摘要
目的 探索成年大鼠成肌细胞的原代培养方法以及逆转录病毒对其的基因转染。方法 取成年SD大鼠的胸大肌 ,利用组织块法进行培养 ,并对培养细胞进行形态学研究和免疫细胞化学鉴定。以逆转录病毒为载体 ,将绿色荧光蛋白基因导入成年大鼠成肌细胞 ,利用倒置荧光显微镜及流式细胞仪对转染细胞进行观察。结果 采用组织块培养法 ,2周后成肌细胞的密度可达 10 7/ml,免疫细胞化学分析显示 90 %以上的细胞呈骨骼肌特异的myogenin抗体染色阳性。荧光显微镜和流式细胞仪检测发现eGFP基因可在成年大鼠成肌细胞中有效地表达 ,其转染效率约为 12 82 %。结论 利用组织块法成功培养成年大鼠原代成肌细胞 ,该方法实用性强 ,所得成肌细胞纯度高 ;逆转录病毒可介导成肌细胞的基因转导 ,为深入研究心肌梗死瘢痕中转基因成肌细胞自体移植奠定了基础。
Objective To explore a experimental method for primary culture of aduli rat skeletal myoblasts and to investigate the gene transfection mediated by retrovirus in myoblasts. Methods Primary skeletal myoblasts were isolated from Sprague-Dawley rats and cultured by tissue culture method.All the cells were numbered and observed by light microscopy combined with immunocytochemical identification.The myoblasts were also transfected by retrovirus carried eGFP gene. The fluorescence microscope and flow cytometer were used to identify the transfected cells.Results After 2 weeks, the number of skeletal myoblasts reached 10 7 per millilitre. Immunocytochemical analysis showed that over 90% cells were stained positive for myogenin, a factor specific to skeletal myocytes. The fluorescence microscope and flow cytometer confirmed the expression of eGFP gene in skeletal myoblasts, and the effieiency of transfection was about 12.82%.Conclusion Tissue culiure method conveniently permits the purification and proliferatiom of myoblast which can be successfully transfected mediated by retrovirus.These experiments set the stage for future studies to assess the ahility of gene transferred skeletal myoblasts to repair myocardial infarct by auto-transplantation.
出处
《临床军医杂志》
CAS
2004年第1期1-4,共4页
Clinical Journal of Medical Officers
基金
国家自然科学基金项目资助 ( 3 0 0 70 75 0
3 960 0 0 63 )
国家重点基础研究发展规划 973项目资助 (G2 0 0 0 0 5 690 5 )
