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目的 探讨野生型Smad3基因对大鼠骨髓间充质干细胞 (MSC)增殖和成骨分化的影响以及作用机理。方法 脂质体介导稳定转染Smad3和Smad3△C ,间接免疫荧光试验鉴定 ;采用噻唑蓝 (MTT)检测Smad3转染对MSC增殖的影响 ;采用逆转录聚合酶链反应检测转染细胞中ALP和核心结合因子 (cbfa1)mRNA的表达 ,用对硝基苯磷酸盐 (PNP)法检测细胞ALP活性 ,用茜素红染色法检测细胞矿化能力 ,观察Smad3对MSC成骨分化的影响 ;用PD980 5 9选择性阻断非细胞外信号调节激酶(ERK)通路 ,观察ERK通路在Smad3调节MSC成骨分化中的作用。结果 稳定转染Smad3和Smad3△C的细胞中c Myc抗原阳性表达 ;Smad3 MSC增殖缓慢 ,细胞群体倍增时间延长 ,没有明显的对数生长期 ;Smad3 MSC中ALP和cbfa1mRNA的表达水平、ALP活性以及矿化能力明显高于Smad3△C MSC与V MSC ;加入PD980 5 9后 ,Smad3 MSC中ALP活性以及矿化能力有所减低 ,但与未加入PD980 5 9组相比 ,差异无显著意义 (P >0 0 5 )。结论 野生型Smad3基因抑制MSC的增殖 ,并通过非ERK通路促进MSC向成骨分化和成熟 ,是促进骨形成的重要成分。
Objective To investigate the effect and mechanism of wild-type Smad3 on proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (MSCs). Methods Liposomes were transfected into Smad3 and Smad3ΔC cells by indirect immunofluorescence assay. The effect of Smad3 transfection on the proliferation of MSC was detected by MTT assay. The expression of ALP in transfected cells was detected by reverse transcription polymerase chain reaction And core binding factor (cbfa1) mRNA expression. The cell ALP activity was assayed by p-nitrophenylphosphate (PNP) method. The cell mineralization ability was detected by alizarin red staining. The effect of Smad3 on osteogenic differentiation was observed. PD9805 selectively blocked the extracellular signal-regulated kinase (ERK) pathway and observed the role of ERK pathway in Smad3-regulated osteogenic differentiation of MSCs. Results The expression of c Myc antigen in Smad3 and Smad3ΔC cells was positive. The proliferation of Smad3 MSCs was slow and the population doubling time was prolonged without obvious logarithmic growth phase. The expressions of ALP and cbfa1 mRNA and ALP in Smad3 MSC and The mineralization ability of Smad3 was significantly higher than that of Smad3ΔCMSCs and VMSCs. After addition of PD98059, the activity of ALP and the mineralization ability of Smad3MSCs were decreased, but there was no significant difference compared with those without PD98059 (P > 0 0 5). Conclusion The wild-type Smad3 gene inhibits the proliferation of MSC and promotes MSC differentiation and maturation into osteoblasts through non-ERK pathway, which is an important component of promoting bone formation.