本文選題：SHP2 + IL-6��； 參考：《浙江大學(xué)》2012年博士論文

【摘要】：目的和意義 正常B細胞分化發(fā)育分子途徑中存在著天然的風(fēng)險�？乖牟粩啻碳�、SHM以及CSR都會導(dǎo)致B細胞DNA處于經(jīng)常性斷裂、轉(zhuǎn)位和修復(fù)過程中,使得B細胞基因組不穩(wěn)定,長期積累將引起B(yǎng)細胞的發(fā)育異常和惡性轉(zhuǎn)化。目前認為這是B淋巴細胞源性淋巴瘤產(chǎn)生的重要分子機制之一。B淋巴細胞可因其微環(huán)境的改變而重新定向分化而改變命運,我們稱之為“B淋巴細胞的重分化”,目前學(xué)術(shù)界已逐步形成共識。淋巴組織的生發(fā)中心(Germinal Center, GC)是B細胞分化發(fā)育的重要微環(huán)境之一,也是導(dǎo)致B細胞基因組不穩(wěn)定的諸多分子事件(如SHM和CSR)集中地,是影響B(tài)淋巴細胞的重分化的關(guān)鍵場所。 近年來的文獻揭示,Burkitt淋巴瘤(Burkitt Lymphoma, BL)、濾泡性淋巴瘤(Follicular Lymphoma, FL)、大部分彌漫性大B細胞性淋巴瘤(Diffuse Large B Cell Lymphoma, DLBCL)和部分霍奇金氏淋巴瘤在形成過程中都與GC有著密不可分的聯(lián)系,它們的分子發(fā)生模式在分子生物學(xué)和遺傳學(xué)上都存在著淵源關(guān)系。已發(fā)現(xiàn)的證據(jù)有：它們的免疫球蛋白重鏈基因(IgH)上都經(jīng)歷了SHM和CSR；它們或多或少都涉及IgH相關(guān)的染色體轉(zhuǎn)位；它們免疫表型譜系具有共性的和相互重疊性,有的甚至可以相互轉(zhuǎn)化。鑒于上述類型的淋巴瘤分子機制與GC有著密不可分的淵源關(guān)系,由此我們提出GC源性淋巴瘤(GC-derived lymphomas)的概念并對其發(fā)育分化的分子機制進行深入研究。GC源性B細胞淋巴瘤(GC-derived B cell Lymphoma)是最易發(fā)生染色體易位的惡性淋巴瘤。GC源性B細胞淋巴瘤在其發(fā)生的分子模式上具有遺傳學(xué)上的淵源關(guān)系,因此具有相似分子特征。而且各類GC源性B細胞淋巴瘤具有共同的免疫表型譜系,有的甚至可以相互轉(zhuǎn)化。因此本研究作者認為對GC源性B細胞淋巴瘤發(fā)育分化的分子機制進行深入研究具有重要的科學(xué)意義。 已有研究證明微環(huán)境中細胞因子IL-6水平的變化是調(diào)節(jié)B淋巴細胞的重分化的關(guān)鍵因素。正常情況下,IL-6通過調(diào)節(jié)胞內(nèi)SHP2的活性來決定GC來源B淋巴細胞的發(fā)育。SHP2是第一個確定的具有酪氨酸磷酸酶活性的原癌基因,其在諾南綜合癥和多發(fā)性黑痣綜合癥及幼年型白血病中的調(diào)控機制較明確。除了作為一種原癌基因,SHP2還在多條促癌信號通路當中起到重要作用。已知SHP2是RAS-ERK、PI3K-AKT和STAT1、STAT3等信號通路上游信號分子。SHP2還參與了B淋巴細胞和T淋巴細胞生長與發(fā)育。已有研究證實SHP2作為IL-6信號通路中關(guān)鍵蛋白,是B細胞分化的重要調(diào)節(jié)分叉點。當IL-6刺激時,其信號通路中關(guān)鍵蛋白SHP2的活化與否決定了B細胞是會生長停止,走向凋亡還是會促進B細胞生長存活兩種截然不同的命運。因此,有必要更加深入研究IL-6信號通路中的SHP2蛋白在B淋巴細胞生長分化中所起到的作用。這一課題研究一方面將有助于人們深入了解IL-6促進GC源性B細胞惡性腫瘤生長發(fā)育的機制,并為GC源性B細胞淋巴瘤的治療提供新的作用靶點；另一方面將促進對IL-6在B淋巴細胞胞漿信號轉(zhuǎn)導(dǎo)途徑中的地位和作用的深入了解。本課題的研究中,首先,我們從具有代表意義的GC源性B-NHL細胞株入手,主要進行以下兩個方面的研究：1)研究SHP2在IL-6促進GC源性B細胞淋巴瘤生長以及調(diào)節(jié)GC標志蛋白表達中的作用；2)探索SHP2在IL-6誘導(dǎo)的GC源性B細胞淋巴瘤的重分化中的作用。 本研究旨在“B淋巴細胞的重分化”視野下,深入研究GC源性B-NHL轉(zhuǎn)化分子機制,這有助于闡明IL-6/SHP2途徑在B細胞重分化中作用和信號通路調(diào)節(jié)關(guān)鍵靶點,進而探討不同GC源性的B-NHL在相應(yīng)的微環(huán)境改變下產(chǎn)生的不同分子調(diào)控模式以及它們的分子節(jié)點,并在此基礎(chǔ)上闡明GC源性的B-NHL的獨特的分子發(fā)病機制,為GC源性B-NHL臨床分子診斷和治療相關(guān)分子靶標提供科學(xué)依據(jù)。 方法 一、SHP2表達沉默對GC來源B-NHL細胞生物學(xué)特性的影響 ①利用SHP2 siRNA抑制GC來源B-NHL細胞中內(nèi)源性SHP2的表達,real time qRT-PCR和Western Blotting檢測轉(zhuǎn)染的SHP2 siRNA對各組細胞內(nèi)源性SHP2表達水平的影響；此外,用IL-6分別刺激SHP2干擾組和陰性對照組GCB-NHL細胞,Western Blotting檢測IL-6刺激后各組細胞中p-ERK1/2、p-SRC、p-STAT3、p-AKT的活化水平；②構(gòu)建了SHP2 PTP結(jié)構(gòu)域上的定點突變(R465E)質(zhì)粒,外源性轉(zhuǎn)染入GC來源B-NHL細胞,再用IL-6分別刺激SHP2R465E定點突變組和陰性對照組GC來源B-NHL細胞,Western Blotting方法檢測IL-6刺激后各組細胞中p-ERK1/2、p-SRC、p-STAT3、p-AKT的活化水平,以探討GC來源B-NHL細胞中SHP2調(diào)控IL-6活化的信號傳導(dǎo)通路的結(jié)構(gòu)基礎(chǔ)；③應(yīng)用MTT法和CFSE去檢測SHP2表達沉默對GC來源B-NHL細胞增殖能力的影響；同時用Western Blotting方法檢測SHP2表達沉默對GC來源B-NHL細胞中PCNA表達水平的影響；我們還應(yīng)用Annexin V法和PU法檢測SHP2表達沉默對GC來源B-NHL細胞周期和凋亡的影響；④應(yīng)用Transwell 5μM孔徑遷移小室檢測SHP2表達沉默對GC來源B-NHL細胞遷移的影響。 二、在IL-6誘導(dǎo)的GCB-NHL細胞的重分化中的作用 ①應(yīng)用Western Blotting方法檢測SHP2表達沉默對GC來源B-NHL細胞GC表型標志蛋白表達水平的影響；此外,用IL-6分別刺激SHP2干擾組和陰性對照組GC來源B-NHL細胞,流式細胞儀檢測IL-6刺激后GCB-NHL細胞中GC表型標志蛋白(CD77)/漿細胞表型標志蛋白(CD138)表達水平的變化；同時Western Blotting去檢測各組細胞中漿細胞分化因子Blimpl和XBP1表達水平的影響；②分別應(yīng)用MEK Inhibitor U0126和SRC Inhibitor PP2PP2處理GC來源B-NHL細胞,Western Blotting去檢測抑制SRC和ERK通路對GCB-NHL細胞GC表型標志蛋白表達水平的影響；③應(yīng)用Western Blotting方法檢測c-myc表達沉默對GC來源B-NHL細胞GC表型標志蛋白表達的影響；④PCR法檢測SHP2表達沉默對GC來源B-NHL細胞中t(8；14)(q24；q32)轉(zhuǎn)位的影響。 結(jié)果①SHP2參與調(diào)控GC來源B-NHL細胞生物學(xué)行為：SHP2對IL-6活化的GC來源B-NHL細胞中的p-ERK和p-SRC磷酸化是必需的,而對IL-6活化的P-STAT3和p-AKT磷酸化程度沒有影響；SHP2通過其PTP酶活性影響IL-6刺激的p-ERK和p-SRC的活化；SHP2表達沉默抑制了IL-6促GC來源B-NHL細胞增殖的效應(yīng)；SHP2表達沉默還會導(dǎo)致GC來源B-NHL細胞GO-G1期延長,S-G2-M期縮短；但是SHP2表達沉默不會影響GC來源B-NHL細胞早期凋亡和晚期凋亡水平；無論有無IL-6刺激,干擾SHP2的表達,都會抑制GC來源B-NHL細胞的遷移。②SHP2參與維持GC來源B-NHL細胞的GC表型：SHP2表達沉默會導(dǎo)致GC來源B-NHL細胞中關(guān)鍵的GC表型蛋白Bcl6、Pax5、E2A、AICDA表達以及c-myc原癌基因蛋白表達減少；SHP2表達沉默抑制了Raji和Ramos細胞中t(8；14)(q24；q32)轉(zhuǎn)位頻率。③SHP2決定了IL-6誘導(dǎo)的GC來源B-NHL細胞的重分化方向：SHP2表達沉默的GC來源B-NHL細胞中漿細胞分化因子Blimp1、XBP1表達水平升高；流式細胞儀檢測發(fā)現(xiàn),IL-6刺激SHP2表達沉默的GC來源B-NHL細胞膜上的活化GCB細胞標志蛋白CD77表達水平降低,而漿細胞表型標志蛋白CD138的表達水平升高,說明SHP2表達沉默可以導(dǎo)致IL-6誘導(dǎo)GC來源B-NHL細胞向漿細胞方向分化。 結(jié)論SHP2參與調(diào)控GC來源B-NHL細胞的增值和遷移,但對其早期和晚期凋亡水平?jīng)]有影響。并且SHP2影響了GC源性B-NHL細胞GC表型標志蛋白的表達。SHP2是IL-6誘導(dǎo)的GC源性B-NHL細胞重分化的樞紐。
[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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R363

【參考文獻】

相關(guān)期刊論文 前1條

1 梁云,周韌,葉月芳,謝云,毛崢嶸;中國浙江地區(qū)漢族正常人群外周血細胞中BCL-2/IgH易位現(xiàn)象的初步研究[J];中華醫(yī)學(xué)遺傳學(xué)雜志;2005年05期

，

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