Notch信号通路阻断剂对人脐带间充质干细胞软骨分化的影响  

A Notch signaling pathway inhibitor affects chondrogenesis of human umbilical cord mesenchymal stem cells

作　　者：张家文[1] 罗二梅[1] 吴颜晖[1] 符淑莹 陈丹纯[1] 宇丽[1] 唐明乔[2] 

机构地区：[1]暨南大学医学院生物化学系,广东省广州市510632 [2]暨南大学理工学院力学和土木工程系,广东省广州市510632

出　　处：《中国组织工程研究》2014年第23期3609-3615,共7页Chinese Journal of Tissue Engineering Research

基　　金：广东省自然科学基金资助项目(9151008901000050);暨南大学大学生创新创业训练计划(Cx12200)~~

摘　　要：背景:Notch信号通路作为一条在细胞增殖和分化过程中起重要作用的信号通路,目前它在人脐带间充质干细胞在体外向软骨细胞诱导分化过程中的作用仍然未知。目的:首次探讨Notch信号通路特异性阻断剂2,4-二氨基-5-苯噻唑(DAPT)对人脐带间充质干细胞向软骨细胞诱导分化的影响。方法:从人脐带中分离获得间充质干细胞,体外向软骨细胞诱导分化。实验分4组:①无诱导组换成含体积分数5%胎牛血清和1%双抗的DMEM高糖培养基培养。②单纯诱导组换成终浓度为6.25 mg/L胰岛素、6.25 mg/L转铁蛋白、10μg/L转化生长因子β1、0.1μmol/L地塞米松、50 mg/L维生素C、体积分数5%胎牛血清和1%双抗的软骨诱导培养基培养。③二甲基亚砜组在软骨诱导培养基中加入终浓度为0.1%的二甲基亚砜培养。④2,4-二氨基-5-苯噻唑组在软骨诱导培养基中加入终浓度为5μmol/L的2,4-二氨基-5-苯噻唑(溶于二甲基亚砜)培养,二甲基亚砜终浓度为0.1%。结果与结论:诱导人脐带间充质干细胞向软骨细胞分化后,细胞形态由长梭形变为多边形,且甲苯胺蓝染色和免疫荧光染色均呈现阳性;软骨诱导分化后Jag-1、PS-1、Notch-1、Hes-1的基因表达均明显下降(P<0.01);添加2,4-二氨基-5-苯噻唑后,与单纯诱导组相比,人脐带间充质干细胞中Jag-1、PS-1、Hes-1(P<0.01)和Notch-1(P<0.05)的基因表达显著降低;蛋白聚糖和Ⅱ型胶原蛋白含量均降低(P<0.01);蛋白聚糖的基因表达显著降低(P<0.01),Ⅱ型胶原蛋白的基因表达也出现一定程度的下降。提示Notch信号存在于人脐带间充质干细胞中,诱导人脐带间充质干细胞向软骨细胞分化后,这种信号强度迅速减弱;2,4-二氨基-5-苯噻唑可能通过Jag-1-Notch-1-Hes-1途径抑制人脐带间充质干细胞向软骨细胞分化。BACKGROUND：Notch singling pathway is very important for cellproliferation and differentiation, but its role is stil unknown during chondrogenesis of human umbilical cord mesenchymal stem cells. OBJECTIVE：To investigate the effect of N-[N-（3,5-difluorophenacetyl-L-alanyl）]-（S）-phenylglycinet-butyl ester （DAPT） on inducing human umbilical cord mesenchymal stem celldifferentiation into chondrocytes. METHODS：Human umbilical cord mesenchymal stem cells were isolated from human umbilical cord, then were induced to differentiate into chondrocytes. There were four experimental groups：non-induced group, high-glucose Dulbecco’s modified Eagle’s medium containing 5%fetal bovine serum and 1%double antibody;induced group, induced medium containing 6.25 mg/L insulin, 6.25 mg/L transferrin, 10μg/L transforming growth factor beta 1, 0.1μmol/L dexamethasone, 50 mg/L vitamin C, 5%fetal bovine serum and 1%double antibody;dimethyl sulfoxide group, induced medium containing 0.1%dimethyl sulfoxide;DAPT group, induced medium containing 5μmol/L DAPT. RESULTS AND CONCLUSION：After chondrogenic induction, the morphology of human umbilical cord mesenchymal stem cells became polygon and positive for toluidine blue and immunofluorescence staining;the expression of Jag-1, PS-1, Notch-1 and Hes-1 decreased significantly （P〈0.01）. After the addition of DAPT, compared with the induced group, the relative gene expression of Jag-1, PS-1 and Hes-1 decreased markedly （P〈0.01）, the relative gene expression of Notch-1 decreased obviously as wel （P〈0.05）, and the contents of proteoglycan and col agen type II proteins decreased significantly （P〈0.01）. At the same time, the relative gene expression of proteoglycan decreased obviously （P〈0.05）, and the relative gene expression of col agen type II decreased in part. Notch signaling pathway exists in human umbilical cord mesenchymal stem cells, once chondrogenesis begins, the signaling strength wil decline rapidly. DAPT may prevent human umbilical co

关 键 词：干细胞 脐带脐血干细胞 分化 人脐带间充质干细胞 DAPT Notch信号通路 蛋白聚糖 Ⅱ型胶原蛋白 甲苯胺蓝染色 免疫荧光染色 qPCR 广东省自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]