配对盒基因6/小鼠胚胎干细胞细胞系的建立及干细胞生物学特性分析

doi:10.16098/j.issn.0529-1356.2019.04.008

解剖学报 ›› 2019, Vol. 50 ›› Issue (4): 451-458.doi: 10.16098/j.issn.0529-1356.2019.04.008

配对盒基因6/小鼠胚胎干细胞细胞系的建立及干细胞生物学特性分析

丁玲¹ 高杰¹李红¹ 胡蓉^1* 苏敏^1,2*

1.贵州医科大学基础医学院组织学胚胎学教研室，贵阳 550004; 2.贵州医科大学贵州省细胞工程生物医药技术国家地方联合工程实验室，贵州省再生医学重点实验室，贵阳 550004

收稿日期:2018-12-25 修回日期:2019-05-08 出版日期:2019-08-06 发布日期:2019-08-06
通讯作者: 胡蓉;苏敏 E-mail:370481959@qq.com
基金资助:
贵州省科技厅基金;贵州省教育厅青年科技人才成长项目;贵州省普通高校科技拔尖人才支持计划

Establishment of paired box gene 6/mouse embryonic stem cell line and its stem cell biological characteristics

DING Ling¹GAO Jie¹ LI Hong¹ HU Rong^1* SU Min^1,2*

1.Department of Histology and Embryology, Guizhou Medical University,Guiyang 550004,China; 2.National and Guizhou Joint Engineering Laboratory for Cell Engineering and Biomedicine Technique，Guizhou Province Key Laboratory of Regenerative Medicine, Guizhou Medical University, Guiyang 550004,China

Received:2018-12-25 Revised:2019-05-08 Online:2019-08-06 Published:2019-08-06
Contact: HU Rong; SU Min E-mail:370481959@qq.com

摘要/Abstract

摘要：

目的建立配对盒基因6（Pax6）/小鼠胚胎干细胞（mESCs）细胞系并鉴定其干细胞生物学特性。方法体外培养mESCs，将重组载体pEF1α-Pax6-IRES-AcGFP和空载体pEF1α-IRES-AcGFP分别用脂质体法转染mESCs，经G418梯度及荧光蛋白双筛选后，使用细胞免疫荧光染色、免疫印迹法及RT-PCR技术检测Pax6的表达情况，流式细胞术检测Pax6/mESCs阳性细胞的比例。将获得正确的细胞系分别采用细胞免疫荧光染色对其干细胞标志物阶段特异性胚胎抗原1（SSEA1）、八聚体结合转录因子4（OCT4）进行检测，碱性磷酸酶（AP）染色法对其多能性进行检测，流式细胞术检测增殖指数Ki67。将Pax6/mESCs进行肾背囊下移植，移植物行HE染色观察其分化能力。结果 Pax6成功在mESCs内表达，经G418筛选后，获得Pax6/mESCs细胞系，流式结果显示，Pax6阳性率为90%，免疫荧光显示，干细胞标志物SSEA1、OCT4表达阳性且AP染色阳性，并且在体内移植后能向3个胚层分化。结论 Pax6成功在mESCs内表达，经G418筛选后，获得细胞系Pax6/mESCs并维持良好的干细胞特性。

关键词: 配对盒基因6, 小鼠胚胎干细胞, 基因转染, G418筛选, 流式细胞术, 小鼠

Abstract:

Objective To investigate the overexpression of paired box gene 6(Pax6) gene in mouse embryonic stem cells and obtain cell line, which is the basis for further differentiation of Pax6/mouse embryonic stem cells (mESCs). Methods mESCs were cultured in vitro, and the recombinant vector pEF1α-Pax6-IRES-AcGFP and the empty vector pEF1α-IRES-AcGFP were transfected into mESCs by liposome method respectively. The cells were screened by G418 gradient and fluorescent protein. The expression of Pax6 was detected by RT-PCR, immunofluorescence and Western blotting and the proportion of Pax6/mESCs positive cells was detected by flow cytometry. The obtained cell line was detected by cell immunofluorescence for stem cell markers stage specific embryonic antigen 1(SSEA1) and octamer binding transcription factor 4(OCT4), and the pluripotency was detected by alkaline phosphatase staining. Pax6/mESCs cells were subcutaneously transplanted, and the grafts were observed by HE staining to observe their differentiation ability. Results Pax6 was successfully expressed in mESCs. After screening by G418, the cell line Pax6/mESCs was obtained, and the flow rate showed positive rate about 90%. Immunofluorescence showed that stem cell markers SSEA1 and OCT4 were positively expressed and alkaline phosphatase stained cells were stained brownish black, and transplantation in vivo could differentiate into three germ layers. Conclusion Pax6 is successfully expressed in mESCs. After screening by G418, the cell line Pax6/mESCs is obtained and shows the good stem cell characteristics.

Key words: Paired box gene 6, Mouse embryonic stem cell, Gene transfection, G418 screening, Flow cytometry, Mouse

丁玲高杰李红胡蓉苏敏. 配对盒基因6/小鼠胚胎干细胞细胞系的建立及干细胞生物学特性分析[J]. 解剖学报, 2019, 50(4): 451-458.

DING Ling GAO Jie LI Hong HU Rong SU Min. Establishment of paired box gene 6/mouse embryonic stem cell line and its stem cell biological characteristics[J]. Acta Anatomica Sinica, 2019, 50(4): 451-458.

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配对盒基因6/小鼠胚胎干细胞细胞系的建立及干细胞生物学特性分析

Establishment of paired box gene 6/mouse embryonic stem cell line and its stem cell biological characteristics

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