Objective: To investigate the effect of the L10P mutation on the cellular mitochondrial disfunction.
Methods: Spectrophotometer, flow cytometry and electron microscope was utilized to examine cell viability, reactive oxygen species (ROS), mitochondrial transmembrane potential, complex I activity and mitochondrial morphous of the HEK293 monoclone cell lines, in which wild-type and L10P mutant DJ-1 protein are stably expressed.
Results: Compared with the cell lines expressing empty vector, we found the ROS levels were elevated, the cell viability, mitochondrial transmembrane potential, complex I activity were reduced in the cells expressing L10P mutant DJ-1 protein (P<0.05). We also found mitochondria in these cells were swelling and some mitochondria were vacuolar degeneration. These phenomena were more obvious when rotenone was used. But in the cells expressing wild-type DJ-1, ROS levels were lower, the cell viability, mitochondrial transmembrane potential, and complex I activity were higher than other cell lines (P<0.05), especially under the induction of rotenone. These results suggested that L10P mutant DJ-1 protein probably lost the ability of anti-oxidative stress and affect the normal function of mitochondria.
Conclusion: The L10P DJ-1 mutation results in a toxic protein, which lacks the protective function of wild-type protein on mitochondria due to the decrease in the ability of anti-oxidative stress.
目的：探讨DJ-1基因L10P突变对细胞线粒体功能的影响。方法：应用流式细胞仪、分光光度计、电镜等技术对稳定表达空载体、野生型DJ-1蛋白、L10P突变型DJ-1蛋白的HEK293单克隆细胞株的生长活力、活性氧、膜电位、线粒体复合体酶I活性及线粒体形态进行分析。结果：与空载体组相比较，L10P突变型DJ-1组细胞内活性氧增高，细胞活力、膜电位、线粒体复合体I活性降低，线粒体含量减少(P<0.05)，出现线粒体肿胀，甚至线粒体空泡变性，特别是在鱼藤酮的诱导下更明显；野生型DJ-1组细胞内活性氧均较低，细胞活力、膜电位、线粒体复合体I活性均较高(P<0.05)，特别是在鱼藤酮的诱导下更明显。结论：L10P突变后使野生型DJ-1蛋白的抗氧化应激能力丧失，并对线粒体的正常功能存在影响。.