阿尔茨海默病胞质杂交细胞的构建与其线粒体的结构功能变化

Construction of Alzheimer＇s disease cybrids and changes of their mitochondrial structure and function

目的：探讨AD胞质杂交细胞的构建方法及其线粒体形态功能变化情况，为后续研究提供细胞模型。方法：利用融合剂将AD患者及健康老人血小板分别与线粒体DNA缺失细胞（ρ^0细胞）进行融合，建立AD和对照细胞质杂交细胞，透射电镜及PCR法鉴定；透射电镜观察胞质杂交细胞线粒体结构；MTT检测细胞活力；荧光探针DCFH—DA检测细胞内活性氧（ROS）水平。结果：ρ^0细胞与血小板融合后重新出现线粒体和mtDNA与同期对照组胞质杂交细胞比较，到培养晚期，AD胞质杂交细胞中异常线粒体显著增多，细胞活力明显降低，ROS水平明显增高。结论：成功构建出AD胞质杂交细胞和对照组胞质杂交细胞，为下一步的研究奠定了细胞模型基础。

Objective： To explore the construction methods of Alzheimer＇s disease （AD） cybrids and the changes of the mitochondrial structure and function. Methods： To create AD and normal （CTL） cybrids for this study, ρ^0 cells were repopulated with mitochondria containing platelet mtDNA from volunteer AD patients or age-matched controls using fusogenic agent. Identification of AD and CTL cybrids was measured by electron microscopy and mtDNA PCR amplification. The mitochondrion structure, cell viability and ROS levels in AD and CTL cybrids were respectively detected by electron microscopy, MTT assay and fluorescent probe DCFH-DA. Results： The mitochondrial repopulation and mtDNA in AD and CTL cybrids was observed by electron microscopy and PCR amplification after ρ^0 cells being fused with donor platelets. Compared with the CTL cybrids, abnormal mitochondria were markedly increased, cell viability was decreased and ROS levels were significantly increased in late passage AD eybrids. Conclusion ： Succeeding in construction of AD cybrids and CTL cybrids may provide a cell model for future in-depth research.