骨质疏松小鼠模型骨髓间充质干细胞特性的研究

Research of biological properties of MSCs from osteoporotic mouse model

目的探讨骨质疏松小鼠模型骨髓间充质干细胞(mesenchymal stem cells,MSCs)生物学特性的改变。方法通过皮下注射地塞米松构建小鼠骨质疏松动物模型(OP组),同时设立对照(NC组),分离OP组及NC组骨髓MSCs进行体外培养;通过流式细胞术检测细胞表面分化抗原;运用CKK-8实验检测细胞增殖活力;通过脱氧核苷酸末端转移酶介导的dUTP缺口末端标记法(TUNEL)检测细胞凋亡水平;运用碱性磷酸酶(ALP)活性检测、油红O染色及Western blot法检测两组细胞成骨分化及脂肪分化能力。结果经过骨组织学检测证明模型建立成功,体外培养OP组及NC组骨髓MSCs细胞形态无差异。OP-MSCs细胞表面分化抗原Scal1及CD44为阳性,CD34及CD11b为阴性,与NC-MSCs相似,但OP-MSCs增殖活力下降(P<0.05);此外两组细胞凋亡水平类似(P>0.05)。成骨分化诱导7 d后OP-MSCs的ALP活性显著低于对照组(P<0.01),21 d后矿化小结明显减少(P<0.001);脂肪分化诱导8 d后OP-MSCs形成更多脂滴(P<0.05)。Western blot结果显示OP-MSCs在成骨分化中低表达转录因子Runx2(P<0.05)及Osterix(P<0.001),但在脂肪分化早期高表达PPARγ(P<0.001)及C/EBPα(P<0.01)。结论骨质疏松小鼠骨髓MSCs的增殖及分化潜能显著下降。

Objective This work discussed the changes of biological characters of mesenchymal stem cells(MSCs)in a osteoporotic mouse model.Methods Osteoporotic mouse model(OP group)and negative control(NC group)were established by subcutaneous dexamethasone(Dex)and saline injection,respectively.MSCs isolated from bone marrow of both groups were cultured in vitro.Flow cytometry was used to detect cell surface markers.The proliferation rate of MSCs was detected by CKK-8 assay.Cell apoptosis was detected by TUNEL assay.ALP activity assay,oil red O staining and western blot assay were used to detect the osteogenic and adipogenic properties of MSCs from both groups.Results Bone morphologic analysis showed that osteoporotic models had been successfully established.No difference was observed in cell morphology of MSCs isolated from bone marrow of OP and NC groups.Like NC-MSCs,OP-MSCs were Scal1 and CD44 positive,but lowly expressed CD34 and CD11 b.The proliferation rate of OP-MSCs was significantly lower than NC-MSCs(P<0.05).However,no significant difference was noted in the apoptosis rate of MSCs from both OP and NC groups(P>0.05).We further found that OP-MSCs had much lower ALP activity(P<0.01),and formed much less mineralized nodules during osteoblastic differentiation(P<0.001).However,more lipid droplets were noticed in OP-MSCs during adipocytic differentiation(P<0.05).The Western blot result showed that osteoblastic transcription factors Runx2(P<0.05)and Osterix(P<0.001)were lowly expressed in OP-MSCs during osteoblast differentiation.However,the expression levels of PPARγ(P<0.001)and C/EBPα(P<0.01)were significantly higher in OP-MSCs during adipocyte differentiation.Conclusion The proliferation and differentiation properties of osteoporotic MSCs were significantly down-regulated.