喉黏膜间充质干细胞肌向分化的实验研究

Myogenic differentiation of laryngeal mucosal mesenchymal stem cells

目的 将喉黏膜间充质干细胞（laryngeal mucosal mesenchymal stem cells,LM-MSC）向成肌细胞诱导分化,为声带部分或全部缺失的组织工程修复寻求优势种子细胞.方法 从正常会厌舌面黏膜分离培养获得LM-MSC,流式细胞仪检测细胞表面分子标志CD44、CD105、CD90、CD29、CD34和CD45;向成脂、成骨细胞分化鉴定细胞多向分化潜能,油红O、茜素红染色鉴定成脂、成骨分化情况;加入成肌诱导液向成肌细胞方向分化,于成肌诱导分化后1周和3周时行免疫荧光化学染色,诱导后1周、3周和6周行实时定量RT-PCR分析,以分别定性和定量分析成肌分化相关蛋白及基因Myod1、Myogenin、肌球蛋白重链（myosin heavy chain,MyHc）的表达.结果 本实验分离培养获得的LM-MSC可贴壁生长,呈长梭形;高表达间充质干细胞表面分子标志物：CD44（100.0％）,CD105（90.4％）,CD90（99.9％）,CD29 （93.0％）,低表达造血系干细胞表面分子标志物：CD34（0.4％）和CD45（1.3％）;LM-MSC在体外定向向成脂、成骨分化14 d和21 d后有大量串珠样脂滴和矿化结节生成;成肌细胞初始标志蛋白Myod1和特异性标志蛋白Myogenin,MyHc分别于成肌诱导分化后1周和3周出现,RT-PCR结果与免疫荧光化学染色结果一致,并且相关基因持续表达于成肌诱导分化后6周.结论 LM-MSC符合间充质干细胞的特性,并可定向分化为成肌细胞,有潜能成为声带组织工程修复的优势种子细胞.

Objective To investigate the myogenic differentiation of laryngeal mucosal mesenchymal stem cells （LM-MSCs） and the possibility of LM-MSCs as new alternative seed cells for laryngeal tissue engineering.Methods LM-MSCs were separated from normal epiglottis mucosa and the cell surface markers including CD44,CD105,CD90,CD29,CD34 and CD45 were analyzed through flow cytometry.The osteogenesis and adipogenesis differentiation of LM-MSCs were investigated by oil red staining and alizarin red S staining.Immunofluorescence staining and RT-PCR were used to detect the expressions of myogenic differentiation markers including Myod1,Myogenin and myosin heavy chain （MyHc）.Results The separated LM-MSCs were in a fibrocyte-like form with long fusiform shape and grew adherently.The expression rates of cell surface markers LM-MSCs were CD44 （100.0％）,CD105 （90.4％）,CD90 （99.9％）,CD29 （93.0％）,CD34 （0.4％） and CD45 （1.3％） respectively.A number of beaded lipid drops and mineral deposition were observed after 14 days of adipogenesis differentiation and 21 days of osteogenesis differentiation.Myod1,Myogenin and MyHc genes appeared after 1 week and 3 weeks of myogenesis differentiation respectively.Conclusions The LM-MSCs have the properties of mesenchymal stem cells and could be differentiated into myoblasts,providing with the possibility to repair the damaged vocal cords with LM-MSCs through tissue engineering techniques.