Blebbistatin在成年小鼠心肌细胞原代培养以及GFP基因转导中的作用及其机制

The Effect of Blebbistatin on Primary Adult Mouse Cardiac Myocytes Culture and GFP Gene Transfer

目的成年小鼠心肌细胞原代培养是一个有价值的研究工具,用以解决心脏疾病的细胞机制问题。该文旨在寻求一种高效特异的细胞收缩抑制剂(Blebbistatin,BS)来提高成年小鼠心肌细胞原代培养的细胞成活率并延长细胞培养时间,从而进一步提高转基因实验的效率。方法通过肌细胞收缩抑制剂干预成年小鼠心肌细胞原代培养,运用生理学、药理学、细胞形态学和生物化学等学科的研究手段,从细胞成活率,腺病毒转导GFP基因并表达相应蛋白产物的效率等方面进行比较研究。结果研究显示,25μM的BS较10mM的BDM而言,前者能够更大程度地提高成年小鼠心肌细胞原代培养的细胞成活率,延长细胞培养时间,以及能够稳定地维系培养过程中良好的细胞形态,并且高效地表达腺病毒转导GFP基因的蛋白产物。结论BS是一种特异性的细胞收缩抑制剂,能够提高成年小鼠心肌细胞原代培养的细胞成活率并延长培养时间,从而提高由病毒介导的基因转导效率。

Objective Primary adult mouse cardiac myocytes culture is a valuable system to address questions on cellular mechanisms of cardiac disorders.We tried to figure out an efficient and specific inhibitor, Blebbistatin （BS）, that inhibits myocyte contraction and increases the cell viability and extends culture life of adult mouse cardiac myocytes, furthermore,improves the transgenic efficiency.Methods The comparative studies in cell viability, GFP gene transfer and its correlated protein expression as well were performed in the primary adult mouse cardiac myocytes culture by the means of physiology, pharmacology, cell morphology and biochemistry,etc.Results The results showed that BS of 25 μM compared with BDM of 10 mM, the former was more efficient than the latter in increasing cell viability,extending culture life, stably maintaining the morphology and highly expressing GFP following the transgene as well.Conclusion In summary,BS is a specific contraction inhibitor.that improves cell viability, culture life and the efficiency of adenovirus-mediated gene transfer in cultured adult mouse cardiac myocytes.