1.Zhengzhou Shuqing Medical College;2.Huiji District People's Hospital;3.The First Affiliated Hospital of Zhengzhou University
目的 研究解整合素金属蛋白酶 10（ADAM10）基因通过Notch1通路调控骨髓间充质干细胞（BMSCs）成骨分化及胫骨骨折愈合。方法 培养SD大鼠BMSCs，建立稳定转染阴性对照（NC）shRNA或ADAM10 shRNA pGFP-V-RS载体的BMSCs，进行成骨诱导14d，诱导过程中分别加入对照溶剂DMSO或10μmol/L Notch1激动剂丙戊酸（VPA），检测茜素红染色的405nm波长吸光值、ALP活性及ADAM10、OCN、Runx2、Col-I、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或ADAM10 shRNA的pCMV5.0载体，4周后观察骨折愈合情况及基因表达水平。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405nm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405nm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通过抑制Notch1通路促进BMSCs的成骨分化及胫骨骨折的愈合。
Objective To study the regulation of a disintegrin and metalloproteinase 10 (ADAM10) gene on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and tibial fracture union. Methods BMSCs of SD rats were cultured, BMSCs stably transfected with negative control (NC) shRNA or ADAM10 shRNA pGFP-V-RS vector were established, the osteogenesis was induced for 14 days. During the induction process, DMSO or 10 μmol/L Notch1 agonist valproic acid (VPA) were added. absorbance at 405nm of alizarin red staining, ALP activity and the expression levels of ADAM10, OCN, Runx2, CoL-I, NICD and Hes1 were detected. The tibial fracture model of SD rats was established and NC shRNA or ADAM10 shRNA pCMV5.0 vector was injected locally. The fracture healing and gene expression were observed for 4 weeks later. Results The expression level of ADAM10 in BMSCs in sh-ADAM10 group was lower than that in sh-NC group. After osteogenesis induction, the absorbance value of alizarin red staining at 405nm, ALP activity and the expression levels of OCN, Runx2, CoL-I, NICD and Hes1 of sh-ADAM10 group were higher than those in sh-NC group (P<0.05). the absorbance value of alizarin red staining at 405nm, ALP activity and the expression levels of OCN, Runx2, CoL-I of sh-ADAM10 VPA group after osteogenesis induction were lower than those in sh-ADAM DMSO group (P < 0.05). The fracture healing of tibial fracture rats in sh-ADAM10 group was better than that in sh-NC group, and the expression levels of OCN, Runx2, CoL-I, NICD and Hes1 were higher than those in sh-NC group (P<0.05). Conclusion Knockdown of ADAM10 can promote the osteogenic differentiation of BMSCs and the healing of tibial fractures by inhibiting Notch1 pathway.