Objective To study the effect of sex-determining region Y-frame protein 3(SOX3)on proliferation and estradiol secretion in human ovarian granulosa cells(KGN cell line).Methods The gene sequence of human SOX3(NM_005634.3)was searched in Gene-Bank,an NCBI database,and the target gene SOX3 was amplified by PCR,which was cloned into lentiviral vector pLV-EF1a-GFP-2A-Puro to obtain the overexpression lentiviral re-combinant plasmid pLV-EF1a-GFP-2A-Puro-SOX3;the correctly sequenced overexpressed lentiviral recombinant plasmid as well as packaging plasmids(pGag/Pol,pRev,pVSV-G)were co-transfected into human embryonic kidney cell line(HEK 293T)cells(pLV-SOX3 group),and pLV-EF1a-GFP-2A-Puro and packaging plasmids(pGag/Pol,pRev,pVSV-G)were co-transfected into HEK 293T cells(pLV-NC group),the lentiviral particles of both groups were collected and the titers of the viruses were measured after 48 h of transfection,the lentiviruses of the two groups were infected into KGN cells,and the stably expressed cell lines were obtained after puromycin screening for 2 weeks;real-time fluorescence quantitative PCR(RT-qPCR)and Western blot were used to detect the SOX3 mRNA and protein levels in the two groups;CCK-8 assay was used to detect the proliferative ability of the cells in the two groups;ELISA was used to determine the concentration of estradiol in the two groups.Results The identification of PCR products and sequencing results showed that the SOX3 gene fragment was amplified successfully,and the enzyme digestion and sequencing results indicated that the construction of overexpression lentiviral recombinant plasmid was completed;green fluorescence could be detected after lentiviral infection of HEK 293T cells,which indicated that lentiviral packaging was successful;the lentivirus was screened by puromycin after lentiviral infection of KGN cells,and the cells were observed to express green fluorescence under the fluorescence microscope;RT-qPCR and Western blot assays both showed that the expression level of SOX3 in the pLV-SOX3 group was significantly higher than that in the pLV-NC group(P<0.05).CCK-8 assay results showed that the proliferation ability of the cells in the pLV-SOX3 group significantly increased compared with that in the pLV-NC group(P<0.01).ELISA results showed that estradiol concentration was elevated in the pLV-SOX3 group com-pared with the pLV-NC group(P<0.05).Conclusion Overexpression of the transcription factor SOX3 can pro-mote the proliferation and estradiol secretion of human ovarian granulosa cells KGN.
关键词
SOX3/卵巢颗粒细胞/慢病毒载体/增殖/雌二醇/绿色荧光蛋白
Key words
SOX3/ovarian granulosa cell/lentiviral vector/proliferation/estradiol/green fluorescent protein
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