摘要
目的探讨骨细胞Wnt信号通路对骨髓间充质干细胞(BMSCs)成骨分化的作用及其分子机制。方法采用条件性基因敲出Cre/loxp技术制备特异性激活骨细胞Wnt/β-Catenin信号通路的小鼠;体外分离其和野生对照组小鼠的骨细胞,并分别与野生型小鼠骨髓间充质干细胞共培养;碱性磷酸酶(ALP)染色及活性定量、茜素红(Alizarin red)染色检测共培养早期成骨分化和晚期钙盐沉积;Real-Time PCR检测骨细胞Notch信号配体Jag1、Jag2、Dll1、Dll4及共培养后成骨分化特异标志物Runx2、ALP、Osteocalcin的mRNA表达水平。随后于共培养中加入Notch信号通路化学抑制剂DAPT(对照组加DMSO),再次行碱性磷酸酶(ALP)染色及活性定量和Real-Time PCR检测成骨分化特异标志物表达水平。结果 Wnt/β-Catenin激活的骨细胞其自身Jag1、Jag2、Dll4 mRNA表达较对照组骨细胞明显升高(P<0.05),与BMSCs共培养后成骨转录因子Runx2,成骨分化特异标志物ALP、Osteocalcin mRNA的表达较野生型明显升高(P<0.05),钙盐沉积增加。加入DAPT后,骨细胞Wnt激活组成骨转录因子Runx2、ALP、Osteocalcin mRNA的表达明显下降(P<0.05)。结论骨细胞调控骨的代谢,激活其Wnt/β-Catenin信号通路将通过上调Notch信号而促进BMSCs的成骨分化。
Objective To determine the effect of osteocytic Wnt/β-Catenin signaling on osteoblastic differentiation in bone marrow stromal cells( BMSCs) and its molecular mechanism. Methods Mice with osteocytic Wnt activation( daβcatOt) were generated previously by crossing DMP1-8 kb-Cre mice with catnblox(ex3) mice. The osteocytes were isolated from Wnt activation mice and wild type mice. Then the two types osteocytes were separately co-cultured with wild-type BMSCs. ALP staining and Alizarin red staining were performed to measure osteoblastic differentiation in co-culture. The expressions of osteocytic Notch ligands Jag1,Jag2,Dll1,and Dll4 and osteoblastic marker genes Runx2,ALP,and osteocalcin were detected by qPCR. Furthermore,Notch signaling inhibitor DAPT was applied to the co-culture and osteoblastic marker genes were detected by qPCR again. Results The expression of Jag1,Jag2,and D114 mRNA in daβcatOt osteocytes was significantly higher than that in the control( P 〈 0. 05). In the co-cultures,the expression of osteoblastic makers Runx2,ALP,and osteocalcin mRNA was significantly higher than that in the control( P 〈 0. 05),and the deposition of calcium also increased compared to the control. The expression of osteoblastic transcription factors Runx2,ALP and Osteocalcin mRNA decreased significantly after addition of Notch inhibitor DAPT( P 〈0. 05). Conclusion Osteocyte regulates bone metabolism. The activation of Wnt/β-Catenin signaling pathway in osteocytes promotes osteogenic differentiation of BMSCs via upregulation of Notch signaling.
作者
任磊
代光明
林枭
张东力
田冕
贺尧
许文娟
涂小林
黄伟
REN Lei;DAI Guangming;LIN Xiao;ZHANG Dongli;TIAN Mian;HE Yao;XU Wenjuan;TU Xiaolin;HUANG Wei(Department of Orthopedics, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China;Laboratory of Skeletal Development and Regeneration, Institute of Life Science, Chongqing Medical University, Chongqing 400016, China)
出处
《中国骨质疏松杂志》
CAS
CSCD
北大核心
2018年第5期600-605,共6页
Chinese Journal of Osteoporosis
基金
国家自然科学基金(81371972
81572142)