线粒体DNA3243A > G突变糖尿病外周血单个核细胞生物力学变化特征

Biomechanical Characteristics of Peripheral Blood Mononuclear Cells in Mitochondrial Diabetes Caused by mt.3243A > G Mutation

目的探索线粒体DNA3243A>G(mt.3243A>G)突变糖尿病患者和正常对照外周血单个核细胞(PBMC)表面形貌和力学性能差异。方法采集5例mt.3243A>G突变糖尿病患者和5例年龄、性别匹配的正常对照外周血2ml,然后利用聚蔗糖-泛影葡胺(Ficoll-hypaque)密度梯度离心法分离出PBMC。应用原子力显微镜(AFM)对突变患者和正常对照的PBMC进行表面形貌和力学性能测量。结果运用AFM测量和分析发现,在表面形貌方面,mt.3243A>G突变糖尿病患者的PBMC高度(0.73±0.24μm vs 2.49±1.17μm,P=0.011)低于正常对照组;但其表面粗糙度(Ra:161.8±33.2nm vs 66.4±16.3nm,P=0.000;Rq:202.2±40.9nm vs 85.4±17.1nm,P=0.000)高于正常对照组。在力学性能方面,mt.3243A>G突变糖尿病患者PBMC黏附力约比正常对照组高3倍(779.6±190.0p N vs 161.1±83.1p N,P=0.000)。与正常对照组相比,mt.3243A>G突变糖尿病患者PBMC杨氏模量(138.3±77.2k Pa vs 421.4±140.0k Pa,P<0.01)显著增加,病变细胞表面硬度增加。结论本研究利用AFM从单细胞水平上揭示了mt.3243A>G突变糖尿病患者PBMC的表面形貌和力学性能变化,有助于加深对该疾病病理生理机制的理解。

Objective To explore the surface morphological and biomechanical properties differences of peripheral blood mononuclear cells （PBMCs） between groups of patients with mitochondrial diabetes caused by mt.3243A〉G mutation and healthy controls. Methods 2 milliliters blood were obtained from each subject of the mitochondrial diabetes group （ n =5） and the control group （ n =5）. The PBMCs were separated from the blood using the standard Ficoll-Hypaque density-gradient centrifugation method and detected by atomic force microscope （AFM）. Results The morphological analysis revealed that compared with control group, the PBMCs of diabetic patients tended to have a lower cell height （0.73±0.24μm vs 2.49±1.17μm, P =0.011） and a much rougher cell membrane （Ra：161.8±33.2nm vs 66.4±16.3nm, P =0.000;Rq：202.2±40.9nm vs 85.4±17.1nm, P =0.000）. The adhesion force distribution was nearly three times higher in PBMCs of diabetic patients than that of the control group （779.6±190.0pN vs 161.1±83.1pN, P =0.000）. The Young′s modulus of PBMCs was significantly increased in diabetic patients （421.4±140.0kPa vs 138.3±77.2kPa, P 〈0.01）, indicating that diabetic PBMCs were stiffer than control cells. Conclusion Our study demonstrated the surface morphological and biomechanical properties changes in mitochondrial diabetes caused by mt.3243A〉G mutation at PBMCs level, which was beneficial to the better understanding of the pathophysiological mechanisms of mitochondrial diabetes associated with mt.3243A〉G mutation.