不同组织匀浆法对心肌组织线粒体提取质量的影响

Effects of different tissue homogenization methods on the quality of mitochondria extracted from myocardial tissue

目的:探讨不同的组织匀浆法对小鼠心肌组织线粒体提取质量的影响。方法:分别用研磨珠匀浆法、乳化分散仪法、玻璃匀浆器法对C57BL/6J小鼠的心肌组织进行匀浆,差速离心法分离线粒体;Western blot检测细胞质蛋白及线粒体蛋白表达水平;透射电镜观察分离后线粒体的形态结构;JC-1法检测线粒体膜电位。结果:3种组织匀浆法均能获得高纯度的线粒体,且提取效率差异无统计学意义(P>0.05)。与研磨珠匀浆法相比,乳化分散仪法和玻璃匀浆器法得到的线粒体膜电位降低,差异均有统计学意义(P<0.05)。研磨珠匀浆法得到的线粒体超微结构完整,而乳化分散仪法与玻璃匀浆器法得到的线粒体部分结构不完整或破碎。结论:研磨珠匀浆法结合差速离心法分离的心肌组织线粒体,其形态结构完整、功能良好。

Objective:To investigate the effect of different tissue homogenization methods on the quality of mitochondria extracted from mouse myocardial tissue.Methods:The myocardial tissue of C57BL/6J mice was homogenized by grinding bead homogenization method,emulsification disperser method,and glass homogenizer method,and mitochondria were separated by differential centrifugation.The protein expressions of cytoplasm and mitochondria were detected by Western blot,the morphological structure of the mitochondria after separation was observed by transmission electron microscope;the mitochondrial membrane potential was measured by the JC-1 method.Results:All the three methods could obtain high-purity mitochondria,and the difference in extraction efficiency was not statistically significant(P>0.05).Compared with the grinding bead homogenization method,the membrane potential of the mitochondria obtained by the emulsification disperser method and the glass homogenizer method decreased,and the difference was statistically significant(P<0.05).The ultrastructure of mitochondria obtained by the grinding bead homogenization method was undamaged,while the ultrastructure of some mitochondria obtained by the emulsification disperser method and the glass homogenizer method was incomplete or broken.Conclusion:The mitochondria of myocardial tissue separated by the grinding bead homogenization combined with the differential centrifugation method have complete ultrastructure and good function.