雷帕霉素对大鼠骨髓源平滑肌祖细胞增殖及分化的影响

Effects of Sirolimus on Proliferation and Differentation of Rat Smooth Muscle Progenitor Cells in Vitro

目的观察雷帕霉素对骨髓源性平滑肌祖细胞增殖及分化的影响,探讨影响动脉粥样硬化的骨髓源性平滑肌祖细胞与雷帕霉素洗脱支架抑制新内膜增生的关系。方法采用密度梯度离心法从大鼠骨髓获取单个核细胞,接种于纤维连接素包被的培养板,加入不同浓度雷帕霉素（0.01-100μg/L）,培养12天后,-αSMA免疫荧光染色鉴定骨髓源性平滑肌祖细胞,并在倒置荧光显微镜下计数。收集培养8天贴壁细胞,分别加入不同浓度雷帕霉素（0.1-200μg/L）培养24 h。分别采用MTT比色法、改良的Boyden小室和粘附能力测定实验观察平滑肌祖细胞的增殖、迁移和粘附能力。结果雷帕霉素显著抑制骨髓单个核细胞分化为平滑肌祖细胞,0.1μg/L雷帕霉素作用12天,平滑肌祖细胞减少了70.9%±4.5%（P〈0.01）。雷帕霉素也显著抑制平滑肌祖细胞增殖,其抑制作用随雷帕霉素浓度增加而增加,1μg/L雷帕霉素作用24 h使平滑肌祖细胞数量减少27.1%±4.5%（P〈0.01）。0.1~200μg/L雷帕霉素显著抑制平滑肌祖细胞的粘附和迁移能力。结论雷帕霉素可抑制平滑肌祖细胞分化,并抑制平滑肌祖细胞增殖及迁移能力。

Aim To investigate the effects of sirolimus on proliferation and differentiation of rat smooth muscle progenitor cells （SPC）. Methods Total mononuclear cells （MNC） were isolated from marrow of rats by Ficoll density gradient centrifugafion method, and then plated on fibronectin-coated culture dishes. Fresh isolated MNC were treated with sirolimus （0.01 - 100 μg/L） or vehicle control for 12 days. SPC were characterized as adherent cells positive for a-SMA by indirect immunofluorescentstaining. After8dayscuhured, attached cells were treated with sirolimus （ to make a series of final concentrafiom ： 0 .1 - 200 μg/L） or vehicle control for 24 h. The proliferation and migration of SPC were assayed with MTr and modified Boyden chamber assay respectively. STC adhesion assay was performed by replating those on fibroneetin-coated dishes and counting the adherent cells. Results Sirolimus potently inhibited SPC outgrowth. The number of SPC at 12th day was dramatically decreased by sirolimus. At a concentration as low as0.1 μg/L, sirolimus significanfly reduced （70.9±4.5）% of SPC compared with control （ P 〈 0.01 ）. In addition, sirolimus also inhibited SPC proliferative, migratory and adhesive capacity in a concentration-dependent manner. Conclusion Sirolimus could inhibit the proliferation and differentiation of rat smooth muscle progenitor cells.