单 位：
河南省农业科学院畜牧兽医研究所;国家干细胞工程技术研究中心陕西分中心;陕西省农业分子生物学重点实验室;西北农林科技大学动物医学院
关键词：
诱导性多能干细胞;神经细胞;诱导分化;发育多能性;牛
摘 要：
【目的】通过类胚体介导体外自由分化与诱导分化,验证所建立的牛诱导性多能干细胞(Bovine induced pluripotent stem cells,biPSCs)能否分化为外胚层的神经细胞。【方法】将biPSCs悬浮培养以制备类胚体,通过类胚体介导使biPSCs在添加全反式维甲酸(RA)和β巯基乙醇(β-Me)诱导体系中分化为神经细胞,比较其诱导效率;提取不同诱导体系的分化细胞与类胚体的总RNA,利用RT-PCR方法鉴定分化细胞中神经细胞特异性基因的表达。【结果】培养的biPSCs集落呈球形,中央隆起,周围界限清晰,与胚胎干细胞集落形态类似。biPSCs碱性磷酸酶(AP)染色为阳性,并表达SSEA-4干细胞特异性表面蛋白。biPSCs通过类胚体介导分化,在未添加化学诱导物的条件下,可自由分化为Nestin、GFAP与NSE阳性神经细胞,其阳性细胞率分别为(15.14±1.13)%,(6.25±0.35)%和(5.45±0.62)%;biPSCs在RA诱导组中分化的神经细胞的数量最多,诱导后表达Nestin、GFAP和NSE细胞的阳性率分别为(49.56±2.33)%,(16.58±1.28)%和(13.66±2.21)%;在β-Me诱导组中,表达Nestin的阳性细胞率为(42.23±1.25)%。2个诱导组的诱导效率与无化学诱导物组有显著差异(P<0.05)。RT-PCR检测结果显示,不同诱导体系获得的分化细胞均能扩增到神经细胞特异性基因Nestin和βⅢ-Tubulin片段,产物长度与预期结果完全一致。【结论】biPSCs在体外具有向神经细胞发育的能力;RA是诱导biPSCs向神经细胞分化的良好诱导物。
译 名：
Differentiation potential of neural lineage cells from bovine induced pluripotent stem cells
作 者：
L Chang-rong1,CHEN Dong-mei 1,XIN Xiao-ling1,2,DOU Zhong-ying1(1 College of Veterinary Medicine,Northwest A&F University,Shaanxi Key Laboratory of Molecular Biology for Agriculture,Shaanxi Branch of National Stem Cell Engineering&Technology Center,Yangling,Shaanxi 712100,China;2 Institute of Animal Husbandry and Veterinary Medicine,He'nan Academy of Agricultural Sciences,Zhengzhou,He'nan,450002,China)
关键词：
induced pluripotent stem cells(iPSCs);neural lineage cells;cell differentiation;developmental potential;bovine
摘 要：
【Objective】Bovine induced pluripotent stem cells(biPSCs)have been generated in the previous study.This study was conducted to verify the differentiation potential of neural lineage cells from the resulting biPSCs under different induction conditions.【Method】The resulting biPSCs were cultured using the hanging drop and suspension culture method for embryoid body(EBs)formation,and EBs were transferred into 0.1% gelatin-coated 24-well plates for attached culture.The attached cells were divided into three induction groups including inducer-free group,supplemented with retinoic acid(RA)and β-Mercaptoethanol(β-Me).The marker proteins of nerve cells including nestin,GFAP,and NSE were examined by immunocytochemistry staining.The induction efficiency was evaluated based on the number of immunocytochemistry positive cells.RT-PCR was performed to examine the neural cell marker genes β Ⅲ-Tubulin and Nestin.【Result】The resulting biPSCs colonies showed a ball-shaped and tightly packed morphology with rough surfaces and clear borders.The cells were strongly positive for alkaline phosphatase(AP)and SSEA-4.BiPSCs could effectively form typical ball-shaped and cavity-like EBs in suspension culture.Under the inducer-free condition,the attached cells randomly differentiated nerve cells,the percentage of positive for Nestin,GFAP and NSE respectively was(15.14±1.13)%,(6.25±0.35)% and(5.45±0.62)% about 6days after attached culture.There were(49.56±2.33)%,(16.58±1.28)% and(13.66±2.21)% of the attached cells to express Nestin,GFAP and NSE in RA medium around 8 days after induction.Approxi-mately(42.23±1.25)% attached cells were positive for Nestin protein in β-Me medium after 3hours induction.RT-PCR further confirmed that the differentiated cells derived from biPSCs could express neural lineage cells marker genes nestin and β Ⅲ-Tubulin.【Conclusion】The results revealed that the established biPSCs can effectively differentiate into neural lineage cells.RA is suitable for biPSCs induced differentiation into nerve cells.
