过表达SHOX2对BMP9诱导的小鼠间充质干细胞系C3H10T1/2成骨分化的影响

Effects of SHOX2 over-expression on BMP9-induced osteogenic differentiation of mouse mesenchymal stem cell line C3H10T1/2

目的研究矮小身材同源框2(SHOX2)对骨形态发生蛋白9(BMP9)诱导的小鼠间充质干细胞系C3H10T1/2成骨分化的影响。方法构建SHOX2重组腺病毒(Ad-SHOX2),实验分为对照(NC)组、Ad-SHOX2组、BMP9组和BMP9+Ad-SHOX2组。碱性磷酸酶(ALP)评估早期成骨;钙盐沉积检测晚期成骨;显微镜观察细胞增殖;Western blot检测成骨相关转录因子(RUNX2)、增殖细胞核抗原(PCNA)、ERK1/2磷酸化及总蛋白表达。结果成功构建Ad-SHOX2。Ad-SHOX2组与BMP9组较NC组ALP活性、钙盐结节及RUNX2蛋白表达明显上升(P<0.05)。相较于以上两组,BMP9+Ad-SHOX2组ALP活性增高(P<0.05),低感染率BMP9+Ad-SHOX2组钙盐结节减少。高感染率组则较BMP9组钙盐沉积增多,与Ad-SHOX2组无明显差异。同时,BMP9+Ad-SHOX2组RUNX2蛋白表达高于BMP9组,低于Ad-SHOX2组(P<0.05)。显微镜观察Ad-SHOX2组与BMP9组细胞密度均高于NC组,PCNA蛋白表达上调(P<0.05)。且BMP9+Ad-SHOX2组PCNA蛋白表达高于BMP9组(P<0.05)。此外,Ad-SHOX2组及BMP9+Ad-SHOX2组较NC组T-ERK1/2和p-ERK1/2蛋白表达增高(P<0.05)。结论无外源性成骨刺激因子诱导条件下,SHOX2可能通过调控MAPK/ERK信号通路促进C3H10T1/2细胞增殖及成骨分化,但其与BMP9对成骨分化的调控作用是否可能互为拮抗,有待进一步研究。

Objective To investigate the effects of short stature homeobox 2(SHOX2)on bone morphogeneticprotein 9(BMP9)-induced osteogenic differentiation of mouse mesenchymal stem cell line C3H10T1/2.Methods After construction of SHOX2 recombinant adenoviruse(Ad-SHOX2),normal control(NC)group,Ad-SHOX2 group,BMP9 group,as well as BMP9+Ad-SHOX2 group were set up.Early osteogenic ability was evaluated with alkaline phosphatase(ALP);Late osteogenic ability was detected by Ca2+deposition;Cell proliferation was observed using microscope;Meanwhile,Western blot was employed to confirm protein expression of proliferating cell nuclear antigen(PCNA),runt-related transcription factor 2(RUNX2),T-ERK1/2 and p-ERK1/2.ResultsAd-SHOX2 was constructed successfully.Ad-SHOX2 and BMP9 groups produced higher ALP activity,more Ca2+accumula-tion,and higher RUNX2 expression in comparison with NC group(P<0.05).Compared with Ad-SHOX2 and BMP9 groups,ALP activity in BMP9+Ad-SHOX2 group was enhanced(P<0.05).Ad-SHOX2(low infection)+BMP9 group dampened Ca2+accumulation remarkably.And that with high infection stimulated Ca2+accumulation compared with BMP9 group,but changeless in contrast with Ad-SHOX2 group.Meanwhile,RUNX2 expression in BMP9+Ad-SHOX2 group was higher compared with BMP9 group's,but no change was found in Ad-SHOX2 group(P<0.05).Cell growth density in Ad-SHOX2 and BMP9 groups was greater than NC group's,consistent with protein level of PCNA(P<0.05).And protein expression of PCNA in BMP9+Ad-SHOX2 group was increased compared with BMP9 group's(P<0.05).Additionally,protein expressions of T-ERK1/2 and p-ERK1/2 in both Ad-SHOX2 group and BMP9+Ad-SHOX2 group were up-regulated compared with NC group(P<0.05).ConclusionsSHOX2 may promote proliferation and osteogenic differentiation of C3H10T1/2 cells via regulating MAPK/ERK signaling pathway in the absence of exogenous osteoinductive factors.However,its effects on osteogenic differentiation with BMP9 potentially function with antagonistic effect and this is target for further research.