肌苷对化学性缺氧损伤的少突胶质细胞保护作用的研究

发布时间：2018-04-30 21:01

本文选题：脊髓损伤 + 少突胶质细胞　；参考：《第四军医大学》2011年博士论文

【摘要】：肌苷是一种在体内广泛存在的小分子物质,它对多个系统、多种细胞都存在较好的保护作用。近年来的有关肌苷对中枢神经系统的保护作用的研究提示,肌苷不仅能够调节免疫细胞的作用、促进神经元轴突广泛的生长,还对损伤后的神经细胞的有较好的保护作用,在体外化学性缺氧环境中肌苷可以保护ROC-1细胞、星形胶质细胞和神经元的活性,在体内肌苷治疗脑缺血损伤大鼠能够减少神经细胞的凋亡和死亡、抑制血小板的聚积、磷酸肌醇的降解和钙离子的形成、减轻脑缺血损伤程度,减小脑梗死的体积、抑制谷氨酸的突触后效应、促进大鼠神经功能恢复。尽管如此,但是ROC-1是一种大鼠少突胶质细胞和C6胶质细胞融合而成的杂交瘤细胞,同时具有这两种细胞的属性,无法肯定肌苷对少突胶质细胞缺氧损伤后有保护作用;另外ROC-1是一种不死的瘤细胞,且没有区分不同的分化阶段,无法真正体现少突胶质细胞对病理损伤的敏感性;最后,虽然肌苷治疗脑缺血模型大鼠能够减少神经细胞的凋亡、促进神经功能的恢复,但肌苷对体内缺血缺氧环境下的少突胶质细胞的保护作用仍然不清楚。因此,调查研究肌苷对缺氧环境下少突胶质细胞的保护作用有着极其重要的意义。本研究分为三部分,第一部分调查研究在化学性缺氧环境中肌苷是否对分化后的少突胶质细胞有保护作用;第二部分调查研究肌苷在化学性缺氧环境中是否对具有增殖能力的少突胶质前体细胞有保护作用;第三部分调查研究肌苷治疗脊髓挫伤模型7天后对大鼠运动功能恢复和临近坏死区的少突胶质细胞存活的影响。第一部分实验通过免疫荧光染色等形态学观察,同时结TUNEL染色、MTT实验和ATP检测实验来评估肌苷对不同分化阶段少突胶质细胞在化学性缺氧损伤环境下细胞存活和活性的影响。结果表明:采用相同浓度的鱼藤酮损伤不同分化阶段的少突胶质细胞24 h所造成的损伤不相同。因而,要达到大致相似的损伤程度所使用的鱼藤酮浓度也就不同,未成熟少突胶质细胞的损伤浓度为5μM鱼藤酮,成熟少突胶质细胞的损伤浓度为20μM鱼藤酮。在未成熟少突胶质细胞和成熟少突胶质细胞大致相似的损伤程度下采用肌苷预处理受损伤的少突胶质细胞,结果肌苷能明显减少未成熟少突胶质细胞的死亡和凋亡,并且肌苷呈剂量依赖性提高受损的未成熟少突胶质细胞的活性,但是肌苷只能减少成熟少突胶质细胞的死亡,不能减少成熟少突胶质细胞的凋亡,并且10 mM肌苷能提高受损成熟少突胶质细胞的活性。肌苷促进分化后少突胶质细胞的存活、提高分化后少突胶质细胞的活性和肌苷减少鱼藤酮介导细胞内产生的过亚硝酸盐、提供ATP有一定的相关性。对未成熟和成熟少突胶质细胞的保护效果的差异可能与这两个阶段少突胶质细胞上的腺苷受体的表达差异有关。第二部分实验通过免疫荧光染色等形态学观察,同时结合MTT实验来评估肌苷对少突胶质前体细胞在化学性缺氧损伤环境下细胞存活和活性的影响。结果表明:20μM鱼藤酮能够明显地诱导OPCs的死亡、细胞活性和细胞总数下降,阻止NG2抗原的表达,但并没有诱导OPCs的凋亡,10 mM肌苷预处理鱼藤酮损伤的OPCs并不能够减少鱼藤酮所诱导的OPCs的死亡和活性下降,却能提高受损细胞上NG2抗原的表达。除此之外,值得注意的是10 mM肌苷能够显著降低正常培养的OPCs(也就是空白对照组的OPCs)的活性。第三部分实验通过行为学和免疫组织化学来观察大鼠腹腔注射肌苷对脊髓挫伤模型大鼠运动功能恢复和临近坏死区的少突胶质细胞存活数目的影响。结果表明:给脊髓挫伤大鼠模型腹腔注射肌苷(75mg/kg,每8 h一次)治疗对大鼠的运动功能和临近坏死区的少突胶质细胞存活数目并没有改善作用。在体外实验中我们确实观察到肌苷能明显促进化学性缺氧损伤的未成熟和成熟少突胶质细胞的存活和活性的提高。对分化后的少突胶质细胞的保护机制和肌苷所提供的ATP、肌苷清除过亚硝酸盐具有一定的相关性。对化学性缺氧损伤的少突胶质前体细胞的存活和活性的提高没有改善作用。同时在用肌苷治疗脊髓挫伤大鼠模型时也没有观察到损伤区周边的少突胶质细胞的数目增加和大鼠运动功能恢复,可能的原因是:脊髓损伤后的给药剂量、给药途径、给药时间和观察时间长度可能对肌苷治疗脊髓损伤大鼠的疗效的存在着不同的影响。
[Abstract]:Inosine is a small molecular substance widely existed in the body. It has a good protective effect on multiple systems and many cells. Recent studies on the protective effect of inosine on the central nervous system suggest that inosine not only regulates the role of immune cells, promotes the extensive growth of neuron axons, but also affects the damaged God. In vitro, inosine can protect ROC-1 cells, astrocytes and neurons in the chemical anoxic environment. In vivo, inosine can reduce the apoptosis and death of neurons, inhibit the accumulation of platelets, degradation of inositol phosphate and the formation of calcium ions, and reduce the formation of calcium ions. The extent of cerebral ischemia, reducing the volume of cerebral infarction, inhibiting the postsynaptic effect of glutamic acid, and promoting the recovery of neural function in rats. However, ROC-1 is a hybridoma cell fused by the oligodendrocytes and C6 glial cells in rats, and has the properties of these two cells, which can not confirm the anoxia of the oligodendrocytes. In addition, ROC-1 is an undead tumor cell, and it does not differentiate between different stages of differentiation and can not truly reflect the sensitivity of oligodendrocytes to pathological damage. Finally, although inosine treatment of cerebral ischemia model rats can reduce the apoptosis of nerve cells and promote the recovery of nerve function, inosine is ischemic in the body. The protective effect of oligodendrocytes in anoxic environment is still not clear. Therefore, it is of great significance to investigate the protective effect of inosine on oligodendrocytes in anoxic environment.
This study is divided into three parts. The first part is to investigate whether inosine has protective effect on the differentiated oligodendrocytes in chemical anoxic environment. The second part is to investigate whether inosine has protective effect on oligodendrocytes with proliferative ability in chemical anoxic environment and the third part of the investigation and study of inosine treatment. The effects of 7 days after spinal cord contusion on the recovery of motor function and the survival of oligodendrocytes in the necrotic area were observed.
In the first part, the effects of inosine on the survival and activity of oligodendrocytes in different stages of hypoxia injury were evaluated by the morphological observation of immunofluorescence staining, TUNEL staining, MTT test and ATP test.
The results showed that the damage caused by 24 h of oligodendrocytes with the same concentration of rotenone at different stages of differentiation was different. Therefore, the concentration of rotenone used to achieve roughly similar damage was also different. The damage concentration of immature oligodendrocytes was 5 UG, and the damage concentration of mature oligodendrocytes was strong. The degree is 20 u M rotenone. Inosine can pretreat damaged oligodendrocytes under the roughly similar damage degree of immature oligodendrocytes and mature oligodendrocytes. Inosine can significantly reduce the death and apoptosis of immature oligodendrocytes, and the inosine dose dependent increase of immature oligodendrocytes Cell activity, but inosine can only reduce the death of mature oligodendrocytes, and can not reduce the apoptosis of mature oligodendrocytes, and 10 mM inosine can improve the activity of damaged mature oligodendrocytes. Inosine promotes the survival of oligodendrocytes after differentiation, improves the activity of oligodendrocytes after differentiation and inosine reduction of the inosine. The difference in the protective effect of immature and mature oligodendrocytes may be related to the difference in the expression of adenosine receptors on oligodendrocytes in these two stages, which is related to the protective effect of the immature and mature oligodendrocytes.
In the second part, the effects of inosine on the survival and activity of oligodendrocyte precursor cells in the environment of chemical anoxic injury were evaluated by immunofluorescence staining and MTT experiments.
The results showed that 20 M rotenone could obviously induce the death of OPCs, the cell activity and the total number of cells decreased, which prevented the expression of NG2 antigen, but did not induce the apoptosis of OPCs. 10 mM inosine pretreated by the pretreatment of rotenone did not reduce the death and activity decline of the rotenone induced OPCs, but could improve the NG2 antigen on the damaged cells. In addition, it is worth noting that 10 mM inosine can significantly reduce the activity of OPCs in normal culture (that is, OPCs in blank control group).
In the third part, the effects of inosine intraperitoneal injection of inosine on the recovery of motor function and the number of oligodendrocytes near the bad dead zone were observed by intraperitoneal injection of inosine in rats.
The results showed that the intraperitoneal injection of inosine (75mg/kg, every 8 h) to the rat model of spinal cord contusion did not improve the motor function of rats and the number of oligodendrocyte survival near the necrotic area.
In vitro experiments we did observe that inosine could significantly promote the survival and activity of immature and mature oligodendrocytes with chemical anoxic damage. The protective mechanism of oligodendrocytes after differentiation and the ATP provided by inosine and the removal of Nitrites by inosine are related to chemical anoxia damage. There is no improvement in the survival and activity of oligodendrocyte precursor cells. At the same time, the number of oligodendrocytes around the injured area and the recovery of motor function are not observed when using inosine to treat the rat model of spinal contusion. The possible reason is the dosage, the way of administration, the time of administration and the time after the injury of the spinal cord. The length of observation time may have different effects on inosine in the treatment of spinal cord injury in rats.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

【参考文献】