胚胎面突外胚间充质细胞的培养鉴定及向平滑肌细胞的自分化研究

IDENTIFICATION OF ECTOMESENCHYMAL STEM CELLS OF HUMAN FETAL FACIAL PROCESSES AND SPONTANEOUS DIFFERENTIATION TO SMOOTH MUSCLE CELLS

目的 探讨胚胎面突外胚间充质细胞在体外的生长特性、表型特征及在无分化抑制剂 -白血病抑制因子 (L IF)的 DMEM/ F12培养基中向平滑肌细胞自分化的过程。 方法 取孕 5 0天人胚面突 ,用消化法培养获得外胚间充质细胞 ,倒置显微镜下观察细胞形态和生长情况 ,免疫组织化学鉴定细胞分化表型。用无 L IF的 DMEM/ F12培养基培养 ,2天后进行抗α-平滑肌肌动蛋白 (α- SMA)单克隆抗体免疫组织化学检测 ,通过 RT- PCR在 m RNA水平检测α- SMA的表达 ,透射电镜观察细胞超微结构。 结果 培养的细胞呈单层生长 ,成纤维状 ,长梭形 ,有 2～ 4个突起 ,抗人自然杀伤细胞标志、波形丝蛋白、S- 10 0蛋白、神经元特异性烯醇化酶、肌红蛋白及 因子染色阳性 ,抗神经胶质纤维酸性蛋白、神经纤维细丝蛋白、α- SMA、角蛋白染色阴性。去除 L IF2天后 ,抗 α- SMA免疫组织化学检测呈阳性 ,RT- PCR显示细胞在 RNA水平表达平滑肌细胞特异的 α- SMA,透射电镜可见超微结构与平滑肌细胞相类似。 结论 所获得的外胚间充质细胞具有干细胞特性。在无分化抑制剂存在时 ,面突外胚间充质细胞呈明显向平滑肌细胞的自然分化。

Objective To investigate the characteristic and phenotype of ectomesenchymal stem cells of human fetal facial processes and the procedure of spontaneous differentiation to smooth muscle cells. Methods The primary ectomesenchymal cells of E 50 human fetal facial processes were isolated by 2.5 g/L trypsin and cultured with DMEM/F 12 with 10 -6 U/L leukemia inhibitor factor(LIF). The morphology and growth rate were observed by inverted microscop. After being withdrawn LIF, the characteristic of cells were identified by immunohistochemistry and RT PCR. Ultrastructure was observed by transmission electron microscope. Results The cultured cells displayed monolayer growth and were fibroblast like with 2 4 processes. The cells were stainely positived for anti human natural killer cell marker 1, Vimentin, S 100, neuron specific enolase, myoglobin and Ⅷ factor, but negatively for glial fibrillary acidic protein, neural fiblament, α SMA and cytokeratin in immunohistochemistry. Two days after being withdrawn the LIF, cells expressed α SMA in protein and mRNA levels. The cells were rich in muscular filament like structure and dense bodies under transmission electron microscope. Conclusion Cultured cells are undifferentiated ectomesenchymal stem cells. The cells have the potential for differentiating spontaneously to smooth muscle cell.