人源性成肌细胞和肌管细胞中β-TubulinⅢ的表达

Class Ⅲ Beta-Tubulin is expressed in human myoblasts and myotubes

目的 :确定来源于成年人骨骼肌的干细胞在离体条件下能否分化为神经细胞 ,为骨骼肌干细胞应用于移植治疗神经系统损伤和疾病打下基础 .方法 :采用适用于大鼠的神经细胞诱导分化液诱导贴壁的人成肌细胞向神经细胞的分化 ;采用免疫细胞化学染色并结合WesternBlot及RT PCR等技术对不同培养液作用后的人成肌细胞进行 β TubulinⅢ ,NeurofilamentMr6 8× 1 0 3 和GFAP表达的研究 .结果 :用诱导分化液作用后 ,未见小的、伴有突起的放射状形态的神经细胞 ;抗β TubulinⅢ对经神经元 胶质细胞诱导分化液作用后的人成肌细胞、增殖培养液培养后的各代人成肌细胞及仅含 2 0mL/LHS分化液分化形成的肌管细胞染色均为阳性 ;C2C1 2细胞 β TubulinⅢ抗体染色阴性 ;上述所有细胞抗NeurofilamentMr6 8× 1 0 3 和抗GFAP染色均为阴性 .RT PCR和WesternBlot验证上述β TubulinⅢ染色阳性的人成肌细胞和肌管细胞中 β TubulinⅢmRNA和蛋白质分子的表达 .结论 :成年人骨骼肌干细胞不能被有效诱导分化成神经细胞 ;并证实人源性培养的成肌细胞和肌管细胞表达 β TubulinⅢ ,但是不表达Neurofil amentMr6 8× 1 0 3 .两种蛋白神经元标志物在人成肌细胞的表达不相吻合 ,我们对 β TubulinⅢ能否作为人体组织横向分化研究的神经?

AIM: To determine whether pluripotent stem cells isolated from adult human muscle could be induced to differentiate into neural cells in vitro as a prelude to future skeletal muscle stem cells studies in therapy for nervous injury and diseases. METHODS: We employed neural induction medium which had been used in rats to induce human adherent myoblasts to differentiate into neural cells and investigated the expression of β Tubulin Ⅲ, neurofilament and GFAP in human adherent myoblasts cultured by different medium by immunocytochemical, western blotting and RT PCR techniques. RESULTS: After differentiation of human adherent myoblasts by neural induction medium, no cells with a neural cell morphology (ie., small, refractile cell body with dendritic cell extensions) were seen. All remaining myoblasts cultured with neural induction medium, myoblasts with proliferation medium and myotubes with differentiation medium containing 20 mL/L HS were positive for β Tubulin Ⅲ. C2C12 myoblasts were negative for β Tubulin Ⅲ. In contrast, all the above cells were negative for the markers Neurofilament M r 68×10 3 and GFAP. Human myoblasts and myotubes positive for β Tubulin Ⅲ immunoreactivity were verified with RT PCR and western blotting techniques. CONCLUSION: We failed in inducing adult human adherent myoblasts to differentiate into neurons or glial cells. Human myoblasts and myotubes express neuron marker β Tubulin Ⅲ but not neurofilament M r 68×10 3. The expression of β Tubulin Ⅲ is not consistent with that of neurofilament M r 68×10 3 in human myoblasts. Thus, we question the justification of using β Tubulin Ⅲ expression as a specific neuron marker for transdifferentiation studies. human; muscle stem cells; myoblasts and myotubes; β-Tubulin Ⅲ; immunocytochemistry