本文選題：TIP30 + Oligl��； 參考：《第二軍醫(yī)大學(xué)》2015年博士論文

【摘要】：在脊椎動物中樞神經(jīng)系統(tǒng)(central nervous system, CNS)中,軸突外圍不連續(xù)的髓鞘片段結(jié)構(gòu)的形成使得神經(jīng)元興奮周期得到了大幅縮減,從而極大地提高了軸突傳導(dǎo)速率。在CNS中,髓鞘是由少突膠質(zhì)細胞(oligodendrocyte, OL)逐圈致密包繞軸突而成的結(jié)構(gòu)。而OL是由少突膠質(zhì)前體細胞(oligodendrocyte precursor cell, OPCs)經(jīng)歷三個主要階段最終分化而來,主要包括A285+幼稚期,PDGFa+和04+不完全成熟OL階段,以及髓鞘堿性蛋白MBP+成熟且具有成髓鞘能力的OL階段。OPC產(chǎn)生自非常局限的室管膜區(qū),自胚胎期開始生成,出生后早期階段也有少量發(fā)育。OPC分化對CNS髓鞘化及成年后再髓鞘化都發(fā)揮至關(guān)重要作用。近年來,關(guān)于OL分化的研究取得了許多新發(fā)現(xiàn),尤其是相關(guān)堿性螺旋—環(huán)一螺旋結(jié)構(gòu)(bHLH)的轉(zhuǎn)錄因子的研究�，F(xiàn)有研究表明,bHLH轉(zhuǎn)錄因子Olig1和Olig2在OPC發(fā)育早期發(fā)揮關(guān)鍵作用。其中,Oligl無論在OL發(fā)育的分化過程中還是脫髓鞘病變后的再髓鞘化過程中,都呈現(xiàn)出由胞核轉(zhuǎn)移至胞漿的動態(tài)變化過程。這一動態(tài)性胞核與胞質(zhì)定位改變與OL分化之間有著怎樣的關(guān)系,目前尚未得到確切結(jié)論。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,首先在變異性小細胞肺癌中被發(fā)現(xiàn),具有抑制侵襲的功能。TIP30基因定位于人類染色體上11p15.1的位置,其表達水平與多種不同器官的腫瘤發(fā)生呈負相關(guān)。后續(xù)研究發(fā)現(xiàn),其可以抑制Importin β介導(dǎo)的具有核定位信號底物的入核過程。OL轉(zhuǎn)錄調(diào)控因子Olig1具有核定位信號,其核漿定位過程能否被TIP30進行調(diào)控,目前尚未見報道。在本研究工作中,我們發(fā)現(xiàn)TIP30對OL發(fā)育起負性調(diào)控作用。免疫細胞化學(xué)染色顯示TIP30表達于少突膠質(zhì)細胞譜系(oligodendrocyte lineage cells, OLs)所有分化時期。實時定量PCR顯示TIP30在髓鞘發(fā)育關(guān)鍵時期,出生后第二十一天表達顯著降低。與野生型小鼠相比,TIP30-/-小鼠在出生后十四天和二十一天均表現(xiàn)出顯著升高的髓鞘化水平。進一步利用利用體外OPC培養(yǎng)的方法,將TIP30過表達后進行分化培養(yǎng)發(fā)現(xiàn)TIP30過表達導(dǎo)致OPC體外成熟過程嚴(yán)重受阻,而將TIP30基因敲減之后,OPC分化水平則顯著提高。隨后,我們探討了TIP30抑制OPC分化的機制。利用核漿蛋白分離和免疫細胞化學(xué)染色發(fā)現(xiàn),在OPC啟動分化早期,TIP30過表達恰可以將Olig1大量阻滯在胞漿,從而實現(xiàn)阻止Olig1入核調(diào)控髓鞘相關(guān)基因轉(zhuǎn)錄過程。而TIP30敲減之后,我們發(fā)現(xiàn)大量Olig1進入核內(nèi),參與OPC分化啟動。進一步利用免疫共沉淀實驗發(fā)現(xiàn),TIP30可以和OL分化調(diào)控的一個重要轉(zhuǎn)錄因子Olig1發(fā)生相互結(jié)合。這在某種程度上是導(dǎo)致OPC分化加速的主要原因之一。接下來,我們利用脫髓鞘動物模型和人類多發(fā)性硬化腦組織樣本研究了病理情況下TIP30調(diào)控Olig1胞核胞質(zhì)定位的意義。利用cuprizone誘導(dǎo)的脫髓鞘動物模型,我們發(fā)現(xiàn)發(fā)生髓鞘脫失病變的胼胝體部位有大量NG2-細胞募集,而這些NG2+細胞中的Olig1主要聚集在細胞核中,隨著再髓鞘化的進行,Olig1在核內(nèi)行使功能結(jié)束后逐漸轉(zhuǎn)移至胞漿,并且在此過程中未觀察到NG2+細胞中有TIP30表達上調(diào)。而在多發(fā)性硬化癥(multiple sclerosis, MS)慢性非活動性的脫髓鞘病灶處,可見大量NG2+細胞浸潤,但是在NG2+細胞亦見TIP30表達異常升高,并且?guī)缀跷匆娺@些NG2+細胞中Oligl入核。上述研究結(jié)果表明,TIP30在OL發(fā)育過程中起著負性調(diào)控的作用。OL內(nèi)源性調(diào)控因子TIP30通過調(diào)控Olig1核漿轉(zhuǎn)運過程以實現(xiàn)其對OPCs分化過程的負性調(diào)控。這一研究結(jié)果,有助于進一步提高我們對脫髓鞘相關(guān)疾病中有效的髓鞘再生機制的認識。
[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.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R741

【共引文獻】

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