【摘要】 目的探讨 Sonic hedgehog(shh)信号通路对小鼠主动脉-中肾-肾上腺(AGM)区来源的成血-血管细胞增殖、分化和迁移的影响。方法联合应用抗小鼠 CD34、Flk1单克隆抗体(单抗)的磁珠,从孕11 d BALB/c 鼠胚胎 AGM 区分离培养成血-血管干细胞,并同时培养 AGM 区来源基质细胞,用流式细胞术对所分离细胞进行表型鉴定。用免疫组织化学法检测基质细胞表达 Shh 蛋白的情况;借助 Transwell 细胞共培养体系,观察在 AGM 的基质细胞共培养体系中,不同浓度的 Shh 活性肽及其单抗对成血-血管干细胞增殖、细胞周期、迁移及向内皮细胞定向分化的影响。结果 AGM 区基质细胞高表达 Shh 蛋白(阳性率高达90%以上),并可促进成血-血管干细胞增殖,其增殖效应可被10.00 μg/mlShh 单抗阻断;0.10～10.00 μg/ml 外源性 Shh 活性蛋白与 AGM 区基质细胞共培养的成血-血管干细胞,可使成血-血管干细胞长时间增殖,并促进细胞迁移,但并不诱导细胞凋亡与分化;而当0.10～10.00 μg/ml 外源性 Shh 活性蛋白作用于单独培养的成血-血管干细胞,细胞经过短时间增殖后,随后发生凋亡并向内皮细胞分化。结论 Shh 信号通路参与了成血-血管干细胞的增殖、分化、凋亡及迁移等生物学特性的调控,但其具体作用存在时空的差异,AGM 区微环境对 Shh 信号通路具有整合和调试的作用。更多还原

【Abstract】 Objective To explore the role of sonic hedgehog(Shh)pathway in regulating the prolif- eration,migration and differentiation of hemangioblasts derived from aorta-gonad-mesonephros(AGM). Methods The hemangioblasts were isolated from AGM region of 11-day postcoitum(dpc)murine embryos by using the immuno-magnetic beads cooted with CD34 and Flk1 monoclonal antibodies.The phenotypic ana- lysis of hemangioblasts and AGM-derived stromal cells were detected by flow cytometry.The secretion of Shh was examined by immunohistochemical staining.The roles of Shh in regulating the proliferation,migration and differentiation of hemangioblasts in the transwell non-contact coculture system with AGM-derived stromal cells were observed by adding exogenous Shh N-Terminus and its antibody.Results The protein of Shh was highly expressed on AGM-derived stromal cells.The proliferation of hemangioblasts was promoted when co-cultured with AGM-derived stromal cells,and the effects of the latter could be blocked by antibody of Shh.The prolif- eration of hemangioblasts was strengthened further and kept for a long time without differentation and apoptosis when exogenous Shh N-Terminus was added into the transwell non-contact co-culture system with AGM-de- rived stromal cells.When exogenous Shh N-Terminus was added into the cultural supernatant of hemangio- blasts without AGM-derived stromal cells,the hemangioblasts were observed to be induced to apoptosis or dif- ferentiation after a short time of proliferation.Furethmore,the ability of migration could be promoted in the co-cuhured hemangioblasts by adding exogenous Shh N-Terminus.Conclusion Shh pathway probably in- volves in the regulation of the proliferation,differentiation,apoptosis and migration of hemangioblasts,and is regulated by the AGM microenvironment.更多还原