本文選題：Nlp　切入點(diǎn)：中心體　出處：《北京協(xié)和醫(yī)學(xué)院》2017年博士論文

【摘要】：中心體相關(guān)蛋白Nlp是一個(gè)細(xì)胞周期和有絲分裂中重要的調(diào)控蛋白,其在中心體成熟、微管生發(fā)成核、有絲分裂過(guò)程中紡錘體形成、前中期/后期轉(zhuǎn)換、后期/末期轉(zhuǎn)換和胞質(zhì)分裂等過(guò)程中發(fā)揮著重要作用。Nlp表達(dá)異常導(dǎo)致有絲分裂異常和細(xì)胞周期紊亂,導(dǎo)致染色體異常分離及異倍體核型的產(chǎn)生,從而加劇染色體不穩(wěn)定性,在腫瘤發(fā)生發(fā)展中發(fā)揮著重要作用。Nlp高表達(dá)能促進(jìn)腫瘤發(fā)生,在多種人類癌癥如肺癌、乳腺癌、卵巢癌、頭頸癌、腎癌、甲狀腺癌等中均存在高表達(dá)現(xiàn)象,且在肺癌中有明顯基因擴(kuò)增現(xiàn)象。然而,異常低水平的Nlp也會(huì)導(dǎo)致異倍體核型,已有前期研究發(fā)現(xiàn)其在人淋巴瘤組織中呈低表達(dá)。人源Nlp轉(zhuǎn)基因小鼠模型不僅自發(fā)乳腺癌、卵巢癌、睪丸癌等多種腫瘤,而且對(duì)化學(xué)誘癌因素7,12-二甲基苯并(a)蒽(DMBA)和物理誘癌因素IR均更加敏感。體外培養(yǎng)其MEF細(xì)胞,不僅發(fā)現(xiàn)細(xì)胞生長(zhǎng)與抗凋亡能力均增強(qiáng),還發(fā)現(xiàn)廣泛的中心體擴(kuò)增現(xiàn)象。盡管Nlp在有絲分裂中的功能和高表達(dá)促癌的研究已較為充分,而Nlp功能缺失對(duì)腫瘤發(fā)生發(fā)展的影響及其在有絲分裂調(diào)控之外的生物學(xué)功能還亟待研究。為深入研究Nlp基因功能與腫瘤發(fā)生發(fā)展的關(guān)系,在本研究中,我們構(gòu)建了Nlp基因缺陷小鼠模型,發(fā)現(xiàn)其易自發(fā)淋巴瘤。我們對(duì)收集到的Nlp基因缺陷小鼠自發(fā)淋巴瘤標(biāo)本進(jìn)行免疫組化實(shí)驗(yàn),結(jié)果顯示其為B細(xì)胞來(lái)源。我們進(jìn)一步對(duì)Nlp基因缺陷小鼠進(jìn)行血液學(xué)研究,結(jié)果顯示其各型造血細(xì)胞比例未受影響,但相對(duì)正常野生型小鼠,缺陷小鼠的造血重建B細(xì)胞能力較弱,并且骨髓中pre-B細(xì)胞和外周血中B細(xì)胞比例降低。隨后我們檢測(cè)了 Nlp基因缺陷小鼠中多種免疫細(xì)胞因子的分泌,結(jié)果顯示Nlp-/-小鼠骨髓中IL-13、IL-17和IL-21分泌量均顯著降低。研究結(jié)果提示Nlp基因缺陷除造成染色體不穩(wěn)定性之外,還可能通過(guò)調(diào)控免疫細(xì)胞因子影響B(tài)細(xì)胞發(fā)育,從而特異性自發(fā)B細(xì)胞淋巴瘤。另一方面,為研究Nlp基因缺陷小鼠在化學(xué)誘癌因素下的腫瘤發(fā)生,我們利用經(jīng)典的化學(xué)致癌物DMBA通過(guò)腹腔注射的方式構(gòu)建誘癌模型。對(duì)一共234只小鼠的誘癌實(shí)驗(yàn)中,我們發(fā)現(xiàn),小鼠在DMBA誘導(dǎo)下發(fā)生肝癌、卵巢癌等多種腫瘤,Nlp基因缺陷小鼠肝癌發(fā)生率和肝癌惡性程度均高于野生型,其肝臟重量及器官系數(shù)、肝癌結(jié)節(jié)數(shù)、最大肝癌結(jié)節(jié)體積均高于野生型。Nlp基因缺陷引起的染色體不穩(wěn)定性導(dǎo)致小鼠肝臟對(duì)DMBA誘導(dǎo)腫瘤更為敏感。為尋找Nlp蛋白的新功能,我們通過(guò)查閱文獻(xiàn)并結(jié)合實(shí)驗(yàn)室前期對(duì)Nlp功能的研究,此次還探究了 Nlp在細(xì)胞內(nèi)囊泡運(yùn)輸系統(tǒng)中的作用。囊泡運(yùn)輸系統(tǒng)是胞內(nèi)分子貨物的運(yùn)輸網(wǎng)絡(luò),通過(guò)調(diào)控包裹著蛋白質(zhì)等其它分子的內(nèi)體在胞內(nèi)的轉(zhuǎn)運(yùn),以完成蛋白分子的加工、降解及細(xì)胞的內(nèi)吞和外排等生物學(xué)過(guò)程。囊泡運(yùn)輸系統(tǒng)不僅對(duì)細(xì)胞內(nèi)物質(zhì)和信息交流至關(guān)重要,還與細(xì)胞自噬、免疫應(yīng)答、細(xì)胞因子分泌等過(guò)程息息相關(guān)。本研究中,我們首先通過(guò)免疫熒光實(shí)驗(yàn)發(fā)現(xiàn)Nlp與細(xì)胞晚期內(nèi)體標(biāo)志物L(fēng)AMP2共定位,提示其可能與晚期內(nèi)體相關(guān)。為研究Nlp對(duì)晚期內(nèi)體的調(diào)控,我們重點(diǎn)觀察Nlp與晚期內(nèi)體分選蛋白R(shí)ab7的相互作用。免疫熒光實(shí)驗(yàn)發(fā)現(xiàn)Nlp不僅與Rab7在細(xì)胞漿內(nèi)存在共定位,而且Nlp可以促進(jìn)Rab7與其下游效應(yīng)蛋白FYCO1相互作用,從而驅(qū)動(dòng)晚期內(nèi)體沿微管正向移動(dòng)。免疫共沉淀實(shí)驗(yàn)也證實(shí)Nlp與Rab7和FYCOI確實(shí)存在相互作用,同時(shí)Nlp高表達(dá)可以促進(jìn)Rab7與FYCO1結(jié)合。研究結(jié)果表明Nlp是一個(gè)新的囊泡運(yùn)輸中晚期內(nèi)體轉(zhuǎn)運(yùn)調(diào)控蛋白,Nlp與Rab7結(jié)合并調(diào)控其功能,影響晚期內(nèi)體轉(zhuǎn)運(yùn)。本研究發(fā)現(xiàn)了 Nlp基因缺陷對(duì)腫瘤的發(fā)生發(fā)展有促進(jìn)作用并探討了其發(fā)病機(jī)制,這對(duì)于中心體蛋白相關(guān)動(dòng)物模型的研究有重要意義。另一方面,本研究首次發(fā)現(xiàn)Nlp是一個(gè)新的囊泡運(yùn)輸調(diào)控蛋白,從而為中心體和囊泡運(yùn)輸之間的關(guān)聯(lián)研究打開新的思路,對(duì)進(jìn)一步完善胞內(nèi)物質(zhì)信息調(diào)控網(wǎng)絡(luò)有重要意義。
[Abstract]:Centrosome associated protein Nlp is a cell cycle and has an important regulatory protein in mitosis, the centrosome maturation, germinal microtubule nucleation, a spindle formation during mitosis, before the mid / late anaphase / telophase conversion, conversion and cytokinesis process play an important role in the abnormal expression of.Nlp in mitosis abnormal cell cycle disorder, lead to abnormal chromosome separation and aneuploid karyotype, thereby increasing chromosome instability in tumor development plays an important role in the high expression of.Nlp can promote tumorigenesis in many human cancers such as lung cancer, breast cancer, ovarian cancer, head and neck cancer, renal cell carcinoma, high expression phenomenon presence of thyroid cancer, and obvious gene amplification phenomenon in lung cancer. However, abnormally low levels of Nlp will lead to aneuploid karyotype, previous research has found that in human lymphoma tissues Low expression. Human Nlp transgenic mice model not only spontaneous breast cancer, ovarian cancer, testicular cancer and other tumors, and the chemical carcinogen 7,12- two methyl benzo (a) anthracene (DMBA) and physical factors were more sensitive to IR induced cancer. The MEF cells were cultured in vitro, not only found that cell growth and anti apoptosis the ability was enhanced, also found widespread phenomenon. Although Nlp centrosome amplification in mitosis function and high expression of cancer has been fully, and the loss of function of Nlp on the occurrence and development of tumor and its effect in the biological functions of mitotic regulation can also be studied. For the function of Nlp gene and tumor development study on the relationship between occurrence and development, in this study, we constructed the Nlp gene deficient mice, found its easy spontaneous lymphoma Nlp gene deficient mice. We collected the spontaneous lymphoma specimens were immunohistochemically experimental results. The display from B cell. We further in Nlp gene deficient mice were hematologic studies, the results showed that the proportion of each type of hematopoietic cells was not affected, but relatively normal wild-type mice and mice deficient in B cells of hematopoietic reconstitution ability is weak, and the B cells and pre-B cells in peripheral blood decreased. Then the percentage of bone marrow we examined the secretion of various cytokines in Nlp deficient mice, the results showed that IL-13 Nlp-/- mice bone marrow, IL-17 and IL-21 secretion were significantly decreased. The results suggest that the Nlp gene defects in addition to cause chromosomal instability, possibly through the regulation of immune cell factors affecting the development of B cells, and the specific spontaneous B cell lymphoma on the other hand, for the tumor in the chemical carcinogenesis factors of Nlp deficient mice, we use the classical chemical carcinogen DMBA by intraperitoneal injection. To construct the induced cancer model induced cancer. A total of 234 experiments on mice, we found that mice liver cancer induced by DMBA, ovarian cancer and other tumors, Nlp deficient mice liver cancer and liver malignancy rate was higher than the wild type, the liver weight and organ coefficient of liver nodules number, maximum liver nodules the volume is higher than that of wild type.Nlp gene defects caused by chromosomal instability in mice liver induced by DMBA tumor is more sensitive. For finding new functions of Nlp protein, we combined the previous research on the function of Nlp and through literature, this also explores Nlp global transportation system role in cell vesicle internal capsule. The transport system is the intracellular molecular cargo transportation network, through the regulation of proteins and other molecules wrapped in intracellular transport within the body, to complete the processing of the protein molecule, and endocytosis and degradation Efflux and other biological processes. Vesicular transport system not only on cellular material and information exchange is essential, and autophagy, immune response, cytokine secretion is closely related to the process. In this study, we first by immunofluorescence test showed that Nlp cells with late endosome marker co localization of LAMP2, suggesting that it may be associated with late within the body. For the regulation of Nlp on the late endosomes, we focus on the interaction effect of Nlp and the late endosomal sorting protein Rab7. Immunofluorescence test showed that Nlp and Rab7 not only in the cytoplasm were colocalized, and Nlp can promote the Rab7 and its downstream effector protein FYCO1 interactions, thus driving the late endosome along microtubule migration. Experiments also confirmed that Nlp and Rab7 and FYCOI exist the interaction of CO immunoprecipitation, and high expression of Nlp can promote the Rab7 combined with FYCO1. The results show that Nlp is a The new body transport vesicles transport in advanced regulatory proteins, Nlp binds to Rab7 and regulate its function and influence of the late endosomal transport. This study found Nlp gene defects on the development of the cancer promoting effect and discuss its pathogenesis, it is important to study the centrosomal protein related animal model. On the other on the one hand, this is the first study found that Nlp is a new regulation of vesicle transport protein, which opens up a new way of association study between centrosome and vesicle transport, to further improve the regulatory network of intracellular material information has important significance.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R730.2

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