山羊胎肝内人脐带间充质干细胞的存活与分化研究

doi:10.3969/j.issn.1674-5817.2019.05.005

实验动物与比较医学 ›› 2019, Vol. 39 ›› Issue (5): 364-370.DOI: 10.3969/j.issn.1674-5817.2019.05.005

山羊胎肝内人脐带间充质干细胞的存活与分化研究

鄢东海^1,2, 黄小昆¹, 向雪萍¹, 卿德科¹, 方伟¹, 李自安¹, 王强¹, 王金祥¹, 庞荣清¹

1.联勤保障部队第920医院干细胞与免疫细胞生物医药技术国家地方联合工程实验室、云南省细胞治疗技术转化医学重点实验室、云南省干细胞工程实验室, 昆明 650032;
2.昆明医科大学920医院临床学院, 昆明 650031

收稿日期:2019-01-16 出版日期:2019-10-25 发布日期:2021-01-05
作者简介:鄢东海(1987-), 男, 硕士研究生。E-mail: 1072346722@qq.com
基金资助:
云南省重点项目(2015FA039); 云南省面上项目 (2017FB042); 云南省后备人才项目(2018HB002)

Study on in Vivo Survival and Differentiation of Human Umbilical Cord Mesenchymal Stem Cells in Fetal Goat Liver

YAN Dong-hai^1,2, HUANG Xiao-kun¹, XIANG Xue-ping¹, QING De-ke¹, FANG Wei¹, LI Zi-an¹, WANG Qiang¹, WANG Jin-xiang¹, PANG Rong-qing¹

1. National and Local Joint Engineering Laboratory of Stem Cell and Immune Cell Biomedical Technology, Key Laboratory of Cellular Therapy and Translational Medicine of Yunnan Province, Yunnan Provincial Stem Cell Engineering Laboratory, The 920th Hospital of Joint Logistics Support Force, Kunming 650032, China;
2. The 920th Hospital Clinical College, Kunming Medical University, Kunming 650031, China

Received:2019-01-16 Online:2019-10-25 Published:2021-01-05

摘要/Abstract

摘要： 目的观察人脐带间充质干细胞(hUCMSC)在胎羊肝脏内的存活与分化。方法妊娠2个月时在胎羊肝脏实质区注射绿色荧光蛋白(GFP)转染标记的hUCMSC(GFP-hUCMSC),出生1月龄时取羔羊肝脏组织,制作切片后在荧光显微镜下观察GFP阳性细胞的分布,采用PCR方法检测人雄性性别决定基因(SRY)的存在; RT-PCR方法检测人肝细胞特异基因的表达; 免疫组织化学染色检测人肝细胞特异蛋白的表达。注射同体积生理盐水的同月龄羔羊为对照。结果在注射了GFP-hUCMSC的1月龄羔羊肝脏组织中可观察到绿色荧光的规则或散在分布,并可检测到人SRY基因的存在。RT-PCR方法可检测到人肝细胞核因子4α(HNF4α)、肝细胞核因子3β(HNF3)和白蛋白(ALB)基因的阳性表达;免疫组织化学染色可检测到人ALB和HNF4α蛋白的阳性表达,而对照羔羊肝脏检测不到绿色荧光阳性细胞和人SRY基因以及人肝细胞特异基因和蛋白的表达。结论 hUCMSC可以在胎羊肝脏内存活并分化为人肝细胞样细胞,并可在胎羊出生后至少持续一个月。

关键词: 人脐带间充质干细胞(hUCMSC), 山羊, 肝细胞, 分化

Abstract: Objective To observe the survival and differentiation of human umbilical cord mesenchymal stem cells (hUCMSC) in fetal goat liver. Methods Liver tissues of 1 month old lambs was harvest which was injected with GFP-hUCMSC into liver at 2 month fetal phase, and the distribution of green fluorescent protein (GFP)-positive cells were observed under fluorescence microscope. The presence of human sex-Determining Region Y (SRY) gene was detected by PCR, while the expression of human hepatocyte-specific genes were detected by RT-PCR, and the expression of human hepatocyte-specific proteins were detected by immunohistochemical staining. The same-month-old lambs injected with the same volume physiological saline during fetal phase as control. Results The regular or scattered distribution of green fluorescence and the presence of human SRY gene were observed in the liver tissue of 1 month old lambs injected with GFP-hUCMSC. The human hepatocyte nuclear factor 4α(HNF4α), HNF3β and albumin (ALB) gene were positive detected by RT-PCR, and the ALB and HNF4α proteins were positive detected by immunohistochemical staining. No green fluorescence, human SRY genes, human hepatocyte specific genes and proteins were detected in the control lamb liver. Conclusion hUCMSC can survive and differentiate into human hepatocyte-like cells in fetal sheep liver, which can last for at least one month after birth.

Key words: Human umbilical cord mesenchymal stem cells (hUCMSC), Goat, Hepatocyte, Differentiation

中图分类号:

Q95-33

鄢东海, 黄小昆, 向雪萍, 卿德科, 方伟, 李自安, 王强, 王金祥, 庞荣清. 山羊胎肝内人脐带间充质干细胞的存活与分化研究[J]. 实验动物与比较医学, 2019, 39(5): 364-370.

YAN Dong-hai, HUANG Xiao-kun, XIANG Xue-ping, QING De-ke, FANG Wei, LI Zi-an, WANG Qiang, WANG Jin-xiang, PANG Rong-qing. Study on in Vivo Survival and Differentiation of Human Umbilical Cord Mesenchymal Stem Cells in Fetal Goat Liver[J]. Laboratory Animal and Comparative Medicine, 2019, 39(5): 364-370.

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山羊胎肝内人脐带间充质干细胞的存活与分化研究

Study on in Vivo Survival and Differentiation of Human Umbilical Cord Mesenchymal Stem Cells in Fetal Goat Liver

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