Nlp基因缺陷小鼠肿瘤表型及其在囊泡运输中功能的研究

发布时间：2018-04-06 06:10

本文选题：Nlp　切入点：中心体　出处：《北京协和医学院》2017年博士论文

【摘要】：中心体相关蛋白Nlp是一个细胞周期和有丝分裂中重要的调控蛋白,其在中心体成熟、微管生发成核、有丝分裂过程中纺锤体形成、前中期/后期转换、后期/末期转换和胞质分裂等过程中发挥着重要作用。Nlp表达异常导致有丝分裂异常和细胞周期紊乱,导致染色体异常分离及异倍体核型的产生,从而加剧染色体不稳定性,在肿瘤发生发展中发挥着重要作用。Nlp高表达能促进肿瘤发生,在多种人类癌症如肺癌、乳腺癌、卵巢癌、头颈癌、肾癌、甲状腺癌等中均存在高表达现象,且在肺癌中有明显基因扩增现象。然而,异常低水平的Nlp也会导致异倍体核型,已有前期研究发现其在人淋巴瘤组织中呈低表达。人源Nlp转基因小鼠模型不仅自发乳腺癌、卵巢癌、睾丸癌等多种肿瘤,而且对化学诱癌因素7,12-二甲基苯并(a)蒽(DMBA)和物理诱癌因素IR均更加敏感。体外培养其MEF细胞,不仅发现细胞生长与抗凋亡能力均增强,还发现广泛的中心体扩增现象。尽管Nlp在有丝分裂中的功能和高表达促癌的研究已较为充分,而Nlp功能缺失对肿瘤发生发展的影响及其在有丝分裂调控之外的生物学功能还亟待研究。为深入研究Nlp基因功能与肿瘤发生发展的关系,在本研究中,我们构建了Nlp基因缺陷小鼠模型,发现其易自发淋巴瘤。我们对收集到的Nlp基因缺陷小鼠自发淋巴瘤标本进行免疫组化实验,结果显示其为B细胞来源。我们进一步对Nlp基因缺陷小鼠进行血液学研究,结果显示其各型造血细胞比例未受影响,但相对正常野生型小鼠,缺陷小鼠的造血重建B细胞能力较弱,并且骨髓中pre-B细胞和外周血中B细胞比例降低。随后我们检测了 Nlp基因缺陷小鼠中多种免疫细胞因子的分泌,结果显示Nlp-/-小鼠骨髓中IL-13、IL-17和IL-21分泌量均显著降低。研究结果提示Nlp基因缺陷除造成染色体不稳定性之外,还可能通过调控免疫细胞因子影响B细胞发育,从而特异性自发B细胞淋巴瘤。另一方面,为研究Nlp基因缺陷小鼠在化学诱癌因素下的肿瘤发生,我们利用经典的化学致癌物DMBA通过腹腔注射的方式构建诱癌模型。对一共234只小鼠的诱癌实验中,我们发现,小鼠在DMBA诱导下发生肝癌、卵巢癌等多种肿瘤,Nlp基因缺陷小鼠肝癌发生率和肝癌恶性程度均高于野生型,其肝脏重量及器官系数、肝癌结节数、最大肝癌结节体积均高于野生型。Nlp基因缺陷引起的染色体不稳定性导致小鼠肝脏对DMBA诱导肿瘤更为敏感。为寻找Nlp蛋白的新功能,我们通过查阅文献并结合实验室前期对Nlp功能的研究,此次还探究了 Nlp在细胞内囊泡运输系统中的作用。囊泡运输系统是胞内分子货物的运输网络,通过调控包裹着蛋白质等其它分子的内体在胞内的转运,以完成蛋白分子的加工、降解及细胞的内吞和外排等生物学过程。囊泡运输系统不仅对细胞内物质和信息交流至关重要,还与细胞自噬、免疫应答、细胞因子分泌等过程息息相关。本研究中,我们首先通过免疫荧光实验发现Nlp与细胞晚期内体标志物LAMP2共定位,提示其可能与晚期内体相关。为研究Nlp对晚期内体的调控,我们重点观察Nlp与晚期内体分选蛋白Rab7的相互作用。免疫荧光实验发现Nlp不仅与Rab7在细胞浆内存在共定位,而且Nlp可以促进Rab7与其下游效应蛋白FYCO1相互作用,从而驱动晚期内体沿微管正向移动。免疫共沉淀实验也证实Nlp与Rab7和FYCOI确实存在相互作用,同时Nlp高表达可以促进Rab7与FYCO1结合。研究结果表明Nlp是一个新的囊泡运输中晚期内体转运调控蛋白,Nlp与Rab7结合并调控其功能,影响晚期内体转运。本研究发现了 Nlp基因缺陷对肿瘤的发生发展有促进作用并探讨了其发病机制,这对于中心体蛋白相关动物模型的研究有重要意义。另一方面,本研究首次发现Nlp是一个新的囊泡运输调控蛋白,从而为中心体和囊泡运输之间的关联研究打开新的思路,对进一步完善胞内物质信息调控网络有重要意义。
[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.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R730.2

【相似文献】