發(fā)布時(shí)間：2018-01-02 01:25

  本文關(guān)鍵詞：Notch信號(hào)通路與淋巴細(xì)胞發(fā)育的相關(guān)研究　出處：《第四軍醫(yī)大學(xué)》2007年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： Notch/RBP-J信號(hào)通路 T細(xì)胞發(fā)育 條件性剔除 邊緣帶B細(xì)胞 濾泡B細(xì)胞 基因芯片 CD36 CXCR7

【摘要】： T,B淋巴細(xì)胞是機(jī)體獲得性免疫應(yīng)答的重要組成部分,她們的正常發(fā)育是機(jī)體對(duì)外來微生物入侵產(chǎn)生有效免疫應(yīng)答的基礎(chǔ),其異常的發(fā)育則是自身免疫性疾病的基本病因之一。解開他們的發(fā)育過程及分子機(jī)制之謎一直是免疫學(xué)家孜孜以求的目標(biāo)。其發(fā)育過程是細(xì)胞分化,增殖和凋亡等多種基本生命現(xiàn)象協(xié)同作用的結(jié)果,涉及多種信號(hào)轉(zhuǎn)導(dǎo)通路的參與。 Notch信號(hào)途徑是通過膜表面受體介導(dǎo)細(xì)胞與細(xì)胞間相互作用,來調(diào)節(jié)前體細(xì)胞和干細(xì)胞的分化方向,從而調(diào)節(jié)相鄰細(xì)胞間分化的基本信號(hào)途徑之一。它在進(jìn)化中高度保守,在從線蟲到人,從胚胎發(fā)育到成年個(gè)體的多種系統(tǒng)中都發(fā)揮著重要作用。Notch信號(hào)通路通過調(diào)控前體細(xì)胞的分化方向,在淋巴細(xì)胞發(fā)育中同樣發(fā)揮著重要作用。這已在淋巴細(xì)胞發(fā)育的多個(gè)環(huán)節(jié)如T/B細(xì)胞的分化、脾臟中邊緣帶B細(xì)胞和濾泡B細(xì)胞的分化中得到證實(shí)。但Notch信號(hào)途徑在淋巴細(xì)胞發(fā)育其他環(huán)節(jié)中的作用并不清楚,而在已知可發(fā)揮作用的環(huán)節(jié)中的分子機(jī)制也為未解之謎。值得注意的是,哺乳類的四種Notch受體都需要轉(zhuǎn)錄因子RBP-J介導(dǎo)其轉(zhuǎn)錄激活活性,將其敲除可以阻斷4種Notch受體的信號(hào),從而獲得完全的Notch信號(hào)通路的loss of function的模型。 為進(jìn)一步研究Notch信號(hào)途徑在T淋巴細(xì)胞發(fā)育和功能中的作用,基于組織特異性剔除Cre-LoxP系統(tǒng)的原理,我們建立了在T細(xì)胞中特異性剔除RBP-J的條件性剔除小鼠—Lck-Cre x RBP-Jflox/flox小鼠,并對(duì)它們進(jìn)行了初步的表型分析。為此,我們構(gòu)建了在T細(xì)胞中特異性表達(dá)Cre重組酶的轉(zhuǎn)基因表達(dá)載體pLck-Cre,通過顯微注射方法,建立了共20系Lck-Cre轉(zhuǎn)基因小鼠。我們將這些小鼠分別與本室擁有的RBP-J條件性剔除小鼠進(jìn)行交配,并采用Southern Blot對(duì)不同Lck-Cre來源的Lck-Cre x RBP-Jflox/+雜合子小鼠的胸腺細(xì)胞中RBP-J的剔除效率進(jìn)行了鑒定,最終鑒定得到胸腺細(xì)胞中RBP-J被完全剔除的兩系小鼠-M5 Lck-Cre,M19 Lck-Cre。我們進(jìn)一步將這兩系小鼠來源的基因型為Lck-Cre x RBP-Jflox/+的雜合子小鼠進(jìn)行交配,以獲得Lck-Cre x RBP-Jflox/flox小鼠進(jìn)行表型分析。 對(duì)M19 Lck-Cre x RBP-Jflox/flox小鼠的表型分析結(jié)果顯示,剔除小鼠胸腺細(xì)胞中αβT細(xì)胞發(fā)育和γδT細(xì)胞發(fā)育均未見異常。AnnexinV染色顯示剔除小鼠胸腺中DP細(xì)胞(CD4+CD8+)的凋亡增加,提示了Notch/RBP-J信號(hào)通路在胸腺細(xì)胞中的抗凋亡作用。另發(fā)現(xiàn)脾臟中RBP-J缺失的CD4 T細(xì)胞的體外增殖能力減弱,提示Notch信號(hào)通路可促進(jìn)外周CD4 T細(xì)胞的增殖,而且此過程是RBP-J依賴的。M5 Lck-Cre x RBP-Jflox/flox小鼠的表型分析結(jié)果顯示,純合子小鼠胸腺中αβT細(xì)胞發(fā)育受阻,但γδT細(xì)胞的發(fā)育未見異常,這提示Notch/RBP-J信號(hào)通路對(duì)αβT細(xì)胞的早期發(fā)育具有促進(jìn)作用。 本課題組前期的研究發(fā)現(xiàn)Notch/RBP-J信號(hào)通路還決定了脾臟中B細(xì)胞的分化命運(yùn),可促進(jìn)邊緣帶B細(xì)胞的發(fā)育而抑制前體細(xì)胞向?yàn)V泡B細(xì)胞的分化,但下游靶基因并不清楚。為此,我們對(duì)脾臟中這兩群來自于共同前體細(xì)胞的B細(xì)胞亞群-邊緣帶B細(xì)胞和濾泡B細(xì)胞進(jìn)行了基因表達(dá)譜的對(duì)比分析,結(jié)果顯示,共有1226個(gè)基因在濾泡B細(xì)胞高表達(dá),而1754個(gè)基因在邊緣帶B細(xì)胞中高表達(dá)。我們對(duì)所有差異表達(dá)的基因進(jìn)行了注釋、功能分類以及進(jìn)一步的分析,發(fā)現(xiàn)Notch信號(hào)通路的一系列分子包括膜表面受體Notch2,胞漿內(nèi)的調(diào)節(jié)蛋白Deltex及負(fù)調(diào)控分子MINT,在兩群B細(xì)胞中均呈差異表達(dá)。與以往的分析一致,Notch信號(hào)通路表現(xiàn)為在邊緣帶B細(xì)胞中的高度活化。另外,已知Hes家族分子是Notch信號(hào)通路的主要的下游靶基因,芯片結(jié)果提示Hes家族成員分子之一Hes5只表達(dá)于邊緣帶B細(xì)胞,在濾泡B細(xì)胞并不表達(dá),這提示我們Hes5很可能是Notch/RBP-J信號(hào)通路調(diào)控外周B細(xì)胞分化的直接的下游靶基因。 隨后,我們圍繞兩群B細(xì)胞差異表達(dá)的膜表面分子和受體展開了一些工作。首先,基于芯片結(jié)果的提示和后續(xù)的流式細(xì)胞分析,我們發(fā)現(xiàn)CD36,一種B型清道夫受體,在邊緣帶B細(xì)胞中高表達(dá),而在濾泡B細(xì)胞中幾乎不表達(dá);CD68,CD49e在濾泡B細(xì)胞中不表達(dá),在邊緣帶B細(xì)胞中以較低水平表達(dá);CD44在兩群B細(xì)胞中都表達(dá),在邊緣帶B細(xì)胞中的表達(dá)水平高于濾泡B細(xì)胞。其中CD36因?yàn)槠涮禺惖谋磉_(dá)模式,可以作為邊緣帶B細(xì)胞的新的表面標(biāo)志。CD36參與脂肪代謝,參與巨噬細(xì)胞對(duì)P. falciparum感染的紅細(xì)胞,凋亡的單核細(xì)胞和粒細(xì)胞的吞噬,它在邊緣帶B細(xì)胞中的特異表達(dá)可能與這群B細(xì)胞在先天性免疫應(yīng)答中對(duì)血源播散性病原體的清除有關(guān)。 另外,我們發(fā)現(xiàn)兩群B細(xì)胞呈現(xiàn)多種細(xì)胞因子受體的差異表達(dá),這與兩群B細(xì)胞對(duì)細(xì)胞因子的差異應(yīng)答有關(guān)。其中,與在對(duì)胸腺依賴性抗原應(yīng)答的作用相一致,濾泡B細(xì)胞高表達(dá)CD124分子;而半定量RT-PCR的結(jié)果顯示邊緣帶B細(xì)胞具有較高水平的CD131的mRNA,可能與邊緣帶B細(xì)胞受到刺激后的快速活化有關(guān)。 最后,芯片結(jié)果提示趨化因子受體CXCR7在兩群B細(xì)胞中呈差異表達(dá)。為進(jìn)一步闡明該受體在介導(dǎo)邊緣帶B細(xì)胞定位中的作用,我們制備了抗趨化因子受體CXCR7的抗血清,發(fā)現(xiàn)CXCR7在骨髓,脾臟,淋巴結(jié),腹腔等多種淋巴器官的B細(xì)胞上均有表達(dá),但在T細(xì)胞表面沒有表達(dá)。在脾臟中,在新生B細(xì)胞的表面沒有表達(dá),在濾泡B細(xì)胞和邊緣帶B細(xì)胞中均有表達(dá),在后者中的表達(dá)較高于前者。CXCR7在腹腔中的兩群B細(xì)胞中呈現(xiàn)差異性表達(dá),在B1細(xì)胞中的表達(dá)高于在B2細(xì)胞中的表達(dá),可能和B1細(xì)胞的特殊定位以及在天然免疫應(yīng)答中的作用有關(guān)。 綜上,我們建立了在T細(xì)胞中特異性剔除Notch信號(hào)途徑關(guān)鍵轉(zhuǎn)錄因子RBP-J的條件性剔除小鼠,在此基礎(chǔ)上發(fā)現(xiàn)Notch/RBP-J信號(hào)通路在體內(nèi)可抑制胸腺細(xì)胞的凋亡,促進(jìn)外周CD4 T細(xì)胞的增殖,這些研究進(jìn)一步揭示了該信號(hào)通路的體內(nèi)功能。另外,我們對(duì)濾泡B細(xì)胞和邊緣帶B細(xì)胞進(jìn)行了基因表達(dá)譜的對(duì)比分析,進(jìn)一步的注釋分析提示Hes家族成員分子之一Hes5很可能是Notch/RBP-J信號(hào)通路調(diào)控外周B細(xì)胞分化的直接的下游靶基因。后續(xù)的工作鑒定出CD36,一種B型清道夫受體,可作為邊緣帶B細(xì)胞的新的表面標(biāo)志。我們還發(fā)現(xiàn)了趨化因子受體CXCR7在脾臟中濾泡B細(xì)胞和邊緣帶B細(xì)胞及腹腔中的B1細(xì)胞和B2細(xì)胞中的差異表達(dá)。這些發(fā)現(xiàn)在一定程度上豐富了外周B淋巴細(xì)胞發(fā)育的基本理論,也為深入研究Notch/RBP-J信號(hào)通路決定外周B細(xì)胞分化的分子機(jī)制奠定了基礎(chǔ)。
[Abstract]:T , B lymphocytes are an important part of the acquired immune response of the organism . Their normal development is the basis of the effective immune response of the organism invasion of the organism . The abnormal development is one of the basic causes of autoimmune diseases . The development process is the result of the synergistic effect of various basic life phenomena , such as cell differentiation , proliferation and apoptosis , and involves the participation of various signal transduction pathways . Notch signaling pathway plays an important role in the differentiation of T / B cells and differentiation of B cells and follicular B cells in the development of lymphocytes . The Notch signaling pathway plays an important role in the differentiation of T / B cells and the differentiation of B cells and follicular B cells in the development of lymphocytes . In order to further study the role of Notch signaling pathway in the development and function of T lymphocytes , we established a transgenic expression vector pLck - cre , which specifically excluded the cells from the T cells . The results showed that the growth of 偽 - 尾 - T cells and the growth of 緯未T cells were not abnormal in the thymus cells of mice . The results showed that the proliferation of 偽 - 尾 - T cells in the thymus of mice was inhibited by AnnexinV staining . In this study , we found that the signal pathway of Notch signaling pathway also determines the differentiation of B cells in the spleen . The results show that there are 1226 genes which are highly expressed in B cells in the marginal zone , but the downstream target gene is not clear . In addition , we find that the Notch signal pathway is a primary downstream target gene in the marginal band B cells . At first , we found that CD36 , a B - type scavenger receptor , was highly expressed in B - cells of the marginal zone and expressed in B - cells at lower levels . CD68 and CD49e could be used as a new surface marker for B - cells in the marginal zone . In addition , we found that two groups of B - cells exhibit differential expression of multiple cytokine receptors , which is related to the differential response of two groups of B - cells to cytokines . The results of semi - quantitative RT - PCR show that the edge band B cells have a higher level of CD131 mRNA and may be associated with rapid activation of the edge band B cells after stimulation . In order to further clarify the role of the receptor in mediating the localization of B - cell , we have prepared an anti - chemokine receptor - 1 , which has not been expressed in B - cells of bone marrow , spleen , lymph node , abdominal cavity and so on , but the expression in the latter is higher than that of the former .





緇間笂,鎴戜滑寤虹珛浜嗗湪T緇嗚優(yōu)涓壒寮傛，

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