人脐血间充质干细胞移植调节兔局灶性脑缺血再灌注炎性反应和神经保护的研究
发布时间：2018-10-22 13:04
【摘要】：目的 研究人脐血间充质干细胞（human umbilical cord blood mesenchymal stemcells, hUCB-MSCs）移植调节兔局灶性脑缺血再灌注损伤早期炎性反应和神经保护的机制。 方法 采集脐带血80ml-100ml，体外分离hUCB-MSCs并传代培养，取第6代培养的细胞做治疗移植用。健康新西兰大白兔64只，随机分为假手术组、缺血再灌注组，MSC组，生理盐水组，每组16只。采用线栓法建立兔大脑中动脉缺血再灌注模型（middle cerebral artery occlusion, MCAO），MSC组通过右腿的股静脉移植内含3mL5×106个hUCB-MSCs悬液，生理盐水组则采用同样的方式注射等量的生理盐水。早期(缺血30min，，缺血再灌注后2h,4h，6h)检测各组实验动物血清及脑组织中的白细胞介素-1β（interleukinIL-1β）、IL-6、IL-10、肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）和脑组织内炎性细胞，评价hUCB-MSCs移植调节炎性反应的效果。神经元凋亡、神经生长因子（nerve growth factor,NGF）、脑源性神经营养因子（brain derived neurotrophic factor, BDNF）、微管相关蛋白2（microtubule associated protein2，MAP2）、purdy评分的检测用于评估hUCB-MSCs移植对实验动物的神经保护作用。 结果 缺血再灌注组和生理盐水组血清中的IL-1β和IL-6在缺血后升高并在再灌注后2小时达到高峰，IL-10呈下降趋势。MSC组IL-1β和IL-6在早期各时间点较缺血再灌注组和生理盐水组明显降低而IL-10显著升高（P＜0.05）。第3d时脑组织匀浆检测炎性因子也有相似的变化，且MSC组脑组织内炎性细胞浸润情况较缺血再灌注组和生理盐水组明显减轻，浸润的炎性细胞数减少（P＜0.05）。 第3d时，缺血再灌注组和生理盐水组脑组织中有大量凋亡细胞且凋亡指数（apoptosis index, AI）较高，而MSC组AI较缺血再灌注组和生理盐水组明显降低（P＜0.05）。MSC组血清NGF、BDNF含量较缺血再灌注组和生理盐水组升高更为显著（P0.05)而Purdy评分降低（P＜0.05）,缺血皮层区的MAP2含量亦增加（P0.05）。 结论 1、脐血间充质干细胞移植可以调节兔脑缺血早期的炎性反应。 2、脐血间充质干细胞移植能抑制兔脑缺血皮层神经元凋亡。 3、脐血间充质干细胞移植能增加脑梗死兔血清中神经营养因子，促进神经元再生。 4、脐血间充质干细胞移植能促进兔脑缺血神经功能缺损恢复。
[Abstract]:Objective to study the mechanism of early inflammatory response and neuroprotection after transplantation of human umbilical cord blood mesenchymal stem cells (human umbilical cord blood mesenchymal stemcells, hUCB-MSCs) in rabbits with focal cerebral ischemia-reperfusion injury. Methods umbilical cord blood was collected from 80 ml to 100 ml, hUCB-MSCs was isolated and cultured in vitro, and the cells cultured in the 6th passage were used for treatment and transplantation. 64 healthy New Zealand rabbits were randomly divided into three groups: sham operation group, ischemia reperfusion group, MSC group and saline group. The rabbit middle cerebral artery ischemia-reperfusion model was established by thread embolization. The (middle cerebral artery occlusion, MCAO), MSC group received 3mL5 脳 106 hUCB-MSCs suspensions through the femoral vein of the right leg, while the saline group received the same amount of normal saline in the same way. The levels of interleukin-1 尾 (interleukinIL-1 尾), IL-6,IL-10, tumor necrosis factor- 伪 (TNF- 伪) and inflammatory cells in brain tissue were detected at the early stage (30 min after ischemia and 4 h after reperfusion). The effect of hUCB-MSCs transplantation on the regulation of inflammatory response was evaluated. Neuronal apoptosis, nerve growth factor (nerve growth factor,NGF) and brain-derived neurotrophic factor (brain derived neurotrophic factor, BDNF),) microtubule-associated protein 2 (microtubule associated protein2,MAP2), purdy) score were used to evaluate the neuroprotective effect of hUCB-MSCs transplantation on experimental animals. Results the levels of IL-1 尾 and IL-6 in the serum of ischemia reperfusion group and normal saline group increased after ischemia and reached a peak at 2 hours after reperfusion, and IL-10 showed a decreasing trend. IL-1 尾 and IL-6 in MSC group were higher than those in ischemia reperfusion group at different time points. And normal saline group decreased significantly, but IL-10 increased significantly (P < 0. 05). On the 3rd day, the level of inflammatory cytokines in brain homogenate was similar, and the infiltration of inflammatory cells in MSC group was significantly less than that in ischemia reperfusion group and normal saline group (P < 0. 05), and the number of inflammatory cells in brain tissue homogenate was lower than that in ischemia reperfusion group and normal saline group (P < 0. 05). On the 3rd day, there were a large number of apoptotic cells in the brain tissues of ischemia reperfusion group and normal saline group, and the apoptotic index (apoptosis index, AI) was higher. However, AI in MSC group was significantly lower than that in ischemia reperfusion group and normal saline group (P < 0. 05). The serum NGF,BDNF content in MSC group was significantly higher than that in ischemia reperfusion group and normal saline group (P < 0. 05), and the Purdy score was decreased (P < 0. 05). The MAP2 content in ischemic cortex was also increased (P0.05). Conclusion 1. Umbilical cord blood mesenchymal stem cell transplantation can regulate the early inflammatory response of rabbit cerebral ischemia. 2. Umbilical cord blood mesenchymal stem cell transplantation can inhibit the apoptosis of rabbit cerebral ischemic cortical neurons. Transplantation of blood mesenchymal stem cells can increase neurotrophic factors in the serum of cerebral infarction rabbits. 4. Transplantation of mesenchymal stem cells from umbilical cord blood can promote the recovery of neural function defect after cerebral ischemia in rabbits.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743
本文编号：2287238
[Abstract]:Objective to study the mechanism of early inflammatory response and neuroprotection after transplantation of human umbilical cord blood mesenchymal stem cells (human umbilical cord blood mesenchymal stemcells, hUCB-MSCs) in rabbits with focal cerebral ischemia-reperfusion injury. Methods umbilical cord blood was collected from 80 ml to 100 ml, hUCB-MSCs was isolated and cultured in vitro, and the cells cultured in the 6th passage were used for treatment and transplantation. 64 healthy New Zealand rabbits were randomly divided into three groups: sham operation group, ischemia reperfusion group, MSC group and saline group. The rabbit middle cerebral artery ischemia-reperfusion model was established by thread embolization. The (middle cerebral artery occlusion, MCAO), MSC group received 3mL5 脳 106 hUCB-MSCs suspensions through the femoral vein of the right leg, while the saline group received the same amount of normal saline in the same way. The levels of interleukin-1 尾 (interleukinIL-1 尾), IL-6,IL-10, tumor necrosis factor- 伪 (TNF- 伪) and inflammatory cells in brain tissue were detected at the early stage (30 min after ischemia and 4 h after reperfusion). The effect of hUCB-MSCs transplantation on the regulation of inflammatory response was evaluated. Neuronal apoptosis, nerve growth factor (nerve growth factor,NGF) and brain-derived neurotrophic factor (brain derived neurotrophic factor, BDNF),) microtubule-associated protein 2 (microtubule associated protein2,MAP2), purdy) score were used to evaluate the neuroprotective effect of hUCB-MSCs transplantation on experimental animals. Results the levels of IL-1 尾 and IL-6 in the serum of ischemia reperfusion group and normal saline group increased after ischemia and reached a peak at 2 hours after reperfusion, and IL-10 showed a decreasing trend. IL-1 尾 and IL-6 in MSC group were higher than those in ischemia reperfusion group at different time points. And normal saline group decreased significantly, but IL-10 increased significantly (P < 0. 05). On the 3rd day, the level of inflammatory cytokines in brain homogenate was similar, and the infiltration of inflammatory cells in MSC group was significantly less than that in ischemia reperfusion group and normal saline group (P < 0. 05), and the number of inflammatory cells in brain tissue homogenate was lower than that in ischemia reperfusion group and normal saline group (P < 0. 05). On the 3rd day, there were a large number of apoptotic cells in the brain tissues of ischemia reperfusion group and normal saline group, and the apoptotic index (apoptosis index, AI) was higher. However, AI in MSC group was significantly lower than that in ischemia reperfusion group and normal saline group (P < 0. 05). The serum NGF,BDNF content in MSC group was significantly higher than that in ischemia reperfusion group and normal saline group (P < 0. 05), and the Purdy score was decreased (P < 0. 05). The MAP2 content in ischemic cortex was also increased (P0.05). Conclusion 1. Umbilical cord blood mesenchymal stem cell transplantation can regulate the early inflammatory response of rabbit cerebral ischemia. 2. Umbilical cord blood mesenchymal stem cell transplantation can inhibit the apoptosis of rabbit cerebral ischemic cortical neurons. Transplantation of blood mesenchymal stem cells can increase neurotrophic factors in the serum of cerebral infarction rabbits. 4. Transplantation of mesenchymal stem cells from umbilical cord blood can promote the recovery of neural function defect after cerebral ischemia in rabbits.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743
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