既往研究认为，线粒体病的临床表现取决于mtDNA的突变比例，然而大量体外实验和我们的病例研究发现，与野生型mtDNA拷贝数更相关。我们提出线粒体能量供应的"木桶"假说，即呼吸链水平的高低取决于短板- - 野生型mtDNA的水平。我们从4方面取证：以q-PCR方法定量患者外周血和尿液细胞中突变型和野生型mtDNA的拷贝数，对比临床表型，明确突变比例和野生型拷贝数孰轻孰重；以微流控(microfluid)技术定量检测微量白细胞中呼吸链酶复合体I、III~V的活性，确认生化表型是否与野生型更相关；以无核的血小板与无mtDNA的Rho0细胞融合的实验，将患者突变定量导入，了解野生型mtDNA的对呼吸链功能的恢复能力；最后将CRISPR/Cas转基因系统用于核外遗传物质mtDNA，快速便捷地建立动物模型，进一步研究线粒体病的发生发展机制。这是核外基因治疗的新思路- - 不必"去除"突变，只要"补充"正常"。

A series of clinical abnormalities are caused by mitochondrial DNA (mtDNA) damage, and mtDNA mutation load or heteroplasmic ratio has long been considered to be important in this process. There is substantial evidence that total amount of wild-type mtDNAs may play more critical role than mutation loads do. To obtain a more accurate evaluation on importance of the percentage ratio of mutated to wild-type mtDNA, or merely wild-type mtDNA level, a cask principle on source of energy is proposed. Which is the shortest plate of mitochondrial respiration? It might be the left level of wild-type mtDNAs in patients who harbor mitochondrial cytopathies, even if the double contribution of nuclear and mitochondrial genome. To make sure that the wild-type mtDNA level is one of the determinants, a series of missense mutations, such as A3243G, A8344G, T8993G/C, G11778A, G3460A,T14484C, A1555G and a large fragment deletion called common deletion, either from peripheral leukocytes or cells from urinary sediment, are quantificationally measured. Both mutant and wild-type mtDNA copy numbers would be severe quantitatively correlated with clinical phenotype. We also correlated mtDNA genotype with non-neglectful biochemical phenotype, the mitochondrial respiratory chain enzyme activities in leukocytes from patients. Complex I, III, IV and V will be determined by a so-called CellSAIC microfluid technique developed by Merck-Millipore, or a Seahorse XF 24 analyzer. Platelet-mediated transformation of mtDNA-less human cells(rho0), with the prolonged viability of platelet mitochondria and the simplicity and efficiency of the mitochondria-transfer procedure, will be applied for patients with different mutation loads, and also for several unrelated normal human individuals after quantification of the amount of the wild-type mtDNA in platelets. A certain recovery in respiratory capacity is supposed to be observed among the platelet-derived rho0 cell transformants, especially in wild-type mtDNA-rich cell. Furthermore, we would like to introduce a system called clustered regularly interspaced short palindromic repeats, CRISPR/Cas, on mitochondrial genome to produce knock-out mice with mtDNA defects. If an overall recovery of mitochondrial function were checked out after the wild-type were knocked-in back, a new way of gene therapy for extra-nuclear genome would be find out. Then, the mitochondrial defects could be cured by 'add in', instead of 'move out'.