ADAM10在骨髓间充质干细胞成骨分化及胫骨骨折愈合中的作用及可能机制

Role and possible mechanism of ADAM10 in osteogenic differentiation of bone marrow mesenchymal stem cells and tibial fracture union

目的 探索解整合素金属蛋白酶10(ADAM10)在骨髓间充质干细胞(BMSCs)成骨分化及胫骨骨折愈合中的作用及可能机制。方法 培养SD大鼠BMSCs,建立稳定转染阴性对照(NC)shRNA pGFP-V-RS载体的BMSCs并分为sh-NC组和sh-NC+DMSO组,建立稳定转染ADAM10 shRNA pGFP-V-RS载体的BMSCs并分为sh-ADAM10组、sh-ADAM10+DMSO组、sh-ADAM10+丙戊酸(VPA)组。成骨诱导14 d后进行茜素红染色并检测405 nm波长吸光值、ALP活性及ADAM10、成骨标志物基因(骨钙素(OCN)、Runt相关转录因子2(Runx2)、胶原蛋白-I(Col-I))、Notch1信号通路分子(Notch细胞内片段(NICD)、Hes1)的表达水平。建立SD大鼠的胫骨骨折模型,局部注射NC shRNA、ADAM10 shRNA的pCMV5.0载体,4周后观察骨折愈合的情况以及基因表达水平。结果 sh-ADAM10组BMSCs中ADAM10的表达水平低于sh-NC组,成骨诱导后茜素红染色的405 nm波长吸光值、ALP活性及OCN、Runx2、Col-I、NICD、Hes1的表达水平均高于sh-NC组(P<0.05);sh-ADAM10+VPA组BMSCs成骨诱导后茜素红染色的405 nm波长吸光值、ALP活性及OCN、Runx2、Col-I的表达水平均低于sh-ADAM10+DMSO组(P<0.05);sh-ADAM10组胫骨骨折大鼠的骨折愈合情况优于sh-NC组,骨折部位OCN、Runx2、Col-I、NICD、Hes1的表达水平均高于sh-NC组(P<0.05)。结论 敲低ADAM10表达促进BMSCs的成骨分化及胫骨骨折愈合,这一作用与抑制Notch1通路有关。

Objective To explore the role and possible mechanism of a disintegrin and metalloproteinase 10(ADAM10)in osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)and tibial fracture union.Methods BMSCs of SD rats were cultured,stably transfected with a negative control(NC)shRNA pGFP-V-RS vector,and divided into sh-NC and sh-NC+DMSO groups.BMSCs were stably transfected with an ADAM10 shRNA pGFP-V-RS vector and divided into sh-ADAM10,sh-ADAM10+DMSO,and sh-ADAM10+valproic acid(VPA)groups.After 14 days of osteogenic induction,absorbance at 405 nm after alizarin red staining,ALP activity,and ADAM10,OCN,Runx2,CoL-I,NICD,and Hes1 expression levels were analyzed.A tibial fracture model was established in SD rats,and NC shRNA or ADAM10 shRNA pCMV50 vectors were injected locally.Fracture healing and gene expression were then observed for 4 weeks.Results The ADAM10 expression level in BMSCs of the sh-ADAM10 group was lower than that in the sh-NC group.After osteogenic induction,the absorbance value of alizarin red staining at 405 nm,ALP activity,and OCN,Runx2,CoL-I,NICD,and Hes1 expression levels of the sh-ADAM10 group were higher than those of the sh-NC group(P<005).The absorbance value of alizarin red staining at 405 nm,ALP activity,and OCN,Runx2,and Col-I expression levels of the sh-ADAM10+VPA group after osteogenic induction were lower than those in the sh-ADAM+DMSO group(P<005).Healing of the tibial fracture in the sh-ADAM10 group was better than that in the sh-NC group,and OCN,Runx2,CoL-I,NICD,and Hes1 expression levels were higher than those in the sh-NC group(P<005).Conclusions Knocking down ADAM10 expression promotes osteogenic differentiation of BMSCs and tibial fracture healing,which is related to inhibition of the Notch1 pathway.