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(HNF3β)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。
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.
作者
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
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(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;The 920th Hospital Clinical College,Kunming Medical University,Kunming 650031,China)
出处
《实验动物与比较医学》
CAS
2019年第5期364-370,共7页
Laboratory Animal and Comparative Medicine
基金
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)
