【作者】 李畅；

【导师】 仲人前；

【作者基本信息】 第二军医大学 ， 临床检验诊断学， 2011， 博士

【摘要】 多发性骨髓瘤(multiple myeloma, MM)是一种常见的血液系统恶性肿瘤,它以恶性浆细胞在骨髓中的聚积为特征,并引起多器官系统的损害。由于MM是一种克隆性B-淋巴细胞恶性疾病,在病人的血液及尿液和其他体液当中常检出特征性的单克隆免疫球蛋白(即M-蛋白),因此又是一种代表性的单克隆免疫增殖病。作为B-细胞终末分化期的恶性浆细胞在骨髓中的大量聚积导致了70-80%的MM病人具有骨质破坏及由骨质破坏引起的骨痛、病理性骨折、高钙血症及瘫痪等病理表现,同时因为大量无功能的单克隆免疫球蛋白或其片段的分泌和排泄,引起肾功能不全、贫血、反复感染等继发表现,最终导致病人死亡。近年来的研究表明,骨髓微环境在MM的发生发展过程中发挥了重要作用,骨髓中的基质细胞(bone marrow stromal cells,BMSC)通过分泌多种细胞因子与骨髓瘤细胞发生非常密切的联系,支持着骨髓瘤细胞的生长、增殖和存活,白细胞介素-6(IL-6)就是其中重要的细胞因子,此外还有胰岛素样生长因子-1(IGF-1)、肝细胞生长因子(HGF)等。近几年来大量研究报道了TNF家族B-细胞活化因子(B-cell-activating factor of TNF family, BAFF)同样具有维持骨髓瘤细胞增殖存活效应。BAFF是1999年由Mukhopadhyay等首先克隆成功的TNF家族的新成员,有BCMA、TACI及BAFF-R三种受体,主要表达于巨噬细胞和树突状细胞,对支持B细胞的增殖与活化,增强体液免疫反应,维持B淋巴细胞稳态都有重要作用,同时与自身免疫性疾病、非何杰金氏病及多种B-细胞性恶性疾病密切相关。BAFF在多发性骨髓瘤中的作用机制是近来的研究热点,研究发现MM中BAFF可引起ERK1/2及PI3-K/AKT通路的活化,并有效活化转录因子NF-kB,还可以引起Bcl-2和Mcl-1两种抗凋亡蛋白的上调作用,共同支持骨髓瘤细胞的增殖存活效应。Act1(NFκB activator 1)是新近鉴定出的NFκB信号通路活化蛋白,亦称为CIKS (Connection to IKK-complex and SAPK),说明Act1参与了NFκB和SAPK/JNK两条信号通路中的信号复合物的形成。研究表明Act1通过与TRAF分子之间的相互作用,既是IL-17信号通路中重要的活化因子,又是CD-40L/BAFFR介导的B淋巴细胞活化的负调控因子,因此Act1在不同细胞的免疫反应中发挥了双重作用。Act1对B细胞中CD-40L/BAFFR信号通路的负调控作用已通过基因敲除小鼠实验予以证实,但在信号通路中具体的作用机制还有待研究。BAFF分子对支持骨髓瘤细胞的增殖存活具有重要作用,而Act1又对B细胞中的BAFFR通路有负调节作用,使我们联想到Act1亦可能对恶性的骨髓瘤细胞起重要的作用。基于对以上研究的认识和了解,我们拟主要通过对骨髓瘤细胞的研究,探讨Act1对骨髓瘤细胞的增殖存活所产生的作用,主要是研究Act1在对骨髓瘤细胞具有重要影响的BAFF通路中的作用,同时了解Act1在MM病人中的表达情况,以期对MM的发病机理有更深一层的了解,为MM的治疗提供一些新思路第一部分MM病人外周血BAFF及Act1的表达水平骨髓微环境在MM发病机制中的作用近年来被广泛的研究和报道,骨髓基质细胞分泌的多种细胞因子都参与了MM的发生发展过程,BAFF分子是这些细胞因子中研究较多细胞因子。Jerome Moreaux等发现MM中,BAFF可引起ERK1/2及PI3-K/AKT通路的活化,并有效活化转录因子NF-kB而引起瘤细胞的增殖。BAFF还可以引起Bcl-2和Mcl-1两种抗凋亡蛋白的上调作用,Mcl-1持续表达可以减少由IL-6下降引起的细胞凋亡,表明Mcl-1是IL-6引起的瘤细胞存活中的主要抗凋亡蛋白,而过表达Bcl-2的骨髓瘤细胞株则可以保护细胞免受由地塞米松引起的凋亡,证明了BAFF促进MM细胞中信号通路活化及对肿瘤细胞的增殖存活发挥了重要作用。此外,MM细胞可能还存在一种BAFF及其受体之间的自分泌反馈循环,但调节BAFF表达、分泌及结合的基本机制仍然不清楚。而Act1基因缺乏小鼠表现出明显的CD40和BAFFR信号增强的表现,外周血B淋巴细胞大量增加,并引起淋巴结和脾肿大及高免疫球蛋白血症和自身抗体产生。而这种效应在CD40/Act1及BAFF/Act1双敲除鼠身上则完全消失,证明了Act1分子对CD40和BAFF通路的负调控作用,从而有可能对BAFF引起的细胞增殖起拮抗作用。BAFF分子能显著促进B细胞的增殖存活效应,而Act1则对B细胞中的BAFF通路具有负调控作用,但这都是在鼠的身上或细胞试验得出的结论,具体到MM病人的身上,MM病人是否表达这两种分子,这两种作用相反的分子的表达情况又是如何呢?我们收集MM病人外周血20例,分离单个核细胞(PBMC),提取总RNA并逆转录为cDNA,荧光定量PCR法扩增BAFF和Act1基因,并与20例正常样本相对照,了解MM病人BAFF和Act1表达情况,为以后的BAFF和Act1调节机制的研究提供前提条件。结果表明,MM病人外周血BAFF和Act1的基因表达水平相对于正常对照组而言均显著增高(P<0.01),分别为对照组的4.72和3.18倍,而MM病人BAFF和Act1基因表达水平之间也存在明显的相关性,提示这两种分子在MM中可能发挥重要作用,并且二者的作用之间可能存在相互影响,同时也为我们后续进行两种分子的调节机制研究提供了可行性。第二部分Act1对MM细胞BAFF通路负调控作用研究Act1对B细胞中的BAFF通路具有负调控作用,敲除该分子的老鼠发生了类似于BAFF分子刺激B细胞相似的作用,如高免疫球蛋白及自身抗体产生等效应,在细胞内部,Act1基因缺乏小鼠的BAFF信号通路活化明显增强,表现为NF-κB2 (p100/p52)信号通路中的IκB磷酸化显著增强,以及JNK, ERK及p38通路的活化。但是在人的B细胞性的恶性肿瘤MM中,Act1分子的表达情况是怎样的,它是否对骨髓瘤细胞中的BAFF通路具有负调控作用呢?为了解决这个问题,培养人骨髓瘤细胞株U266,提取细胞总蛋白进行western blot(WB)分析Act1蛋白在细胞中的表达情况。然后采用小干扰RNA技术,干扰Act1基因的表达,加入0.1μg/ml的BAFF分子刺激细胞,在0,5,10,25分钟时段提取细胞总蛋白,western blot分析pIkB的变化情况,观察Act1对转录因子NF-kB的负调控作用。同时合成Act1全基因并与适当载体相连接,在U266细胞株中进行过表达,加入同样浓度的BAFF分子刺激细胞,按同样的时段提取细胞总蛋白,进行western blot分析pIkB的变化情况,进一步证实Act1对转录因子NF-kB的负调控作用。从我们的实验结果可以看出,与未经处理的对照组细胞相比,经RNA干扰后缺乏Act1分子的细胞IkB发生了迅速的磷酸化,在BAFF分子刺激时间5分钟的时候达到高峰,25分钟后基本结束。而对照组的细胞IkB磷酸化程度较低,趋势不明显,过表达Act1分子的细胞基本没有磷酸化的发生,说明了大量的Act1分子对BAFF分子的刺激作用产生了明显的抑制,从正反两个方面证实骨髓瘤细胞中Act1分子对BAFF信号刺激具有明显的负调控作用。第三部分Act1在BAFF通路中的作用机制初探通过第二部分的实验,证实了Act1对骨髓瘤细胞中的BAFF通路确实具有负调控作用,文献报道Act1的负调控作用可能通过与TRAF家族(TNF Receptor Associated Factor )分子TRAF3及NFκB诱导激酶NIK的相互作用有关,但是调控机制仍不明确。在TNF受体及IL-1/Toll样受体家族的许多受体的下游信号通路中,TRAF家族的分子都扮演了比较关键的调节角色,TRAF2、5、6可以活化经典的NF-κB信号通路,而TRAF3则是非经典NF-κB信号通路的抑制因子。NIK是非经典的NF-κB信号通路活化的重要分子,它与IkB激酶(IKK)分子中的IKKα共同作用于p100的C末端,形成转录复合物p52:RelB,进入细胞核中引起相关基因的表达,从而活化非经典的NF-κB信号通路,因此Act1的作用机制可能与这两种分子密切相关。我们采用针对Act1基因的小干扰RNA处理的骨髓瘤细胞,用BAFF分子刺激,收集不同时段的细胞蛋白,用western blot分析TRAF3及NFκB诱导激酶NIK的表达情况,及不同时段的变化关系,了解它们的变化与IkB磷酸化之间的对应关系,并分析TRAF3和NIK分子之间的相互作用,探索该两种分子与BAFF通路活化的关系,进而了解他们在Act1调节机制中的作用。同时用免疫共沉淀技术分析Act1分子与TRAF3和NIK分子之间是否发生直接的作用。结果发现TRAF3在经过RNA干扰后缺乏Act1的细胞中的含量远低于未经处理的细胞,而NIK的水平则是增加的,提示TRAF3分子的是通过抑制或降解NIK分子实现对NF-κB信号通路的抑制作用。同时免疫沉淀的结果显示Act1与TRAF3和NIK之间都有直接作用,而TRAF3与NIK之间的直接作用相对较弱,说明Act1分子的作用机制是通过与TRAF3的结合实现的,可能介导了TRAF3分子对NIK的抑制作用,或与TRAF3共同参与了对NIK的抑制或降解,从而实现对BAFF信号通路的负作用。第四部分MM细胞相关功能检测通过前几部分的实验我们已经证实了在MM病人的外周血中Act1基因表达增高,且Act1缺乏的MM细胞会出现BAFF通路活化增强的表现,因此细胞的增殖情况,细胞抗凋亡蛋白的表达及细胞的分泌功能都可能随之产生一系列的变化。基于以上分析我们采用western blot的方法检测小干扰RNA处理后缺乏Act1的细胞抗凋亡蛋白Bcl-2的表达情况,同时进行MTT法细胞增殖实验,观察细胞的增殖状态的变化,评价Act1分子对细胞增殖存活的影响。同时检测细胞培养上清液的免疫球蛋白含量,了解Act1分子对细胞外分泌功能的变化的影响。结果表明,Act1缺乏细胞Bcl-2蛋白水平比对照组增加,而细胞上清液IgG的浓度变化不明显,可能与我们进行BAFF分子刺激的时间较短有关,而细胞的增殖明显增强,说明了Act1分子对骨髓瘤细胞的宏观表现也有一定的影响。通过这几部分实验,发现Act1分子在MM病人中的表达增高,并与BAFF分子的基因表达水平之间存在相关性,同时证明Act1分子在MM细胞BAFF通路中发挥负调控作用,其作用机制是通过作为TRAF3分子和NIK分子之间作用的桥梁,介导了NIK分子的降解,导致由NIK引起的NF-κB信号通路活化减弱,对MM细胞的生长起抑制作用,为MM的治疗提供了一个新的作用靶点。更多还原

【Abstract】 Multiple myeloma (MM) is a hematological malignancy characterized by the accumulation of malignant plasma cells in the bone marrow (BM), which induces multi-organic injuries. Large numbers of monoclonal gammaglobulin are secreted to the blood, urine and other fluid, so MM is also a kind of immunoproliferative disease. Accumulation of malignant plasma cells in the bone marrow led to osteolytic bone disease and some other diseases induced from the afunctional gammaglobulin, such as infection, anaemia, thrombocytopenia, renal failure and so on.Many studies indicated that the BM microenvironment played an important role in the generation of MM. The bone marrow stromal cells (BMSC) secreted a great deal of cytokines which were very important to the growth, proliferation and living of the myeloma cells. Interleukin-6 (IL-6), insulin-like growth factor-1 (IGF-1) are the popular cytokines. Recently,B-cell-activating factor of the tumor necrosis factor (TNF) family(BAFF) was identified as a critical factor in normal B-cell development and homeostasis too. BAFF was cloned in 1999 by Mukhopadhyay firstly which acted as a new member of TNF family, and it had three receptors named BCMA, TACI and BAFFR respectively. This molecule was expressed mainly by macrophages and dentritic cells and provides a key survival signal for the maturation of peripheral B cells, in addition, it was nearly correlative with autoimmune diseases, Hodgkin’s lymphoma and some other B-cell diseases. Now the mechanism how BAFF act in multiple myeloma is a pop problem. It has been reported that BAFF can activate the ERK1/2 and PI3-K/AKT accesses and then the NFκB, also, it can up-regulate Bcl-2 and Bcl-x proteins who can restrain apoptosis. All these functions sustain the proliferation and living of MM cells together.Act1(NFκB activator 1)is a new molecule which is identified as an activator in the NFκB signal pathway and it is known as the name of CIKS (Connection to IKK-complex and SAPK) too, the name showed that Act1 was included in both NFκB and SAPK/JNK signal passages. Act1 reacted with TRAFs and had dual effects in IL-17 and CD-40L/BAFFR. It activated the IL-17 signal in one hand and was a negative regulator in CD-40L/BAFFR signal pathway in the other hand. The negative regulation of Act1 to CD-40L/BAFFR signal Has been proved by gene-knockout mice, but the essential mechanism is not known. BAFF can promote the proliferation and living of MM cells and Act1 can regulate the BAFFR signal in B cells negatively, but are there Act1 protein in MM cells and what is the relationship between the two molecules in MM?Based on the understanding of the studies above, we plan to find the function of Act1 to the proliferation and living in MM cells. Mainly our research will focus on how the BAFF signal is affected by Act1 and the level of Act1 gene expression in the peripheral blood of MM patients at the same time to supply a new idea to the therapy of MM.Part one. Expression of BAFF and Act1 gene in peripheral blood of MM patientsBAFF can sustain the proliferation and living of B cells, and Act1 is a negative regulator in CD-40L/BAFFR signal passage of B cells, but these conclusions were gained from the mice. What is the situation of the two molecules in human MM cells? We collect the PBMCs of MM patients and extract the mRNA then put up reverse transcription, followed by amplification of the gene with RTQ-PCR. Through these experiments we can know the expression of BAFF and Act1 genes in MM patients, and provide a precondition to the following research.We can see that the mRNA of BAFF and Act1 are all increased in MM patients contrasted to the healthy persons from our experiments, the former shared 4.72 and 3.18 times to the later respectively. The results indicate the importance of BAFF and Act1 in the generation and development of MM.Part two. Research on the negative regulation of Act1 to BAFF signal pathway in MM cellsAct1 has the function of negative regulation to BAFF signal in B cell, and the mice who were knocked out the Act1 gene behave as the B cells which were stimulated with BAFF, such as hypergammaglobulinemia and the generation of autoantibodies. However, what is the expression of Act1 in human MM cells, and if it is of negative regulation to BAFF signal pathway in MM cells? To settle the problem, we culture the U266 Cell Strain and extract the total protein of the cells to perform western blot analysis in finding out the protein expression of Act1 in MM. whereafter we transfer the siRNA of Act1 into the U266 cells and stimulate the cells with o.1μg/ml BAFF, then we extract the total protein of the cells at 0, 5, 10, 25 minutes to perform the western blot assay on the p-IκB. At the same time the Act1 gene were synthesized and transferred to the cells to overexpress the Act1 protein and the cells were deal with by the same methods.From the results we found that the p-IκB increased more highly in the cells transferred with siRNA than in the untreated cells, but the cells overexpressed the Act1 changed very mildly. The level of p-IκB culminated at 5 minutes after the BAFF stimulation in the cells transferred with siRNA, and the molecules disappeared at 25 minutes when they were stimulated. This result proved the negative regulation of Act1 to BAFF signal pathway in MM cells.Part three. The mechanism of Act1 in BAFF signal passageThrough the experiments in part two, we proved the negative regulated function of Act1 to BAFF, but the mechanism is not clear and possibly it is relative with TRAF3 and NIK molecules, because the two molecules are both involved in the BAFF signal passage. So in this part we also used the U266 cells which were transferred with siRNA to Act1 gene and stimulated by BAFF, then the TRAF3 and NIK were analyzed by western blot to find the relationship between the two molecules and the BAFF signal. By this experiment we hope to know the role of the two molecules in the regulative action of Act1, and we want to find if there is direct contact between them by Immunoprecipitation(IP).Results showed that TRAF3 was lower in the cells transferred whit siRNA than in the unperformed cells, but the NIK was increased in the later. The results of IP showed there was not direct contact between TRAF3 and NIK, they both reacted with Act1 in signal passage. So the Act1 molecule is a bridge in the passage of cells’activation.Part four. Detection of the functions in MM cellsThe MM cells lacked Act1 acted as the behavior of fortified BAFF signal passage, so the functions of multiplication, excretion and expression could change in this state. We evaluated the level of the Bcl-2 protein which acted as a protein resisted apoptosis in MM cells lacked Act1 by western blot, and performed the MTT multiplication test and detection of IgG at the same time. Based on the experiments we found higher levels of Bcl-2 protein in Act1 lacked cells , but the IgG had no obvious difference between Act1 lacked cells and normal MM cells. On the other hand, the Act1 lacked cells showed stronger multiplication than the cells untreated.更多还原