人羊水来源间充质干细胞向成肌细胞诱导的体外实验研究

In vitro study of differentiation of human amniotic fluid-derived mesenchymal stem cells into myoblasts

目的 探讨成肌细胞条件培养液体外诱导人羊水来源间充质干细胞向成肌细胞分化的可行性.方法 B超引导下穿刺抽得孕中期羊水,体外培养、分离得到羊水来源间充质干细胞.鼠成肌细胞体外培养后收集上清液,检测上清液中半乳糖凝集素-1（Galectin-1）含量,并制备成肌细胞条件培养液.实验组于成肌细胞条件培养液中培养,对照组于成肌细胞诱导培养液中培养.观察2组细胞形态学变化,免疫荧光染色、RT-PCR检测成肌细胞特异性标志物Pax7、MyoD、肌结蛋白（Desmin）、肌钙蛋白Ⅰ（Tn Ⅰ）及mRNA表达情况.结果 倒置相差显微镜下可见实验组细胞诱导第18天出现折光性强、体积较小的细胞,呈多角形,并带有突起,且逐渐成长条形;可见少量多核细胞.对照组细胞呈扁平多角形,胞体较大.诱导24 d免疫荧光染色及RT-PCR提示实验组细胞不同程度表达Pax7、MyoD、Desmin、TnⅠ及mRNA;对照组呈阴性.成肌细胞培养上清液中半乳糖凝集素-1含量较低.结论 成肌细胞条件培养液能诱导人羊水来源间充质干细胞向成肌细胞样细胞分化.

Objective To investigate the feasibility of myoblast conditioned medium on differentiation of human amniotic fluid-derived mesenchymal stem cells（hAF-MSCs）into myoblasts in vitro. Methods hAFMSCs were isolated from second trimester amniotic fluid（AF） which was backup of amniocentesis specimens.Mouse myoblasts culture supernatant was collected and galectin-1 content was detected by enzyme-linked immunosorbent assay （ELISA） before being made into myoblast conditioned medium. hAF-MSCs were divided and cultured in myoblast conditioned medium and myoblast inductive medium as experimental and control groups, respectively. The differentiated cells were identified by morphological observation, by measuring myogenic markers （Pax7, MyoD, Desmin and Tn Ⅰ）, by immunofluroseenee, as well as mRNA by RT-PCR on the 24th day. Results Being cultured in myoblast conditioned medium for 18 days, hAF-MSCs became more refractive and polygonal. The cells gradually turned into elongated shape. The hAF-MSCs of control group were flat and polygonal. lmmunofluroscence and RT-PCR demonstrated that Pax7, MyoD, Desmin and Tn Ⅰ were only expressed in the experiment group cells after 24 days＇ induction. Low lever concentration of galectin-1 was detected in the myoblasts culture supernatant. Conclusion Myoblast conditioned medium provides an ideal micro-environment which promote the differentiation of hAF-MSCs into myoblast-like cells in vitro. hAF-MSCs will be ideal seed cells for the cytotherapy and skeletal muscle tissue engineering.