TIP30抑制少突胶质前体细胞分化及其机制的研究

发布时间：2018-04-26 05:04

本文选题：TIP30 + Oligl　；参考：《第二军医大学》2015年博士论文

【摘要】：在脊椎动物中枢神经系统(central nervous system, CNS)中,轴突外围不连续的髓鞘片段结构的形成使得神经元兴奋周期得到了大幅缩减,从而极大地提高了轴突传导速率。在CNS中,髓鞘是由少突胶质细胞(oligodendrocyte, OL)逐圈致密包绕轴突而成的结构。而OL是由少突胶质前体细胞(oligodendrocyte precursor cell, OPCs)经历三个主要阶段最终分化而来,主要包括A285+幼稚期,PDGFa+和04+不完全成熟OL阶段,以及髓鞘碱性蛋白MBP+成熟且具有成髓鞘能力的OL阶段。OPC产生自非常局限的室管膜区,自胚胎期开始生成,出生后早期阶段也有少量发育。OPC分化对CNS髓鞘化及成年后再髓鞘化都发挥至关重要作用。近年来,关于OL分化的研究取得了许多新发现,尤其是相关碱性螺旋—环一螺旋结构(bHLH)的转录因子的研究。现有研究表明,bHLH转录因子Olig1和Olig2在OPC发育早期发挥关键作用。其中,Oligl无论在OL发育的分化过程中还是脱髓鞘病变后的再髓鞘化过程中,都呈现出由胞核转移至胞浆的动态变化过程。这一动态性胞核与胞质定位改变与OL分化之间有着怎样的关系,目前尚未得到确切结论。30 KDa HIV-1 Tat相互作用蛋白(The thirty-kDa HIV-1 Tat interacting protein),TIP30,即CC3或者HTATIP2,首先在变异性小细胞肺癌中被发现,具有抑制侵袭的功能。TIP30基因定位于人类染色体上11p15.1的位置,其表达水平与多种不同器官的肿瘤发生呈负相关。后续研究发现,其可以抑制Importin β介导的具有核定位信号底物的入核过程。OL转录调控因子Olig1具有核定位信号,其核浆定位过程能否被TIP30进行调控,目前尚未见报道。在本研究工作中,我们发现TIP30对OL发育起负性调控作用。免疫细胞化学染色显示TIP30表达于少突胶质细胞谱系(oligodendrocyte lineage cells, OLs)所有分化时期。实时定量PCR显示TIP30在髓鞘发育关键时期,出生后第二十一天表达显著降低。与野生型小鼠相比,TIP30-/-小鼠在出生后十四天和二十一天均表现出显著升高的髓鞘化水平。进一步利用利用体外OPC培养的方法,将TIP30过表达后进行分化培养发现TIP30过表达导致OPC体外成熟过程严重受阻,而将TIP30基因敲减之后,OPC分化水平则显著提高。随后,我们探讨了TIP30抑制OPC分化的机制。利用核浆蛋白分离和免疫细胞化学染色发现,在OPC启动分化早期,TIP30过表达恰可以将Olig1大量阻滞在胞浆,从而实现阻止Olig1入核调控髓鞘相关基因转录过程。而TIP30敲减之后,我们发现大量Olig1进入核内,参与OPC分化启动。进一步利用免疫共沉淀实验发现,TIP30可以和OL分化调控的一个重要转录因子Olig1发生相互结合。这在某种程度上是导致OPC分化加速的主要原因之一。接下来,我们利用脱髓鞘动物模型和人类多发性硬化脑组织样本研究了病理情况下TIP30调控Olig1胞核胞质定位的意义。利用cuprizone诱导的脱髓鞘动物模型,我们发现发生髓鞘脱失病变的胼胝体部位有大量NG2-细胞募集,而这些NG2+细胞中的Olig1主要聚集在细胞核中,随着再髓鞘化的进行,Olig1在核内行使功能结束后逐渐转移至胞浆,并且在此过程中未观察到NG2+细胞中有TIP30表达上调。而在多发性硬化症(multiple sclerosis, MS)慢性非活动性的脱髓鞘病灶处,可见大量NG2+细胞浸润,但是在NG2+细胞亦见TIP30表达异常升高,并且几乎未见这些NG2+细胞中Oligl入核。上述研究结果表明,TIP30在OL发育过程中起着负性调控的作用。OL内源性调控因子TIP30通过调控Olig1核浆转运过程以实现其对OPCs分化过程的负性调控。这一研究结果,有助于进一步提高我们对脱髓鞘相关疾病中有效的髓鞘再生机制的认识。
[Abstract]:In the vertebrate central nervous system (central nervous system, CNS), the formation of discontinuous myelin fragments on the periphery of the axon leads to a large reduction in the excitatory cycle of the neuron, which greatly improves the axon conduction rate. In CNS, the myelin sheath is round the axon by the oligodendrocyte (oligodendrocyte, OL). The structure of OL is formed by the final differentiation of the oligodendrocyte precursor cell (OPCs) in three major stages, mainly including the A285+ naive stage, the incomplete mature OL phase of PDGFa+ and 04+, and the OL stage of myelin basic protein MBP+ with the myelin sheath capability in the OL phase of the ependyma In the region, a small amount of development of.OPC differentiation from the embryonic stage to the early stage of birth also plays a vital role in the myelination of CNS and the myelination of the adult. In recent years, many new discoveries have been made about the differentiation of OL, especially the study of the transcription factors related to the basic spiral loop one spiral structure (bHLH). The bHLH transcriptional factor Olig1 and Olig2 play a key role in the early development of OPC. In the process of myelinating in the process of differentiation and demyelination, Oligl shows a dynamic change from the nucleus to the cytoplasm in the process of demyelination. What is the relationship between the dynamic nucleus and the cytoplasmic localization and the differentiation of OL .30 KDa HIV-1 Tat interaction protein (The thirty-kDa HIV-1 Tat interacting protein) has not yet been obtained. TIP30, i.e. CC3 or, is first found in variant small cell lung cancer. The tumorigenesis of different organs is negatively correlated. Subsequent studies have found that it can inhibit the nucleation of Importin beta mediated nucleation signal substrate, and the.OL transcriptional regulator Olig1 has a nuclear location signal, and whether its nuclear location process can be regulated by TIP30 has not yet been reported. In this study, we found that TIP30 to OL Immunocytochemical staining showed that TIP30 was expressed in all stages of differentiation of oligodendrocyte lineage (oligodendrocyte lineage cells, OLs). Real-time quantitative PCR showed that TIP30 was in the critical period of myelin development and decreased significantly at twenty-first days after birth. Compared with wild type mice, TIP30-/- mice were born after birth. The level of myelination was significantly elevated on fourteen and twenty-one days. Further using in vitro OPC culture, TIP30 overexpressed TIP30 overexpression and found that OPC was severely hindered in the process of maturation in vitro, and the level of OPC segregation was significantly increased after the TIP30 gene was knocked down. Then, we explored TIP30 The mechanism of inhibiting the differentiation of OPC. Using the nucleoplasma protein separation and immunocytochemical staining, it is found that at the early stage of OPC initiation and differentiation, the overexpression of TIP30 can block the Olig1 in the cytoplasm so as to prevent the Olig1 entry into the myelin related gene transcription process. And after the TIP30 knockout, we found that a large number of Olig1 enters the nucleus and participates in OPC differentiation. Further use of immunoprecipitation experiments found that TIP30 can combine with an important transcription factor, Olig1, an important transcription factor that regulates OL differentiation. This is one of the main reasons for the acceleration of OPC differentiation to some extent. Next, we have studied the pathological conditions by using the demyelination animal model and human multiple sclerosis brain tissue samples. Using the demyelinating animal model induced by cuprizone, we found that a large number of NG2- cells were raised in the corpus callosum of the myelinating lesion, and the Olig1 in these NG2+ cells was mainly concentrated in the nucleus, and with the progression of myelinating, the Olig1 in the nucleus after function ended after the TIP30. NG2+ cells were gradually transferred to the cytoplasm, and the expression of TIP30 in the NG2+ cells was not up regulated in this process. A large number of NG2+ cells were seen in the chronic inactive demyelinating lesions of multiple sclerosis (multiple sclerosis, MS), but the expression of TIP30 expression in NG2+ cells was also elevated, and Oligl entry in these NG2+ cells was hardly seen. The results suggest that TIP30 plays a negative role in the development of OL, the.OL endogenous regulator TIP30 regulates the negative regulation of OPCs differentiation by regulating the Olig1 nuclear transport process. This study is helpful to further improve our effective myelin regeneration mechanism in demyelinated disease related diseases. Know.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R741

【共引文献】