠颗粒细胞,通过观察绿色荧光蛋白(GFP)检测感染效率;qRT-PCR方法检测SETmRNA表达;Western blot方法检测SET蛋白的表达水平。结果获得的重组腺病毒载体体外感染小鼠颗粒细胞,24h观察到GFP表达。与AdCMV感染组相比,AdCMV-SET感染组SETmRNA表达水平显著增高(P<0.05)。与空白对照组(Mock)及感染AdCMV组相比,AdCMV-SET感染组SET蛋白水平显著增高(P<0.05)。结论构建的人SET基因重组腺病毒载体能高效感染小鼠颗粒细胞、并有效表达,对细胞活率无影响。
OBJECTIVE: To observe the infection efficiency and gene expression of human SET gene recombinant adenovirus vector in ovarian granulosa cells using primary cultured mouse ovarian granulosa cells as a model. Methods Mouse granulosa cells were isolated and cultured. Infection of primary cultured mouse granulosa cells with AdCMV-SET recombinant adenovirus vector expressing human SET gene and control recombinant adenovirus vector was used to detect the infection efficiency by observing green fluorescent protein (GFP); SETmRNA expression was detected by qRT-PCR; Western blot was used to detect the expression of SET protein. Results The recombinant adenovirus obtained in vitro infection of mouse granulosa cells, GFP expression was observed 24h. Compared with AdCMV infection group, SETCMRNA expression in AdCMV-SET infection group was significantly increased (P <0.05). SET protein levels in AdCMV-SET group were significantly higher than those in Mock group and AdCMV-infected group (P <0.05). Conclusion The constructed recombinant adenovirus vector of human SET gene can efficiently infect mouse granulosa cells and express it efficiently, and has no effect on cell viability.