骨髓间充质干细胞通过抑制多种细胞死亡方式保护体外脑缺血模型的神经元

发布时间：2019-05-16 01:38

【摘要】：研究背景:缺血性卒中是世界上引起人类死亡和导致长期残疾的主要原因之一。然而,目前世界上治疗缺血性卒中特别是促进缺失的神经功能重建的方法十分有限。目前,建立卒中单元和用重组组织型纤溶酶原激活物(recombinant tissue plasminogen activator,r-tPA)进行静脉溶栓治疗目前已被公认可以改善缺血性卒中的预后,但是由于其狭窄的治疗时间窗的限制,使得只有一小部分病人能从中获益。功能康复治疗能使卒中患者缺失的神经功能得到一定程度上的恢复,但是,康复治疗的效果目前仍不是很乐观。因此,研究者们正在努力探索降低缺血性卒中的死亡率和促进神经功能重建的新方法。目前干细胞移植成为治疗卒中的新方法。已有报道骨髓间充质干细胞(mesenchymal stem cells,MSCs)对缺血性卒中的神经保护作用与其抑制神经细胞凋亡有关。但是,骨髓MSCs对其他参与卒中发病的细胞死亡方式,如parthanatos、坏死性凋亡和自噬的作用如何目前尚不清楚。目的:探讨骨髓MSCs通过抑制哪些细胞死亡方式保护体外脑缺血模型的神经元。方法:应用全骨髓细胞培养和差速贴壁法纯化培养大鼠骨髓MSCs。对原代培养的皮层神经元通过氧糖剥夺(oxygen-glucose deprivation,OGD)和N-甲基-D-天门冬氨酸(N-methyl-D-aspartate,NMDA)损伤的方法建立体外脑缺血的模型。应用具有0.4μm小孔的聚碳酸酯膜作为隔层的六孔板建立跨室共培养系统,通过骨髓MSCs-脑缺血的神经元共培养的方法建立骨髓MSCs移植治疗缺血性卒中的体外模型。用2-溴化丙陡(propidiumiodide,PI)/Hoechst33342染色的方法测定神经元的死亡率,并对MSCs与各种选择性抑制细胞死亡的化学抑制剂的神经保护作用进行比较。通过免疫荧光染色、蛋白印迹分析和荧光实时定量PCR的方法测定与骨髓MSCs共培养后缺血的神经元的parthanatos死亡方式标志物细胞核凋亡诱导因子(apoptosis-inducing factor,AIF)和多聚二磷酸腺苷核糖(Poly ADP-ribose,PAR)、坏死性凋亡的标志物受体相互作用蛋白1(receptor interacting protein 1,RIP1)和受体相互作用蛋白 3(receptor interacting protein 3,RIP3)、细胞凋亡的标志物cleaved caspase-3以及自噬的标志物Beclin1/轻链3-B(lightchain3-B,LC3B)表达的变化。结果:与骨髓MSCs共培养可以使OGD引起的神经元死亡减少约73%～75%和使NMDA损伤引起的神经元死亡下降约68%,其神经保护作用优于任何一种选择性抑制细胞死亡的化学抑制剂。免疫荧光染色和蛋白印迹分析的结果表明,MSCs共培养通过减少AIF的入核而抑制parthanatos;通过下调RIP1/RIP3的表达而减少坏死性凋亡;通过降低cleaved caspase-3的表达而抑制细胞凋亡,但是对Beclin1的表达没有调节作用。PCR检测也得到了类似的结果。结论:MSCs共培养通过同时抑制parthanatos、坏死性凋亡和细胞凋亡保护缺血缺氧损伤的神经元,而对自噬没有影响。这个研究结果为骨髓MSCs对脑缺血模型的神经保护作用增加了新的解释机制,并且为MSCs移植治疗缺血性卒中将来应用于临床提供了新的理论依据。
[Abstract]:Background: Ischemic stroke is one of the main causes of human death and long-term disability in the world. However, there is a limited number of methods currently in the world for the treatment of ischemic stroke, in particular for promoting the loss of neurological function. At present, a stroke unit and a recombinant tissue type plasminogen activator (r-tPA) are currently recognized to improve the prognosis of ischemic stroke, but due to its narrow treatment time window, So that only a small portion of the patient can benefit from it. Functional rehabilitation therapy can restore the neurological function of stroke patients to a certain extent, but the effect of rehabilitation therapy is still not very optimistic. Therefore, the researchers are working to explore new ways to reduce the death rate in ischemic stroke and to promote neurological recovery. Stem cell transplantation is a new method in the treatment of stroke. It has been reported that the neuroprotective effect of mesenchymal stem cells (MSCs) on ischemic stroke is related to the inhibition of neuronal apoptosis. However, the role of bone marrow MSCs on other types of cell death involved in stroke, such as parthenatos, necrotizing apoptosis and autophagy, is not yet clear. Objective: To study how to protect the neurons of the in vitro cerebral ischemia model by inhibiting which cell death methods. Methods: The bone marrow MSCs were cultured by full-bone marrow cell culture and differential adhesion method. The model of in vitro cerebral ischemia was established by means of oxygen-glucose deprivation (OGD) and N-methyl-D-aspartate (NMDA) injury to the primary cultured cortical neurons. A cross-chamber co-culture system was established by using a polycarbonate film with a small pore of 0.4. m u.m as an interlayer, and an in-vitro model of bone marrow MSCs transplantation for the treatment of ischemic stroke was established by means of co-culture of the bone marrow MSCs-cerebral ischemia. The mortality of neurons was measured by the method of 2-Bromide (PI)/ Hoechst33342, and the neuroprotective effects of MSCs with a variety of chemical inhibitors of selective inhibition of cell death were compared. The apoptosis-inducing factor (AIF) and polydiphosphate (PPAR) were determined by immunofluorescence staining, Western blot analysis and fluorescence real-time quantitative PCR. The expression of the marker-receptor interacting protein 1 (RIP1) and the receptor-interacting protein 3 (RIP3), the marker of apoptosis, and the expression of the self-phagemented marker Bechin1/ light chain 3-B (lightchmain3-B, LC3B) were observed. Results: The co-culture with bone marrow MSCs can reduce the neuronal death caused by OGD by about 73% ~ 75% and the neuronal death caused by NMDA injury by about 68%, and its neuroprotective effect is superior to any chemical inhibitor that can selectively inhibit cell death. The results of immunofluorescent staining and Western blot analysis showed that the co-culture of MSCs inhibited parthenatos by reducing the in-core of AIF, and decreased the apoptosis by down-regulating the expression of RIP1/ RIP3, but did not regulate the expression of Bechin1 by reducing the expression of the clear caspase-3. The PCR assay also results in a similar result. Conclusion: The co-culture of MSCs has no effect on autophagy by inhibiting parthenatos, necrotizing apoptosis and apoptosis. The results of this study provide a new interpretation mechanism for the neuroprotective effect of bone marrow MSCs on the cerebral ischemia model, and provide a new theoretical basis for the application of MSCs in the treatment of ischemic stroke.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R743.3

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