Mitochondrial organelle transplantation (MOT) is an innovative strategy for the treatment of mitochondrial dysfunction such as cardiac ischemic reperfusion injuries, traumatic brain and spinal cord injuries, cerebral ...Mitochondrial organelle transplantation (MOT) is an innovative strategy for the treatment of mitochondrial dysfunction such as cardiac ischemic reperfusion injuries, traumatic brain and spinal cord injuries, cerebral stroke, and neurodegenerative diseases. The earlier MOT results in better efficacy in animal models of urgent diseases such as ischemic stroke, and traumatic brain and spinal cord injuries. There is no long-term method to preserve mitochondria. Routine MOT procedure from cell growth to mitochondrial injection often takes serval weeks and is not satisfactory for urgent use cases. Hypothesis: Cryopreserved cells might be mitochondrial donors for MOT. Methods: We isolated mitochondria from cryopreserved human fibroblasts and mesenchymal stem cells (MSCs) in cell banks and compared the mitochondrial viability and transplantation with the mitochondria from fresh cells. Key findings: We found that mitochondria from fresh and cryopreserved cells are comparable in mitochondrial viability and transplantation. We also obtained data showing that mitochondria of fibroblasts and MSCs had similar membrane potential and transfer ability, but MSC’s mitochondria had higher ATP content than fibroblast’s mitochondria. In addition, oxygen consumption rates (OCRs) were higher in MSC’s mitochondria compared to fibroblast’s mitochondria and did not change between fresh and frozen cells. Conclusion: Cryopreserved fibroblasts and MSCs are alternative mitochondrial donors for MOT to fresh cells. MSCs could provide higher ATP-produced mitochondria than fibroblasts.展开更多
目的观察右归饮联合MSCs介入治疗早期股骨头缺血性坏死(avascular necrosis of the femoral head,ANFH)的疗效及促进再血管化、再骨化的作用。方法取健康雄性成年毕格犬24只,体重(10.0±0.5)kg,随机分为4组:A组(模型组)、B组(右归饮...目的观察右归饮联合MSCs介入治疗早期股骨头缺血性坏死(avascular necrosis of the femoral head,ANFH)的疗效及促进再血管化、再骨化的作用。方法取健康雄性成年毕格犬24只,体重(10.0±0.5)kg,随机分为4组:A组(模型组)、B组(右归饮组)、C组(MSCs介入组)、D组(MSCs介入加右归饮组),每组6只。通过液氮冷冻法建立早期ANFH模型。分离、培养并以BrdU标记MSCs。造模3周后,C、D组动脉灌注标记后的浓度为(0.5～1.0)×106个/mL的MSCs1mL,B、D组每天予右归饮100mL灌胃,A、C组以100mL蒸馏水灌胃。连续治疗4周及8周后行股骨头大体观察,DSA和MRI观察股骨头供血血管的数量及直径的变化情况,行组织学及免疫组织化学染色观察VEGF、BrdU阳性表达情况,实时荧光定量RT-PCR检测VEGF mRNA表达。结果治疗后4、8周,A组股骨头外形扁平,呈蘑菇状;B、C、D组股骨头外形基本正常。DSA观察示C、D组治疗后供血血管分支增粗、增多,原阻塞血管再通。治疗后4、8周,C、D组间血管数和血管直径比较差异均有统计学意义(P<0.05);两组血管直径与治疗前比较差异有统计学意义(P<0.05),D组血管数与治疗前比较差异有统计学意义(P<0.05)。MRI检查示B、C、D组较A组明显改善,D组尤为明显,T1W略低信号,T2W略低信号,STIR略高信号。组织病理学及免疫组织化学染色示:B、C、D组较A组结构有明显改善,D组较其他各组的VEGF阳性表达率均明显增高(P<0.05),D组较C组的BrdU阳性率、阳性成骨细胞数、阳性血管数表达均明显增高(P<0.05)。实时荧光定量RT-PCR检测示:D组较其他各组VEGFmRNA表达量增多(P<0.05),B、C、D组的VEGF表达均高于A组(P<0.05)。结论右归饮联合MSCs介入治疗早期ANFH疗效显著,可改善坏死股骨头的血供、促进修复、预防塌陷。展开更多
目的观察缺氧预处理骨髓间充质干细胞(MSCs)移植对脑缺血再灌注损伤大鼠SDF-1/CXCR4 m RNA和蛋白表达的影响及清热化瘀方的干预作用。方法采用线栓法制备局灶性脑缺血大鼠(MCAO)模型,将216只SD大鼠随机分为6组:假手术组(SO)、模型组(MC...目的观察缺氧预处理骨髓间充质干细胞(MSCs)移植对脑缺血再灌注损伤大鼠SDF-1/CXCR4 m RNA和蛋白表达的影响及清热化瘀方的干预作用。方法采用线栓法制备局灶性脑缺血大鼠(MCAO)模型,将216只SD大鼠随机分为6组:假手术组(SO)、模型组(MCAO)、MSCs移植对照组(N-MSCs)、经HP处理后的MSCs移植组(HP-MSCs)、MSCs移植联合清热化瘀方组(MSCs+QRHY)、HP-MSCs移植联合清热化瘀方组(HP+QRHY)。每组大鼠36只,每组根据取材时间点7,14,28 d又可分为每组3个亚组,每个亚组12只大鼠。采用q RT-PCR和Western blot观察3个时间点SDF-1/CXCR4 m RNA及其蛋白的表达变化,并以TUNEL法检测神经细胞凋亡。结果各组缺血半暗带SDF-1/CXCR4 m RNA及其蛋白的表达均于7d达到高峰,14,28 d表达逐渐下降。其中7,14 d同一时间点组间比较,HP-MSCs组、MSCs+QRHY组及HP+QRHY组二者的表达均明显优于N-MSCs组(P<0.01,P<0.05),而以HP+QRHY组增高最为明显(P<0.05,P<0.01),28 d后,各移植组的表达趋势趋同,但仍高于模型组(P<0.05)。结论缺氧预处理MSCs移植能够显著提高脑缺血再灌注损伤大鼠SDF-1/CXCR4的表达,清热化瘀方能够进一步上调SDF/1CXCR4的表达,减少细胞凋亡。展开更多
文摘Mitochondrial organelle transplantation (MOT) is an innovative strategy for the treatment of mitochondrial dysfunction such as cardiac ischemic reperfusion injuries, traumatic brain and spinal cord injuries, cerebral stroke, and neurodegenerative diseases. The earlier MOT results in better efficacy in animal models of urgent diseases such as ischemic stroke, and traumatic brain and spinal cord injuries. There is no long-term method to preserve mitochondria. Routine MOT procedure from cell growth to mitochondrial injection often takes serval weeks and is not satisfactory for urgent use cases. Hypothesis: Cryopreserved cells might be mitochondrial donors for MOT. Methods: We isolated mitochondria from cryopreserved human fibroblasts and mesenchymal stem cells (MSCs) in cell banks and compared the mitochondrial viability and transplantation with the mitochondria from fresh cells. Key findings: We found that mitochondria from fresh and cryopreserved cells are comparable in mitochondrial viability and transplantation. We also obtained data showing that mitochondria of fibroblasts and MSCs had similar membrane potential and transfer ability, but MSC’s mitochondria had higher ATP content than fibroblast’s mitochondria. In addition, oxygen consumption rates (OCRs) were higher in MSC’s mitochondria compared to fibroblast’s mitochondria and did not change between fresh and frozen cells. Conclusion: Cryopreserved fibroblasts and MSCs are alternative mitochondrial donors for MOT to fresh cells. MSCs could provide higher ATP-produced mitochondria than fibroblasts.
文摘目的观察右归饮联合MSCs介入治疗早期股骨头缺血性坏死(avascular necrosis of the femoral head,ANFH)的疗效及促进再血管化、再骨化的作用。方法取健康雄性成年毕格犬24只,体重(10.0±0.5)kg,随机分为4组:A组(模型组)、B组(右归饮组)、C组(MSCs介入组)、D组(MSCs介入加右归饮组),每组6只。通过液氮冷冻法建立早期ANFH模型。分离、培养并以BrdU标记MSCs。造模3周后,C、D组动脉灌注标记后的浓度为(0.5～1.0)×106个/mL的MSCs1mL,B、D组每天予右归饮100mL灌胃,A、C组以100mL蒸馏水灌胃。连续治疗4周及8周后行股骨头大体观察,DSA和MRI观察股骨头供血血管的数量及直径的变化情况,行组织学及免疫组织化学染色观察VEGF、BrdU阳性表达情况,实时荧光定量RT-PCR检测VEGF mRNA表达。结果治疗后4、8周,A组股骨头外形扁平,呈蘑菇状;B、C、D组股骨头外形基本正常。DSA观察示C、D组治疗后供血血管分支增粗、增多,原阻塞血管再通。治疗后4、8周,C、D组间血管数和血管直径比较差异均有统计学意义(P<0.05);两组血管直径与治疗前比较差异有统计学意义(P<0.05),D组血管数与治疗前比较差异有统计学意义(P<0.05)。MRI检查示B、C、D组较A组明显改善,D组尤为明显,T1W略低信号,T2W略低信号,STIR略高信号。组织病理学及免疫组织化学染色示:B、C、D组较A组结构有明显改善,D组较其他各组的VEGF阳性表达率均明显增高(P<0.05),D组较C组的BrdU阳性率、阳性成骨细胞数、阳性血管数表达均明显增高(P<0.05)。实时荧光定量RT-PCR检测示:D组较其他各组VEGFmRNA表达量增多(P<0.05),B、C、D组的VEGF表达均高于A组(P<0.05)。结论右归饮联合MSCs介入治疗早期ANFH疗效显著,可改善坏死股骨头的血供、促进修复、预防塌陷。
文摘目的观察缺氧预处理骨髓间充质干细胞(MSCs)移植对脑缺血再灌注损伤大鼠SDF-1/CXCR4 m RNA和蛋白表达的影响及清热化瘀方的干预作用。方法采用线栓法制备局灶性脑缺血大鼠(MCAO)模型,将216只SD大鼠随机分为6组:假手术组(SO)、模型组(MCAO)、MSCs移植对照组(N-MSCs)、经HP处理后的MSCs移植组(HP-MSCs)、MSCs移植联合清热化瘀方组(MSCs+QRHY)、HP-MSCs移植联合清热化瘀方组(HP+QRHY)。每组大鼠36只,每组根据取材时间点7,14,28 d又可分为每组3个亚组,每个亚组12只大鼠。采用q RT-PCR和Western blot观察3个时间点SDF-1/CXCR4 m RNA及其蛋白的表达变化,并以TUNEL法检测神经细胞凋亡。结果各组缺血半暗带SDF-1/CXCR4 m RNA及其蛋白的表达均于7d达到高峰,14,28 d表达逐渐下降。其中7,14 d同一时间点组间比较,HP-MSCs组、MSCs+QRHY组及HP+QRHY组二者的表达均明显优于N-MSCs组(P<0.01,P<0.05),而以HP+QRHY组增高最为明显(P<0.05,P<0.01),28 d后,各移植组的表达趋势趋同,但仍高于模型组(P<0.05)。结论缺氧预处理MSCs移植能够显著提高脑缺血再灌注损伤大鼠SDF-1/CXCR4的表达,清热化瘀方能够进一步上调SDF/1CXCR4的表达,减少细胞凋亡。
