摘要
目的研究神经源性聚集蛋白(agrin)基因在原代培养成肌细胞中的表达。方法原代培养成年大鼠的成肌细胞,免疫细胞化学方法进行鉴定,将agrin—Y428基因cDNA片段亚克隆入 pCDNA3真核表达载体,重组子经脂质体介导转染成肌细胞,G418筛选,获得具有抗性的克隆,增殖后以逆转录-聚合酶链反应(RT-PCR)和免疫荧光的方法检测基因的转录和蛋白表达,并初步测定其生物学活性。结果原代培养8周后,90％以上仍为成肌细胞,转染神经源性agrin基因后,成肌细胞可以表达相应的mRNA和有功能活性的蛋白。结论原代培养的成肌细胞可以作为agrin 基因转移的有效载体,可用于进一步肌肉功能减退的基因治疗中。
Objective To investigate the expression of agrin gene in the primary culture of rat skeletal myoblasts. Methods Primary culture of rat skeletal myoblasts by tissue culture method was identiffed by using immunocytochemical method. Agrin-Y4Z8 cDNA was subcloned into pCDNA3 eukaryotic expression vector and the recombinant plasmids were transfected into primary culture of rat skeletal myoblasts by the cationic liposome. Stable transfectants were selected using G418 and were preserved for 14 days. The expression of agrin mRNA and protein was detected by RT-PCR and immunofluorescence methods. Results After 8 weeks of cell culture, immunocytochemical analysis revealed that over 90% cells were stained positively for myogenin. The myoblasts transfected with agrin gene expressed mRNA and active protein of neural agrin. Conclusion Primary culture of rat skeletal myoblasts can be used as an effective vector of agrin gene therapy on muscle dysfunction following the free neurovascular muscle transfer.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2005年第12期1562-1564,共3页
Chinese Journal of Experimental Surgery
基金
上海市"百名跨世纪学科带头人计划"(076)上海市科技发展基金资助项目(024119057)
