下调骨细胞TGF-β/Smad4信号可抑制小鼠BMSCs成骨及破骨分化

Down-regulation of osteocytic TGF-β/Smad4 inhibits the osteoblastic and osteoclastic differentiation in mouse BMSCs

目的检测骨细胞TGF-β/Smad4信号通路对骨髓间充质干细胞(BMSCs)成骨和破骨分化的作用,并初步探讨其相关机制。方法用条件性基因敲减Cre/loxp技术特异性敲减骨细胞Smad4,获得下调骨细胞TGF-β/Smad4信号通路的小鼠;体外分离骨细胞并与野生型小鼠骨髓间充质细胞(BMSCs)共培养;碱性磷酸酶(ALP)染色、茜素红(alizarin red)染色检测早期成骨分化和晚期钙盐沉积,酸性磷酸酶(TRAP)染色检测破骨细胞;real-time PCR检测成骨分化特异标志物Runx2、Osterix(OSX)、ALP、osteocalcin和破骨分化特异标志物RANKL和OPG的mRNA表达水平。Western blot检测成骨分化特异标志物Runx2和osteocalcin和破骨分化特异标志物RANK蛋白表达水平。结果下调骨细胞TGF-β/Smad4信号能够抑制BMSCs成骨转录因子Runx2、Osterix(P<0.01)、成骨分化特异标志物ALP和osteocalcin(P<0.01)以及破骨分化特异标志物RANK(P<0.01)的表达;增加破骨分化抑制物OPG的表达(P<0.05);而RANKL的表达无明显变化;最终下调了RANKL/OPG的比值(P<0.05)。结论终末分化的骨细胞调控骨的代谢,下调其TGF-β/Smad4信号可抑制BMSCs成骨和破骨细胞的分化。

Objective To determine the effect of ostecytic TGF-β/Smad4 signaling on osteoblastic and osteoclastic differentiation in bone marrow stromal cells （BMSCs）. Methods Mice with osteocytic TGF-β/Smad4 conditional knock down （Smad4^ot CKD） were generated as previously by crossing DMP1-8kb-Cre mice with Smad41ox（ ex8）/ lox（ ex8 ） mice. The osteocytes were isolated from tibial and femoral diaphysis and co-cultured with wild-type BMSCs. ALP staining, Alizarin red staining and TRAP staining were performed to show osteoblastic and osteoclastic differentiation. Then, their marker genes were detected by qPCR and proteins measured by Western blot. Results The expression of Runx2 and Osterix were reduced in smad4 CKDot co-cultured with BMSCs compared with controls （ P 〈 0. 01 ）. Similarly, the specific markers of osteoblastic differentiation were decreased （P 〈 0. 01 ）. Additionally, the expression of RANKL was not significantly changed in with BMSCs. However, OPG was highly expressed in control group compared with smad4 CKD in co-cultured group （P 〈 0.05 ）. Thus, the radio of RANKL/OPG was significantly reduced （P 〈 0. 05）. Furthermore, the expression of RANK was inhibited. Conclusions The terminally-differentiated osteocytes are the cells regulating bone metabolism, while down-regulation of osteocytic-TGF-β/ Smad4 inhibits BMSC osteoblastic and osteoclastic differentiation.