酪氨酸磷酸酶SHP2在IL-6诱导的GC源性B-NHL细胞重分化中的研究

发布时间：2018-04-25 22:20

本文选题：SHP2 + IL-6　；参考：《浙江大学》2012年博士论文

【摘要】：目的和意义正常B细胞分化发育分子途径中存在着天然的风险。抗原的不断刺激、SHM以及CSR都会导致B细胞DNA处于经常性断裂、转位和修复过程中,使得B细胞基因组不稳定,长期积累将引起B细胞的发育异常和恶性转化。目前认为这是B淋巴细胞源性淋巴瘤产生的重要分子机制之一。B淋巴细胞可因其微环境的改变而重新定向分化而改变命运,我们称之为“B淋巴细胞的重分化”,目前学术界已逐步形成共识。淋巴组织的生发中心(Germinal Center, GC)是B细胞分化发育的重要微环境之一,也是导致B细胞基因组不稳定的诸多分子事件(如SHM和CSR)集中地,是影响B淋巴细胞的重分化的关键场所。近年来的文献揭示,Burkitt淋巴瘤(Burkitt Lymphoma, BL)、滤泡性淋巴瘤(Follicular Lymphoma, FL)、大部分弥漫性大B细胞性淋巴瘤(Diffuse Large B Cell Lymphoma, DLBCL)和部分霍奇金氏淋巴瘤在形成过程中都与GC有着密不可分的联系,它们的分子发生模式在分子生物学和遗传学上都存在着渊源关系。已发现的证据有：它们的免疫球蛋白重链基因(IgH)上都经历了SHM和CSR；它们或多或少都涉及IgH相关的染色体转位；它们免疫表型谱系具有共性的和相互重叠性,有的甚至可以相互转化。鉴于上述类型的淋巴瘤分子机制与GC有着密不可分的渊源关系,由此我们提出GC源性淋巴瘤(GC-derived lymphomas)的概念并对其发育分化的分子机制进行深入研究。GC源性B细胞淋巴瘤(GC-derived B cell Lymphoma)是最易发生染色体易位的恶性淋巴瘤。GC源性B细胞淋巴瘤在其发生的分子模式上具有遗传学上的渊源关系,因此具有相似分子特征。而且各类GC源性B细胞淋巴瘤具有共同的免疫表型谱系,有的甚至可以相互转化。因此本研究作者认为对GC源性B细胞淋巴瘤发育分化的分子机制进行深入研究具有重要的科学意义。已有研究证明微环境中细胞因子IL-6水平的变化是调节B淋巴细胞的重分化的关键因素。正常情况下,IL-6通过调节胞内SHP2的活性来决定GC来源B淋巴细胞的发育。SHP2是第一个确定的具有酪氨酸磷酸酶活性的原癌基因,其在诺南综合症和多发性黑痣综合症及幼年型白血病中的调控机制较明确。除了作为一种原癌基因,SHP2还在多条促癌信号通路当中起到重要作用。已知SHP2是RAS-ERK、PI3K-AKT和STAT1、STAT3等信号通路上游信号分子。SHP2还参与了B淋巴细胞和T淋巴细胞生长与发育。已有研究证实SHP2作为IL-6信号通路中关键蛋白,是B细胞分化的重要调节分叉点。当IL-6刺激时,其信号通路中关键蛋白SHP2的活化与否决定了B细胞是会生长停止,走向凋亡还是会促进B细胞生长存活两种截然不同的命运。因此,有必要更加深入研究IL-6信号通路中的SHP2蛋白在B淋巴细胞生长分化中所起到的作用。这一课题研究一方面将有助于人们深入了解IL-6促进GC源性B细胞恶性肿瘤生长发育的机制,并为GC源性B细胞淋巴瘤的治疗提供新的作用靶点；另一方面将促进对IL-6在B淋巴细胞胞浆信号转导途径中的地位和作用的深入了解。本课题的研究中,首先,我们从具有代表意义的GC源性B-NHL细胞株入手,主要进行以下两个方面的研究：1)研究SHP2在IL-6促进GC源性B细胞淋巴瘤生长以及调节GC标志蛋白表达中的作用；2)探索SHP2在IL-6诱导的GC源性B细胞淋巴瘤的重分化中的作用。本研究旨在“B淋巴细胞的重分化”视野下,深入研究GC源性B-NHL转化分子机制,这有助于阐明IL-6/SHP2途径在B细胞重分化中作用和信号通路调节关键靶点,进而探讨不同GC源性的B-NHL在相应的微环境改变下产生的不同分子调控模式以及它们的分子节点,并在此基础上阐明GC源性的B-NHL的独特的分子发病机制,为GC源性B-NHL临床分子诊断和治疗相关分子靶标提供科学依据。方法一、SHP2表达沉默对GC来源B-NHL细胞生物学特性的影响 ①利用SHP2 siRNA抑制GC来源B-NHL细胞中内源性SHP2的表达,real time qRT-PCR和Western Blotting检测转染的SHP2 siRNA对各组细胞内源性SHP2表达水平的影响；此外,用IL-6分别刺激SHP2干扰组和阴性对照组GCB-NHL细胞,Western Blotting检测IL-6刺激后各组细胞中p-ERK1/2、p-SRC、p-STAT3、p-AKT的活化水平；②构建了SHP2 PTP结构域上的定点突变(R465E)质粒,外源性转染入GC来源B-NHL细胞,再用IL-6分别刺激SHP2R465E定点突变组和阴性对照组GC来源B-NHL细胞,Western Blotting方法检测IL-6刺激后各组细胞中p-ERK1/2、p-SRC、p-STAT3、p-AKT的活化水平,以探讨GC来源B-NHL细胞中SHP2调控IL-6活化的信号传导通路的结构基础；③应用MTT法和CFSE去检测SHP2表达沉默对GC来源B-NHL细胞增殖能力的影响；同时用Western Blotting方法检测SHP2表达沉默对GC来源B-NHL细胞中PCNA表达水平的影响；我们还应用Annexin V法和PU法检测SHP2表达沉默对GC来源B-NHL细胞周期和凋亡的影响；④应用Transwell 5μM孔径迁移小室检测SHP2表达沉默对GC来源B-NHL细胞迁移的影响。二、在IL-6诱导的GCB-NHL细胞的重分化中的作用 ①应用Western Blotting方法检测SHP2表达沉默对GC来源B-NHL细胞GC表型标志蛋白表达水平的影响；此外,用IL-6分别刺激SHP2干扰组和阴性对照组GC来源B-NHL细胞,流式细胞仪检测IL-6刺激后GCB-NHL细胞中GC表型标志蛋白(CD77)/浆细胞表型标志蛋白(CD138)表达水平的变化；同时Western Blotting去检测各组细胞中浆细胞分化因子Blimpl和XBP1表达水平的影响；②分别应用MEK Inhibitor U0126和SRC Inhibitor PP2PP2处理GC来源B-NHL细胞,Western Blotting去检测抑制SRC和ERK通路对GCB-NHL细胞GC表型标志蛋白表达水平的影响；③应用Western Blotting方法检测c-myc表达沉默对GC来源B-NHL细胞GC表型标志蛋白表达的影响；④PCR法检测SHP2表达沉默对GC来源B-NHL细胞中t(8；14)(q24；q32)转位的影响。结果①SHP2参与调控GC来源B-NHL细胞生物学行为：SHP2对IL-6活化的GC来源B-NHL细胞中的p-ERK和p-SRC磷酸化是必需的,而对IL-6活化的P-STAT3和p-AKT磷酸化程度没有影响；SHP2通过其PTP酶活性影响IL-6刺激的p-ERK和p-SRC的活化；SHP2表达沉默抑制了IL-6促GC来源B-NHL细胞增殖的效应；SHP2表达沉默还会导致GC来源B-NHL细胞GO-G1期延长,S-G2-M期缩短；但是SHP2表达沉默不会影响GC来源B-NHL细胞早期凋亡和晚期凋亡水平；无论有无IL-6刺激,干扰SHP2的表达,都会抑制GC来源B-NHL细胞的迁移。②SHP2参与维持GC来源B-NHL细胞的GC表型：SHP2表达沉默会导致GC来源B-NHL细胞中关键的GC表型蛋白Bcl6、Pax5、E2A、AICDA表达以及c-myc原癌基因蛋白表达减少；SHP2表达沉默抑制了Raji和Ramos细胞中t(8；14)(q24；q32)转位频率。③SHP2决定了IL-6诱导的GC来源B-NHL细胞的重分化方向：SHP2表达沉默的GC来源B-NHL细胞中浆细胞分化因子Blimp1、XBP1表达水平升高；流式细胞仪检测发现,IL-6刺激SHP2表达沉默的GC来源B-NHL细胞膜上的活化GCB细胞标志蛋白CD77表达水平降低,而浆细胞表型标志蛋白CD138的表达水平升高,说明SHP2表达沉默可以导致IL-6诱导GC来源B-NHL细胞向浆细胞方向分化。结论SHP2参与调控GC来源B-NHL细胞的增值和迁移,但对其早期和晚期凋亡水平没有影响。并且SHP2影响了GC源性B-NHL细胞GC表型标志蛋白的表达。SHP2是IL-6诱导的GC源性B-NHL细胞重分化的枢纽。
[Abstract]:Purpose and significance
There is a natural risk in the molecular pathways of normal B cell differentiation and development. The continuous stimulation of antigen, SHM and CSR can cause the DNA in the B cell to be in the regular fracture, transposition and repair process, which makes the genome of B cells unstable and the long-term accumulation will cause the abnormal development of B cells and the malignant transformation of B cells. It is considered to be the lymphocytic lymphocytic lymphocytic lymphoblastic degeneration of B. .B lymphocyte, one of the important molecular mechanisms of the tumor, can be redirected to change fate because of its microenvironment changes. We call it "B lymphocyte redifferentiation", and the academic community has gradually formed a consensus. The Germinal Center (GC) is one of the important microenvironment for the differentiation and development of B cells. Many molecular events, such as SHM and CSR, which cause genomic instability of B cells, are the key places that affect the redifferentiation of B lymphocytes.
In recent years, the literature revealed that Burkitt lymphoma (Burkitt Lymphoma, BL), follicular lymphoma (Follicular Lymphoma, FL), most diffuse large B cell lymphoma (Diffuse Large B Cell) and some Hodge's lymphoma are inseparable in the formation process, their molecular patterns There is a relationship between molecular biology and genetics. The evidence has been found that their immunoglobulin heavy chain gene (IgH) has experienced SHM and CSR; they are more or less involved in IgH related chromosomal transposition; their immunophenotype lineages are common and overlapping, and some can even be converted to each other. The molecular mechanism of these types of lymphoma has an inseparable relationship with GC, thus we propose the concept of GC derived lymphoma (GC-derived lymphomas) and study the molecular mechanism of its development and differentiation, and the study of.GC derived B cell lymphoma (GC-derived B cell Lymphoma) is the most likely malignant lymphoma of chromosome translocation. .GC derived B cell lymphoma has a genetic relationship in its molecular pattern, and therefore has similar molecular characteristics. And all kinds of GC derived B cell lymphoma have a common immunophenotype lineage, and some can even transform each other. Therefore, the authors believe that the molecular mechanism of the development and differentiation of GC derived B cell lymphoma is a molecular machine. It is of great scientific significance to make a thorough study of the system.
Studies have shown that the change in the level of cytokine IL-6 in the microenvironment is a key factor in regulating the redifferentiation of B lymphocytes. Under normal conditions, IL-6 is the first proto oncogene with tyrosine phosphatase activity by regulating the activity of intracellular SHP2 in the GC source B lymphocyte, which is in the nonan syndrome and in the nonan syndrome. The regulatory mechanism in multiple nevus syndrome and juvenile leukemia is clear. In addition to being a proto oncogene, SHP2 plays an important role in multiple cancer promoting signaling pathways. The known SHP2 is known as RAS-ERK, PI3K-AKT and STAT1, STAT3, and the upstream signal molecule.SHP2 is also involved in the growth and development of B and T lymphocytes. Studies have shown that SHP2 is the key protein in the IL-6 signaling pathway and is an important regulatory bifurcation of the differentiation of B cells. When IL-6 is stimulated, the activation of the key protein SHP2 in the signaling pathway determines whether B cells will grow to stop, to apoptosis or to promote the survival of B cells to survive two completely different destinies. Therefore, it is necessary to more Further study the role of SHP2 protein in the growth and differentiation of B lymphocytes in the IL-6 signaling pathway. This topic will help people to understand the mechanism of IL-6 to promote the growth and development of GC derived B cells, and to provide new targets for the treatment of GC derived B cell lymphoma; on the other hand, it will promote the development of GC derived B cell lymphoma. In-depth understanding of the status and role of IL-6 in B lymphocyte cytoplasmic signal transduction pathway. In this study, first of all, we begin with the representative GC derived B-NHL cell lines, mainly to study the following two aspects: 1) the study of SHP2 in IL-6 to promote the growth of GC derived B cell lymphoma and to regulate the GC marker protein The role of expression; 2) to explore the role of SHP2 in the redifferentiation of IL-6 induced GC derived B cell lymphoma.
The purpose of this study is to study the molecular mechanism of GC derived B-NHL transformation in the view of "B lymphocyte redifferentiation", which helps to elucidate the role of IL-6/SHP2 pathway in the redifferentiation of B cells and the key target of signaling pathway regulation, and then to explore the different molecular regulation patterns of the GC derived B-NHL under the corresponding microenvironment changes. Their molecular nodes, and on this basis, elucidate the unique molecular pathogenesis of GC derived B-NHL, providing a scientific basis for the diagnosis and treatment of related molecular targets for GC derived B-NHL.
Method
1. The effect of SHP2 silencing on the biological characteristics of GC derived B-NHL cells.
The expression of endogenous SHP2 in GC derived B-NHL cells was inhibited by SHP2 siRNA, and real time qRT-PCR and Western Blotting were used to detect the effect of SHP2 siRNA on the endogenous expression level of each cell. The activation level of p-ERK1/2, p-SRC, p-STAT3, p-AKT in the cell, and the construction of the fixed-point mutation (R465E) plasmid on the SHP2 PTP domain, transfected into GC source B-NHL cells, and IL-6 stimulation of SHP2R465E point mutation group and negative control group, respectively. The activation level of p-SRC, p-STAT3 and p-AKT was used to explore the structural basis of SHP2 regulating IL-6 activation in GC derived B-NHL cells. (3) the effects of MTT and CFSE on the proliferation of B-NHL cells in GC sources were detected by MTT and CFSE. The effect of NA expression level; we also used the Annexin V method and PU method to detect the effect of SHP2 expression silencing on the cell cycle and apoptosis of GC source B-NHL cells. (4) the effect of Transwell 5 micron pore migration chamber to detect SHP2 expression silencing on GC source B-NHL cell migration.
Two, the role of IL-6 in the redifferentiation of GCB-NHL cells.
(1) Western Blotting method was used to detect the effect of SHP2 expression silencing on the expression level of GC phenotypic protein expression in GC source B-NHL cells. In addition, IL-6 stimulated SHP2 interference group and negative control group GC source B-NHL cells, and the flow cytometry was used to detect the phenotype marker of the phenotype marker protein of the GCB-NHL cells after IL-6 stimulation. The expression level of white (CD138) was changed, and the effect of Western Blotting on the expression level of plasma cell differentiation factor Blimpl and XBP1 in each cell was detected. (2) MEK Inhibitor U0126 and SRC Inhibitor were used to treat GC source cells respectively. The effect of protein expression level; (3) the effect of c-myc expression silencing on the expression of GC phenotypic expression of B-NHL cells from GC derived B-NHL cells by Western Blotting method; and (4) PCR assay was used to detect the effect of SHP2 expression silencing on t (8; 14) in GC source B-NHL cells.
Results (1) SHP2 participates in the regulation of biological behavior of GC source B-NHL cells: SHP2 is necessary for p-ERK and p-SRC phosphorylation in B-NHL cells activated by IL-6, but has no effect on P-STAT3 and p-AKT phosphorylation of IL-6 activation; IL-6 promoted the proliferation of B-NHL cells from GC source, and SHP2 expression silencing could also lead to the prolongation of GO-G1 phase in GC source B-NHL cells and the shortening of S-G2-M phase, but the expression of SHP2 expression silencing would not affect the early apoptosis and late apoptosis level of GC derived B-NHL cells. (2) SHP2 participates in maintaining the GC phenotype of GC derived B-NHL cells: SHP2 expression silencing can lead to the key GC phenotypic protein Bcl6, Pax5, E2A, AICDA expression and the decrease of the expression of the original oncogene protein in GC source B-NHL cells; The redifferentiation direction of GC source B-NHL cells: SHP2 expressed GC source B-NHL cells of plasma cell differentiation factor Blimp1, XBP1 expression level increased; flow cytometer detection found that IL-6 stimulated SHP2 expression silenced GC source B-NHL cell membrane activated GCB cell marker protein expression level decreased, and plasma cell phenotype marked eggs The expression level of white CD138 increased, indicating that SHP2 silencing can induce IL-6 to induce GC derived B-NHL cells to differentiate into plasma cells.
Conclusion SHP2 participates in the regulation of the increment and migration of B-NHL cells in GC source, but does not affect the level of early and late apoptosis, and SHP2 affects the expression of GC phenotypic protein of GC derived B-NHL cells, which is the hub of IL-6 induced GC derived B-NHL cell redifferentiation.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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