首页|转录因子SOX3对卵巢颗粒细胞增殖和雌二醇分泌的影响

转录因子SOX3对卵巢颗粒细胞增殖和雌二醇分泌的影响

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目的 研究性别决定区Y框蛋白3(SOX3)对人卵巢颗粒细胞(KGN)增殖和雌二醇分泌的影响.方法 在NCBI数据库Gene-Bank中搜索人SOX3(NM_005634.3)的基因序列,用PCR方法扩增目的基因SOX3,将其克隆到慢病毒载体pLV-EF1a-GFP-2A-Puro上,获得过表达慢病毒重组质粒pLV-EF1a-GFP-2A-Puro-SOX3;将测序正确的过表达慢病毒重组质粒以及包装质粒(pGag/Pol、pRev、pVSV-G)共同转染到人胚肾细胞系(HEK 293T)细胞中(pLV-SOX3 组),将pLV-EF1a-GFP-2A-Puro及包装质粒(pGag/Pol、pRev、pVSV-G)共同转染到HEK 293T细胞中(pLV-NC组),转染 48h后收集两组慢病毒颗粒并测定病毒的滴度,将两组慢病毒感染KGN细胞,经嘌呤霉素筛选2 周后,获得稳定表达的细胞系;采用实时荧光定量PCR(RT-qPCR)和Western blot法检测两组中SOX3 的mRNA和蛋白水平;CCK-8 法检测两组细胞增殖能力;ELISA测定两组雌二醇的浓度.结果 PCR产物的鉴定和测序结果表明SOX3 基因片段扩增成功,酶切和测序结果表明过表达慢病毒重组质粒构建完成;慢病毒感染HEK 293T细胞后可检测到绿色荧光,表明慢病毒包装成功;慢病毒感染KGN细胞后经嘌呤霉素筛选,荧光显微镜下观察细胞表达绿色荧光;RT-qPCR和Western blot检测结果均表明pLV-SOX3 组中 SOX3 表达水平高于 pLV-NC 组(P<0.05).CCK-8 检测结果表明,pLV-SOX3 组较pLV-NC组细胞增殖能力明显增强(P<0.01).ELISA 结果显示,pLV-SOX3 组雌二醇浓度较pLV-NC组升高(P<0.05).结论 过表达转录因子 SOX3 能够促进 KGN 的增殖和雌二醇分泌.
Effect of the transcription factor SOX3 on ovarian granulosa cell proliferation and estradiol secretion
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.

SOX3ovarian granulosa celllentiviral vectorproliferationestradiolgreen fluorescent protein

蔡睿、张浩、刘壮、陈远华、谢芬芬、洪强

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安徽医科大学基础医学院组织胚胎学教研室,合肥 230032

SOX3 卵巢颗粒细胞 慢病毒载体 增殖 雌二醇 绿色荧光蛋白

国家自然科学基金安徽省自然科学基金安徽省高等学校科研项目安徽医科大学校科研基金安徽医科大学"早期接触科研"训练计划安徽医科大学"早期接触科研"训练计划

822018042208085QH235KJ2021A02172021xkj0072021-ZQKY-1492022-ZQKY-219

2024

10.19405/j.cnki.issn1000-1492.2024.03.001

安徽医科大学学报
安徽医科大学

安徽医科大学学报

CSTPCD北大核心
影响因子：1.095
ISSN：1000-1492
年,卷(期)：2024.59(3)
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