大鼠心肌成纤维细胞体外培养的生物学特性及转基因研究

Biological characteristics and transgenic study of rat cardiac fibroblasts culture in vitro

目的观察大鼠心肌成纤维细胞（CFs）体外培养的生长增殖情况及肌肉转录调节因子（MyoD）基因转染对其生物学特性的影响。方法采用改良的差速贴壁法培养大鼠CFs，利用携带MyoD cDNA的真核表达载体pLenti6／V5-DEST-MyoD转导培养的大鼠CFs，用稻瘟菌素筛选2周，挑选转基因单克隆细胞，传代培养，进行细胞形态观察、生长曲线以及细胞免疫化学鉴定。结果成功建立高纯度大鼠CFs的细胞培养模型，且具有良好的细胞形态、正常的生长增殖等生物学功能。转染细胞的MyoD cDNA基因表达阳性，其下游抗α-肌动蛋白（α-actin）呈阳性表达，胞质呈棕黄色，并且含有与细胞纵轴平行排列的肌丝，细胞核用苏木素复染后呈淡蓝色。在2．0×10^5／mL细胞密度的CFs接受转MyoD基因后，抗α-actin阳性率为（27．04±9．41）％。结论真核表达载体成功介导MyoD cDNA转导入体外培养的大鼠CFs，诱导其转化为具有潜在功能的类骨骼肌细胞，CFs可成为心血管疾病基因治疗的靶细胞。

Objective To observe the biological characteristics of rat cardiac fibroblasts （CFs） cultured in vitro, and evaluate the possibility of converting cultured CFs to skeletal muscle cells by transfection with myogenic determination gene （MyoD eDNA）. Methods CFs of Sprague-Dawley rat were isolated and cultured with an improved technique of differential anchoring velocity; MyoD eDNA was transduced into CFs using the eukaryotic expression vector pLenti6/VS-DEST-MyoD, and the cells were incubated in medium containing Blasticidin for screening for 2 weeks. Resistant colonies were picked and suhcuhured until use. The morphologic characters of those cells were examined by inverted microscope and growth curve, and immunocytochemical staining was used to evaluate cardiac fihrohlasts. Results The model of rat CFs culture was successfully established with high purity, which had satisfactory morphologic characters and proliferation activity in vitro. CFs clones transduced with the eukaryotic expression vector pLenti6/V5-DEST-MyoD were seen about 2 weeks after transduction. The biological characteristics were significantly different between the resistant colonies and the un-transduced cells. Immunocytochemical staining revealed expression of downstream myogenic anti-α-actin （muscle- specific protein） in the transfected cells. The positive rate of α-actin was （27.04±9.41）%. Conclusion MyoD eDNA can be successfully transduced into CFs using eukaryotic expression vector, which can induce CFs differentiate into＂skeletal muscle like＂cells with potential function, indicating that CFs can serve as ideal target cells for genetic therapy. （Shanghai Med J, 2008, 31：314-316）