大鼠颏舌肌成肌细胞的分离和鉴定

Isolation and identification of rat genioglossus myoblast

目的建立大鼠颏舌肌成肌细胞体外培养方法,观察成肌细胞生物学特性。方法取新生3 d内SD大鼠,机械法分离颏舌肌组织,应用胶原酶、胰蛋白酶两步消化法获得成肌细胞,差速贴壁法纯化细胞,细胞计数法绘制生长曲线,α-sarcomeric actin免疫细胞化学鉴定细胞来源。结果成功获得大鼠颏舌肌成肌细胞,超过90%的细胞α-sarcomeric actin染色阳性,证明其为骨骼肌细胞来源。体外培养的成肌细胞呈现良好的增殖和分化能力,在生长培养基中细胞的倍增时间为4~5d,高密度时细胞相互接触融合形成肌管。结论消化法与差速贴壁法结合能够获得足量、纯净的颏舌肌成肌细胞,所得细胞增殖力强,能表达骨骼肌特异性蛋白。

Objective To establish an in vitro culture model of genioglossus myoblast of rat,and to observe the biological character of myoblasts. Methods Genioglossus muscle was from SD neonatal rat no older than 3 days. Two-step enzymatic digestion using collagenase II and trypsine was applied to obtain genioglossus myoblast. The preplate technique was used to purify myoblast. Cytometry was used to draw the cell growth curve. α-sarcomeric actin immunocytochemistry was adopted to identify the myogenic origin of the myoblast. Results Genioglossus myoblast was successfully obtained,and more than 90% of the cells were α-sarcomeric actin positive. The cells were active in proliferation and differentiation. The doubling time of cell numbers was about 4-5 days. The monocyte myoblasts were fused to myotube when cell density was high. Conclusions Sufficient and purified genioglossus myoblast could be obtained by enzymatic digestion and preplate technique. The cells can be proved as myoblast by morphological and immunocytochemistry detection.