摘要
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 observe the effect and mechanism of wild type Smad3 gene on the proliferation and osteoblastic differentiation of rat bone marrow derived mesenchymal stem cell (MSC) Methods Rat bone derived MSC were cultured and transfected with the complexes of pcDNA3 0 Myc Smad3 or pcDNA3 0 Myc Smad3△C and LipofectAMINE reagent Immunofluorescence technique was performed to evaluate the c Myc expression The cell proliferation was detected by MTT method In order to observe the osteoblastic characteristics in stable expression MSC, alkaline phosphatase(ALP) mRNA and core binding factor α1 (cbfa1) mRNA were investigated by RT PCR technique, and ALP activity and mineralization were examined by PNP method and alizarin red staining respectively PD98059 was used to interdict selectively ERK pathway in order to investigate the effect of ERK pathway on Smad3 regulating MSC Results c Myc protein was expressed in Smad3 MSC and Smad3△C MSC The proliferation of Smad3 MSC was slower than that of Smad3△C MSC or V MSC The relative levels of ALP mRNA and cbfa1 mRNA in Smad3 MSC were higher markedly than those in Smad3△C MSC or V MSC, as well as ALP activity and mineralization Howerer, without a significant difference ALP activity and mineralization was after PD98059 treatment (both slightly decreased P >0 05). Conclusion The wild type Smad3 gene, which is a crucial component promoting bone formation, can inhibit the proliferation of MSC and promote the osteoblastic differentiation and maturation of MSC independent of ERK pathway
出处
《中华医学杂志》
CAS
CSCD
北大核心
2004年第18期1523-1527,共5页
National Medical Journal of China
基金
国家自然科学基金资助项目 (3 0 170 2 70
3 0 2 0 0 0 63 )
