探索骨髓基质细胞（MSCs）中线粒体转移在脑缺血后的损伤保护作用。

采用小鼠骨髓MSCs分离与原代培养方法，培养MSCs并通过流式细胞仪进行鉴定；取出生后第0天（P0）SD幼鼠的皮层，进行原代神经元培养，并进行氧糖剥夺（OGD）处理，将含有线粒体的MSCs培养基（MCM）组和不含有线粒体的MSCs培养基（mdMCM）组与OGD神经元进行共培养，另以未经过OGD处理的神经元（Neuron组）和经过OGD处理的神经元（OGD组）作为对照；通过MitoTracker追踪线粒体，分析线粒体从MSCs向OGD神经元的转移情况；通过检测试剂盒对神经元内ATP含量和神经元活性进行分析；通过对线粒体膜电势检测，分析线粒体的功能；采用Western Blot分析线粒体Miro1蛋白的表达水平；通过MCAO造模和计算梗死体积，分析MSCs移植对脑缺血的保护作用。采用方差分析和t检验进行统计学分析。

原代培养的骨髓MSCs纯度达到99﹪以上。取原代培养MSCs的培养基分别去除线粒体（mdMCM）与不去除线粒体（MCM），与OGD神经元共培养，在MCM组，观察到神经元中存在MSCs来源的线粒体；经过OGD处理的神经元，其胞内ATP水平降低至0.634±0.023，给予MCM处理后，神经元胞内ATP水平上升至1.623±0.039，当给予mdMCM处理后，神经元胞内ATP水平降低至0.645±0.011，ATP比率变化的差异具有统计学意义（F?= 3413.62，P?< 0.01）；经过OGD处理，神经元活性降低至（73.7±1.12）﹪；给予MCM处理后，神经元活升高到（83.3±1.57）﹪，当给予mdMCM处理后，神经元活性降低至（72.9±1.25）﹪，与MCM组相比差异具有统计学意义（F?= 654.280，P?< 0.01）。在未经过处理的对照组中，线粒体膜电势丢失1.7﹪；经过OGD处理后，膜电势丢失70.3﹪；添加MCM后的OGD神经元，线粒体膜电势丢失44.7﹪，与OGD组相比，差异具有统计学意义（P?= 0.036）；而添加mdMCM的OGD处理神经元，线粒体膜电势丢失67.7﹪，与OGD+MCM组相比，差异具有统计学意义（P?= 0.041）。给予CCCP处理后的阳性对照神经元，膜电势丢失为99.3﹪。在Miro1表达干预中，空白对照组神经元胞内的ATP平均水平记为1，神经元活性为100﹪，计算其余各组相对空白对照组的ATP水平和神经元活性。在Miro1高表达组，胞内ATP水平为2.304，与对照质粒组（ratio = 1.611）相比，差异具有统计学意义（P?= 0.034）；神经元活性检测中，Miro1高表达组相比对照质粒组（90.4﹪vs 81.7﹪），差异具有统计学意义（P?= 0.040）。在MCAO手术后，小鼠的脑梗死体积达到38.4﹪，而给予MSCs后的小鼠，梗死面积降低到14.4﹪，差异具有统计学意义（P?= 0.004）。

MSCs来源的线粒体可以向损伤神经元转移，提升神经元胞内ATP水平和神经元活性，降低缺血损伤中小鼠的脑梗死体积。线粒体Miro1蛋白参与了线粒体向神经元转移保护过程。

To explore MSCs mitochondria transfer mediated protection after stroke.

Isolation and primary culture of bone MSCs of mice. FACS was used to identify the purity of primary MSCs. Primary neuron cultures were prepared from the cerebral cortex of postnatal day 0 (P0) SD rats. OGD (oxygen-glucose deprivation) was performed on the primary neurons. OGD neurons were grouped into MCM group (MSCs conditioned mitochondria, MCM) , mdMCM group (depleted extracellular mitochondria in MCM, mdMCM) , OGD group and normal neurons which labeled as Neuron group. MitoTracker was used to track the mitochondria transfer from MSCs to OGD neurons. Detection kits was used to analyze the ATP level and viability of neurons. Membrane potential of mitochondria was analyzed by detection kit to measure the function of mitochondria. The expression of Miro1 protein was performed by Western Blot. The protection of MSCs transplant was performed by MCAO and MSCs injection. Statistical analysis was performed using analysis of variance and t-tests.

The purity of primary cultured MSCs was higher than 99﹪. Mitochondria transfer from MSCs to OGD neurons was observed. The transfer of mitochondria protected OGD neurons by increasing the ATP level, and the mdMCM group ATP level were significant lower than MCM group. (0.634±0.023 in OGD group, 1.623±0.039 in MCM group and 0.645±0.011 in mdMCM group, F = 3413.62, P < 0.01) ; And the viability of neurons in MCM group higher than in OGD group, and the viability of neurons in mdMCM group were significant lower than MCM group[ (73.7±1.12) ﹪ in OGD group, (83.3±1.57) ﹪ in MCM group and (72.9±1.25) ﹪ in mdMCM group, F = 654.280, P < 0.01]. And the membrane potential loss of mitochondria in OGD and mdMCM groups were significantly higher than in MCM group. (70.3﹪ in OGD group, 44.7﹪ in MCM group and 67.7﹪ in mdMCM group, P < 0.05) . After intervention of Miro1, the ATP level increased in Miro1-overexpression group (2.304 vs 1.611 in control group, P?= 0.034) ; the neuronal viability also increased in Miro1-overexpression group (90.4﹪ vs 81.7﹪ in control group, P?= 0.040) . After MCAO, the infarct volume of mice reached 38.4﹪, while the MSCs-injected mice decreased to 14.4﹪ (P?= 0.004) .

MSCs transplantation decreased the infarct volume of MCAO mice and the protection may be mediated by Miro1 protein.