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目的观察骨髓间充质干细胞(BMSCs)移植脑缺血大鼠神经细胞及神经营养因子表达的变化以及从大黄苷元对其变化的影响方面探讨二者联合的作用机制。方法体外全骨髓贴壁筛选培养法扩增BMSCs;线栓法制备局灶性脑缺血动物模型(MCAO)模型;术后24h将BMSCs由同侧颈内动脉移植脑内,大黄苷元灌胃用药;免疫组织化学法测定BMSCs移植后7d(早期)、14d(中期)和28d(后期)神经细胞特异性标志兔抗大鼠神经元特异性烯醇化酶抗体(NSE)和兔抗大鼠胶质纤维酸性蛋白抗体(GFAP)及神经营养因子兔抗大鼠神经生长因子抗体(NGF)和兔抗大鼠胶质源性神经营养因子抗体(GDNF)表达变化。结果假手术组大鼠NSE和GFAP表达显著,NGF和GDNF呈弱阳性。模型组7d的GFAP、NGF和GDNF表达增强,14d和28d的GFAP、NGF表达递减,14d的GDNF表达减弱、28d增强。大黄苷元组7d的NSE较模型组表达增强,GFAP减弱,28d的NGF表达增强,7d和14d的GDNF减弱。移植组28d的GFAP、NGF和GDNF表达均较模型组增强。联合组各时间点的NSE表达增强;7d的GFAP表达减弱,14d增强;联合组14d和28d的NGF表达均较大黄苷元组和移植组增强。结论脑缺血再灌注损伤后随时间延长神经元细胞呈递减反应,神经胶质细胞反应先增强再减弱;神经营养因子早期呈反应性增强,此后NGF递减,GDNF出现先减弱再增强的特点。BMSCs移植脑内中后期增强神经营养因子及神经细胞反应的作用明显;大黄苷元可使BMSCs移植后保护神经细胞的时间提前,并使其作用增强,其作用机制可能与上调早期NGF和GDNF及增强中后期NGF的表达有关。
Objective To observe the changes of neural cells and neurotrophic factors expression in rat brain after ischemia-reperfusion of bone marrow mesenchymal stem cells (BMSCs) and to explore the mechanism of action of the combination of them. Methods BMSCs were expanded by in vitro whole bone marrow adherence screening culture method; focal cerebral ischemia animal model (MCAO) model was prepared by suture; 24 hours after transplantation, BMSCs were transplanted into the brain from the same internal carotid artery, and rhubarb aglycone was intragastrically administered. Medication; Immunohistochemical assay of BMSCs 7 days (early), 14 days (middle) and 28 days (later) neuron-specific markers rabbit anti-rat neuron-specific enolase antibody (NSE) and rabbit anti-rat glue Changes of expression of fibrillary acidic protein antibody (GFAP) and neurotrophic factor in rabbits against rat nerve growth factor antibody (NGF) and rabbit anti-rat GDNF. Results The expression of NSE and GFAP in the sham-operated group was significant, and NGF and GDNF were weakly positive. The expression of GFAP, NGF, and GDNF increased in model group 7d, decreased in 14d and 28d, decreased in GDNF 14d, and increased 28d. NSE of rhubarb aglycone group 7d was stronger than that of the model group, GFAP was weakened, NGF expression was enhanced at 28 days, and GDNF was decreased at 7 days and 14 days. The expression of GFAP, NGF and GDNF in the transplanted group was higher than that of the model group on the 28th day. In the combined group, the expression of NSE was increased at each time point; the expression of GFAP was weakened on the 7th day and increased on the 14th day; the NGF expression on the 14th day and the 28th day in the combined group was higher in the xanthene group and the transplantation group. Conclusion The neuronal cells showed diminishing responses with prolonged time after cerebral ischemia-reperfusion injury. The glial response firstly increased and then weakened. The neurotrophic factor showed an increased reactivity in the early stage, after which NGF decreased, and GDNF first weakened and then enhanced. The effect of BMSCs in enhancing the neurotransmitters and neurotransmitters in the mid and late stage of brain transplantation is significant. Rhubarb aglycone can advance the time of protecting neurons after BMSCs transplantation and enhance its effect. The mechanism may be related to the up-regulation of NGF and GDNF in early stage. Enhancing the expression of NGF in the later period.