摘要
背景:脐带间充质干细胞可在体内外诱导分化为肝样细胞,然而确切机制目前尚不清楚,研究表明Wnt/β-catenin信号通路与之密切相关。目的:探讨Wnt/β-catenin信号通路对脐带间充质干细胞肝向分化的影响及其机制。方法:采用组织块贴壁法分离培养人脐带间充质干细胞,将培养至第4-6代脐带间充质干细胞分成4组,分别为对照组,常规诱导肝向分化组,激活剂Wnt3a组(在常规诱导肝向分化基础上加入20μg/L Wnt/β-catenin信号通路激活剂Wnt3a),抑制剂Dkk-1组(在常规诱导肝向分化基础上加入20μg/L Wnt/β-catenin信号通路抑制剂Dkk-1)。分别在诱导第7,14,21,28天收集细胞,采用荧光定量PCR和Western blot检测肝细胞分化相关基因的m RNA和蛋白表达。运用PAS染色、LDL摄取实验、ICG吸收实验在诱导第28天时进行肝样细胞功能检测。结果与结论:(1)与对照组相比,常规诱导组、激活剂Wnt3a组、抑制剂Dkk-1组中除了CK-19 mRNA及蛋白表达均下调外(P<0.01),AFP、ALB、HNF4α mRNA和AFP、HNF4α蛋白表达均显著上调(P<0.05);(2)与常规诱导组相比,激活剂Wnt3a组AFP、ALB、HNF4α mRNA及AFP、HNF4α蛋白表达均显著下调(P<0.05);(3)与常规诱导组、激活剂Wnt3a组相比,抑制剂Dkk-1组AFP、ALB、HNF4α mRNA及AFP、HNF4α蛋白表达显著增高(P<0.05);(4)抑制剂Dkk-1组PAS染色、LDL摄取实验、ICG吸收实验中检测到的阳性细胞最多,常规诱导组其次,阳性细胞最少的是激活剂Wnt3a组;(5)以上结果表明,Wnt/β-catenin信号通路受抑制时可促进脐带间充质干细胞肝向分化,反之,则抑制脐带间充质干细胞肝向分化。
BACKGROUND：Umbilical cord mesenchymal stem cells can be induced to differentiate into hepatocyte-like cells in vitro and in vivo.However,the exact mechanism is still unknown. Existing studies have shown that the Wnt/β-catenin signaling pathway is closely related to this process. OBJECTIVE： To explore the effect of Wnt/β-catenin signaling pathway on the differentiation of umbilical cord mesenchymal stem cells into hepatocyte-like cells and its potential molecular mechanism. METHODS： Human umbilical cord mesenchymal stem cells were extracted from the neonatal umbilical cord by tissue adherent method. After being cultured and purified, the umbilical cord mesenchymal stem cells at passages 4-6 were divided into four groups： control group （DMEM culture group）, hepatocyte-like differentiation group, activator Wnt3a group （adding 20 μg/L Wnt3a, an activator of Wnt/β-catenin signaling pathway, under the differentiation condition）, and inhibitor Dkk-1 group （adding 20 μg/L Dkk-1, an inhibitor of Wnt/β-catenin signaling pathway, under the differentiation condition）. Induced cells were collected respectively on days 7, 14, 21, 28. Their mRNA and protein expressions of α-fetoprotein （AFP）, albumin （ALB）, hepatocyte nuclear factor 4α （HNF4α） and Cytokeratin-19 （CK-19） in the cells were detected by real-time quantitative PCR and western blot respectively. Meanwhile, Periodic Acid-Schiff staining, low-density lipoprotein uptake test and indocyanine green absorption test were applied to detect the function of hepatocyte-like cells. RESULTS AND CONCLUSION： Compared with the control group, expressions of AFP and HNF4α mRNA and protein as well as ALB mRNA were significantly up-regulated in the hepatocyte-like differentiation group, activator Wnt3a group and inhibitor Dkk-1 group （P 〈 0.05）. Whereas, there was a decrease in the CK-19 expression at mRNA and protein levels （P 〈 0.01） in these three groups. Compared with the hepatocyte-like differentiation group, the mRNA and
作者
彭琴
尹燕峰
管峥
吕莎
苏文君
闪海燕
张蕾
Peng Qin;Yin Yan-feng;Guan Zheng;Lv Sha;Su Wen-jun;Shan Hai-yan;Zhang Lei(Kunming Medical University, Kunming 650500, Yunnan Province, China;Affiliated Calmette Hospital of Kunming Medical University （First Hospital of Kunming）, Kunming 650011, Yunnan Province, China)
出处
《中国组织工程研究》
CAS
北大核心
2018年第13期2011-2019,共9页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金(81660303)
云南省科技厅-昆明医科大学联合专项项目~~
