原代人胚成肌细胞体外培养增殖及分化特性

目的 分离培养人胚胎成肌细胞，观察原代细胞体外增殖与分化特性。方法 选择健康妇女损赠的胎儿骨骼肌标本，参照Blau等的胰蛋白酶与胶原酶混合、多步消化法分离成肌细胞。经美速贴壁法纯化后，在含20％胎牛血清的生长培养基中培养，以生长曲线评价及其增殖情况；含5％胎牛血清的融合培养基中培养，以磷酸肌酸激酶－MM型合成量及成肌细胞融合率评价及其分化能力。通过光镜、透射电镜、免疫细胞化学方法（小鼠抗人肌球蛋白单克隆抗体）鉴定所得细胞。结果 电镜下可见所得细胞含大量游离核糖体，肌球蛋白免疫细胞化学染色强阳性，能合成磷酸肌酸激酶，能融合形成肌小管，证明其为骨骼肌成肌细胞。在生长培养基中原代细胞倍增时间为4．8天，在融合培养基作用下，肌小管融合率及磷酸肌酸激酶合成量明显增加。结论 多步酶消化法与差速贴壁法能获得足量、纯净成肌细胞，所得细胞增殖能力强，能表达骨骼肌特异的收缩蛋白；融合培养基能促进其体外分化。

THE PROLIFERATION AND DIFFERENTIATION OF PRIMARY HUMAN EMBRYONIC SKELETAL MYOBLASTS

Objective To observe the proliferation and differentiation properties of primary human embryonic skeletal myoblasts cultured in vitro . Methods The skeletal muscle samples were obtained from 20 to 25 week abortion fetus, the family history of inherited myopathies of parental generation was negative. With a modified method of Blau, the muscle sample was digested with trypsin and collagenase. The isolated cell suspension was a mixture of myoblasts and fibroblasts, the latter was removed by repeated attachment to culture dishes. The morphological, immunohistochemical observation, the proliferation and differentiation of primary myoblasts were studied. Results The isolated myoblasts were spherical in cell suspension and spindle like after attached to culture dishes. The myosin specialized immunohistochemical staining was strongly positive. A large quantity of skeletal muscle specialized creatine kinase (CK MM) was synthesized in cultured myoblasts. Additionally, while the cell density of myoblasts increased, the monocyte myoblasts would fused to form multinucleated myotube. All those indicated that the cultured cells were myoblasts. Primary myoblasts proliferated quickly, the doubling time, measured in growth curve, was 4.8 days. Conclusion A large number of myoblasts can be available with digestion and repeated attachment method.The cultured cells can be proved as myoblasts by morphological and immunohistochemical detection.The cultured myoblasts have good ability of proliferation and differentiation.