重组人骨形态发生蛋白-2对体外培养的肺动脉平滑肌细胞增殖的影响及其机制

Bone morphogenetic protein-2 inhibited proliferation in pulmonary artery smooth muscle cells by downregulating expression of Cyclin D1 gene in vitro

目的探讨重组人骨形态发生蛋白（rhBMP）-2对体外培养的肺动脉平滑肌细胞（PASMC）增殖的影响及机制。方法将用贴块法培养的PASMC传代后分成5组，Ⅰ组：含0．1％FBS培养基培养；Ⅱ组：含10％FBS培养基正常培养；Ⅲ组：10％FBS＋rhBMP-21ug/L；Ⅳ组：10％FBS＋rhBMP-210ug／L；V组：10％FBS＋rhBMP-2100ug／L。倒置相差显微镜及免疫荧光法观察并鉴定培养的PASMC；使用噻唑蓝（MTT）比色法测定PASMC增殖率；流式细胞仪分析PASMC细胞周期的变化；^3H—TdR掺入实验检测DNA合成情况；RT—PCR检测细胞周期素（Cyclin）D1mRNA的表达量；Western blot检测pSmad蛋白量及CyclinD1蛋白表达。结果抗SM—α—actin免疫荧光染色鉴定所培养细胞为PASMC；Ⅱ组引起PASMC增殖率升高（90．26±30．12），S期细胞百分比增加（35．90±0．73），DNA合成增加（4890±372）的作用可以被Ⅲ、Ⅳ组的rhBMP-2抑制（66．29±27．19、15．90±0．61、3390±198，P〈0．01），同时Ⅲ、Ⅳ组的rhBMP-2明显增加了pSmad蛋白量（1．37±0.26，1．68±0．31，P〈0．01），并抑制了CyclinD1mRNA及蛋白表达（1．38±0．13，1．01±0．10，P〈0．01）。结论rhBMP-2可以通过Smad信号转导途径抑制10％FBS培养的PASMC表达CyclinD1。进而抑制血清刺激引起的PASMC增殖。

Objective To investigate the effect and mechanism of rhBMP-2 on pulmonary artery smooth muscle cells in vitro. Methods The pulmonary artery was isolated from the rats and cut into small tissue pieces （ 1 mm^2-2 mm^2）. These explants were seeded onto culture flasks and passaged after 85% confluence of primary cells. The pulmonary artery smooth muscle cells （PASMCs） were divided into 5 groups：group Ⅰ （1% fetal calf serum for 24 h）; group Ⅱ （10% fetal calf serum for 24 h）; group Ⅲ （ 10% fetal calf serum and 1 ug/L rhBMP-2 for 24 h） ; group Ⅳ（ 10% fetal calf serum and 10 ug/L rh- BMP-2 for 24 h） ; group Ⅴ （ 10% fetal calf serum and 100 ug/L rhBMP-2 for 24 h）. The cultured cells were observed by phase-contrast microscope and identified by immunofluorescent stain of smooth muscle cells specific antigen. The proliferation of SMCs was evaluated by the MTT assay. The cell cycle analyses including proliferation index （PI） and S-phase cell fraction （SPF） were performed by the flow cytometry. The DNA synthesis was detected by [ methyl-^3H ]-thymidine incorporation. The expression of Cyclin D1 mRNA was detected by RT-PCR assay. The amount of phosphorylated Smadl （ pSmadl ） and the expression of Cyclin D1 protein were determined by Western blot. Results A typical ＂hill-valley＂ growth pattern was displayed under phase-contrast light microscope. Immunofluorescent stain revealed that 98% of the cultured cells were positive for SM-α-actin. The 10% FBS and 10 or 100 ug/L rhBMP-2 attenuated proliferation rate, SPF, DNA synthesis compared with 10% FBS alone （90.26 ＋- 30.12）, （35.90 ＋- 0.73）, （4890 ＋-372） respectively, P 〈 0.01 ） ; but pSmadl was increased in group Ⅳ and group Ⅴ as compared with 10% FBS alone （ 1.37 ＋- 0.26,1.68 ＋- 0.31, P 〈 0.01 ）. Conclusion The rhBMP-2 could downregulate the expression of Cyclin D1 gene by smad signaling transduction system, then depress seruminduced proliferation of PASMCs in vitro.