本文關(guān)鍵詞：Smad4的條件性缺失對(duì)T細(xì)胞活化的影響　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： TGF-β Smad4條件性缺失 T細(xì)胞活化 李斯特菌 CD8~+CTL

【摘要】：轉(zhuǎn)化生長(zhǎng)因子-β（Transforming growth factor-β，TGF-β)超家族是一類多功能的多肽，在維持T細(xì)胞免疫的過程中發(fā)揮重要的作用，能夠影響T細(xì)胞的發(fā)育、分化和增殖等各個(gè)環(huán)節(jié)。在哺乳動(dòng)物體內(nèi)，TGF-β亞類包括3種亞型，即TGF-β1、TGF-β2和TGF-β3。其中，TGF-β1在機(jī)體免疫系統(tǒng)中廣泛存在并且發(fā)揮著重要的調(diào)節(jié)作用，能夠調(diào)節(jié)免疫系統(tǒng)內(nèi)淋巴細(xì)胞及非淋巴細(xì)胞的活化及效應(yīng)細(xì)胞的功能。TGF-β1發(fā)揮其在細(xì)胞水平的功能是通過與其異質(zhì)性的復(fù)合物組成的TGF-β受體Ⅰ及受體Ⅱ介導(dǎo)的受體偶聯(lián)絲氨酸/蘇氨酸激酶進(jìn)行信號(hào)的轉(zhuǎn)導(dǎo)。Smad（Smaand Mad Homologue）蛋白家族是TGF-β受體的底物之一，目前認(rèn)為Smad蛋白家族包括八個(gè)成員：TGF-β通路中的R-Smad包括Smad2及Smad3；BMP通路中的R-Smad包括Smad1、Smad5及Smad8；Co-Smad即Smad4；兩個(gè)I-Smads包括Smad6和Smad7。 Smad4蛋白是TGF-β/Smad信號(hào)通路在細(xì)胞內(nèi)傳導(dǎo)過程中最重要的中介分子之一。在TGF-β/Smad信號(hào)轉(zhuǎn)導(dǎo)過程中，Smad2和Smad3能夠被其C端受體介導(dǎo)的磷酸化所激活。Smad2和Smad3經(jīng)過受體誘導(dǎo)的磷酸化活化之后，，能夠與Smad4形成雜聚體，轉(zhuǎn)導(dǎo)進(jìn)入細(xì)胞核內(nèi)。在細(xì)胞核內(nèi)該聚合體能夠與轉(zhuǎn)錄因子、轉(zhuǎn)錄因子活化蛋白及阻遏蛋白相結(jié)合，從而調(diào)節(jié)靶基因的轉(zhuǎn)錄。在過去的幾年里有大量的證據(jù)表明，接受TGF-β信號(hào)刺激后Smad4能夠與Smad2和Smad3形成Smad2/Smad2/Smad4、Smad3/Smad3/Smad4以及Smad2/Smad3/Smad4聚合體，并且認(rèn)為Smad4是TGF-β信號(hào)通路中唯一的Co-Smad而發(fā)揮著協(xié)助Smad2和Smad3進(jìn)入細(xì)胞核發(fā)揮調(diào)節(jié)基因轉(zhuǎn)錄的蛋白分子。然而，在2006年He等科學(xué)家發(fā)現(xiàn)了另外一種蛋白分子——轉(zhuǎn)錄調(diào)節(jié)因子1γ（Transcriptional IntermediaryFactor1γ，TIF1γ），能夠與Smad4競(jìng)爭(zhēng)結(jié)合磷酸化活化的Smad2、Smad3，介導(dǎo)該聚合體進(jìn)入細(xì)胞內(nèi)調(diào)節(jié)基因的轉(zhuǎn)錄。除了TGF-β/Smad信號(hào)通路，TGF-β還能夠激活非Smad信號(hào)通路，包括細(xì)胞外調(diào)節(jié)蛋白激酶(extracellular regulated proteinkinase, ERK)、c-Jun氨基端激酶（c-Jun N-terminal kinase，JNK）和p38激活的蛋白激酶信號(hào)通路。 盡管Smad4蛋白是TGF-β/Smad信號(hào)通路中一個(gè)重要的分子，而TGF-β在維持T細(xì)胞免疫過程中起著重要的作用，目前關(guān)于Smad4蛋白缺失對(duì)T細(xì)胞免疫影響的研究卻很少。我們的研究主要的目的就是利用Smad4條件性缺失小鼠模型，探索Smad4對(duì)于T細(xì)胞增殖、活化和效應(yīng)T細(xì)胞的分化過程是否有著與TGF-β同樣重要的作用。鑒于Smad4完全敲除的小鼠由于外胚層形成的缺陷在胚胎早期就會(huì)死亡，我們引進(jìn)了利用Cre-loxP系統(tǒng)的Smad4基因T細(xì)胞條件性敲除的小鼠（Smad4~(Cre/Co/Co)）作為動(dòng)物模型，Smad4條件基因打靶小鼠（Smad4Co/Co）作為對(duì)照。 1.研究目的和內(nèi)容 1) Smad4條件性缺失對(duì)T細(xì)胞發(fā)育和增殖的影響 TGF-β最早被發(fā)現(xiàn)的功能是它能夠?qū)⒓?xì)胞周期阻滯在G1期，從而發(fā)揮它對(duì)各種細(xì)胞的抑制作用，其中也包括了免疫系統(tǒng)的T細(xì)胞。而有研究認(rèn)為，Smad4蛋白在TGF-β發(fā)揮對(duì)細(xì)胞周期的阻滯功能過程中處于中樞地位，Smad4通過與R-Smad聚合體在細(xì)胞核內(nèi)抑制有絲分裂信號(hào)的轉(zhuǎn)導(dǎo)，調(diào)節(jié)細(xì)胞周期。因此，我們的研究目的之一就是探討Smad4對(duì)T細(xì)胞發(fā)育和增殖的影響，以及Smad4條件性缺失小鼠模型是否會(huì)造成T細(xì)胞發(fā)育不完全和對(duì)T增殖抑制作用的消失。 2) Smad4條件性缺失對(duì)T細(xì)胞活化和分化的影響 研究表明，TGF-β通過促進(jìn)CD4~+CD25~+Treg細(xì)胞的發(fā)育和分化來發(fā)揮其抑制其他T細(xì)胞的活化及分化的功能，CD4~+CD25~+Treg能夠分泌TGF-β1或表達(dá)膜結(jié)合型的TGF-β1繼而抑制其它免疫細(xì)胞的活化和分化。因此，我們的研究?jī)?nèi)容之一是觀察Smad4條件性缺失小鼠模型T細(xì)胞活化和分化的過程是否與對(duì)照組小鼠有著顯著性的差異。 3) Smad4條件性缺失對(duì)CD8~+T細(xì)胞活化和增殖的影響 在研究過程中我們發(fā)現(xiàn)，年老的Smad4條件性缺失小鼠CD8~+T細(xì)胞的活化能力有缺陷，而其增殖能力與對(duì)照組小鼠相比沒有明顯的差別。在接下來的工和作中我們利用尾靜脈注射李斯特菌的方法建立動(dòng)物模型，在用李斯特菌免疫0天、5天、7天后研究活化的CD8~+CD44~(hi)T細(xì)胞比例Smad4~(Cre/Co/Co)和Smad4~（Co/Co）小鼠之間有無顯著性差別。 4) Smad4條件性缺失對(duì)CD8~+效應(yīng)CTL的影響 目前，關(guān)于Smad4與抗原特異性CD8~+T細(xì)胞的研究還未見報(bào)道，而CD8~+T細(xì)胞在早期李斯特菌感染的清除過程中起著重要的作用。因此，這部分我們的研究?jī)?nèi)容主要是Smad4條件性缺失對(duì)CD8~+效應(yīng)CTL的影響及其機(jī)制。 2.研究方法 1) PCR鑒定Smad4基因型 小鼠尾部組織提取基因組DNA作為模板，PCR反應(yīng)條件：94°C,1分鐘；68°C,1分鐘20秒；72°C,30秒；94°C,2分鐘；94°C,30秒，60°C,30秒，72°C,1分鐘，30個(gè)循環(huán)；72°C,8分鐘；4°C，保存。 1) Immunoblotting Analysis M2buffer裂解，蛋白樣品經(jīng)SDS-PAGE蛋白電泳、蛋白轉(zhuǎn)印、封閉、一抗孵育、二抗孵育和辣根過氧化物酶-ECL法檢測(cè)其中Smad4蛋白含量。 2)流式細(xì)胞術(shù) 用熒光標(biāo)記的單抗對(duì)待檢測(cè)表面Marker進(jìn)行標(biāo)記，1%多聚甲醛固定，上機(jī)FACS檢測(cè)其表達(dá)水平。 對(duì)細(xì)胞內(nèi)細(xì)胞因子檢測(cè)，則需要將待檢細(xì)胞用PMA/Ino/BFA處理4h后，先對(duì)表面Marker進(jìn)行標(biāo)記，用透化液處理后，用熒光抗體標(biāo)記胞內(nèi)因子，上機(jī)FACS檢測(cè)其表達(dá)水平。 3)統(tǒng)計(jì) 組間差異比較使用t檢驗(yàn),以P0.05為差異有統(tǒng)計(jì)學(xué)意義。 4)其它 本實(shí)驗(yàn)使用了細(xì)胞計(jì)數(shù)、FITC-標(biāo)記的Annexin V、CFSE以及BrdU摻入等方法檢測(cè)細(xì)胞的增殖能力。 3.研究結(jié)果和結(jié)論 1)基因型鑒定 我們用DNA鑒定、Western-Blot和熒光抗體染色的方法證實(shí)了引進(jìn)的確實(shí)是Smad4基因T細(xì)胞條件性敲除小鼠。 2) Smad4條件性缺失對(duì)T細(xì)胞發(fā)育和分化的影響 我們的實(shí)驗(yàn)沒有發(fā)現(xiàn)Smad4基因的T細(xì)胞條件性敲除對(duì)Treg細(xì)胞、CD4~+T細(xì)胞、CD8~+T細(xì)胞發(fā)育有顯著的影響，同樣的，我們發(fā)現(xiàn)Smad4基因條件性缺失對(duì)Th1/Th2/Th17細(xì)胞的分化也沒有顯著性的影響。 3) Smad4條件性缺失對(duì)T細(xì)胞增殖的影響 Smad4條件性缺失對(duì)幼年小鼠CD4~+和CD8~+T細(xì)胞數(shù)目沒有影響，對(duì)年老小鼠脾細(xì)胞總數(shù)和CD4~+T細(xì)胞數(shù)目也沒有影響，而我們發(fā)現(xiàn)Smad4~(Cre/Co/Co)與Smad4Co/Co相比CD8~+T細(xì)胞數(shù)卻有著顯著性的差別，產(chǎn)生這一現(xiàn)象的原因尚不明確。 4) Smad4條件性缺失對(duì)T細(xì)胞活化的影響 對(duì)新生小鼠脾細(xì)胞和年老（52周齡）小鼠的脾、淋巴結(jié)細(xì)胞的分析，我們發(fā)現(xiàn)Smad4Co/Co與Smad4~(Cre/Co/Co)相比CD4~+CD44hi細(xì)胞比例均沒有顯著性差異。而在對(duì)年老小鼠脾細(xì)胞進(jìn)行分析時(shí)發(fā)現(xiàn)，Smad4~(Cre/Co/Co)小鼠脾細(xì)胞中CD8~+CD44hi細(xì)胞低于對(duì)照組小鼠，說明Smad4條件性缺失對(duì)CD8~+T細(xì)胞的活化有一定影響。 5) Smad4條件性缺失對(duì)胃腸道上皮穩(wěn)態(tài)的影響 曾有研究發(fā)現(xiàn)，用Lck-Cre的方法T細(xì)胞條件性敲除Smad4基因小鼠在年老時(shí)會(huì)出現(xiàn)胃腸道多發(fā)性的腫瘤；而另有研究表明，以Lck-Cre或CD4-Cre技術(shù)T細(xì)胞特異性Smad4條件性敲除小鼠在年老時(shí)只會(huì)在十二指腸部位出現(xiàn)上皮細(xì)胞性腫瘤。在我們的研究中，對(duì)52周齡的Smad4條件性敲除小鼠肝、十二指腸和結(jié)腸病理切片，HE染色分析，并未發(fā)現(xiàn)有胃腸道腫瘤的發(fā)生。我們推測(cè)，Smad4基因T細(xì)胞條件性缺失不一定會(huì)造成胃腸道的腫瘤，Smad4基因T細(xì)胞條件性缺失造成胃腸道的腫瘤的發(fā)生與否可能與基因型及飼養(yǎng)環(huán)境有著密切的關(guān)系。 6) Smad4條件性缺失對(duì)CD8~+效應(yīng)CTL的影響 我們使用李斯特菌感染小鼠模型誘導(dǎo)抗原特異性CD8~+T細(xì)胞免疫應(yīng)答，探討Smad4基因T細(xì)胞條件性敲除對(duì)CD8~+T細(xì)胞活化的影響。在研究過程中我們發(fā)現(xiàn)，Smad4基因條件性缺失會(huì)導(dǎo)致CD8~+效應(yīng)CTL細(xì)胞活化明顯滯后，李斯特菌尾靜脈注射5天時(shí)Smad4~(Cre/Co/Co)小鼠脾細(xì)胞中CD8~+CD44hi細(xì)胞低于對(duì)照組小鼠，而在7天時(shí)這種顯著性差異消失。我們?cè)谘芯恐�，發(fā)現(xiàn)李斯特菌感染Smad4~(Cre/Co/Co)小鼠脾細(xì)胞中CD8~+T細(xì)胞的Gran B、IFN-γ等殺傷Marker水平要遠(yuǎn)遠(yuǎn)低于對(duì)照組小鼠，同時(shí)Smad4~(Cre/Co/Co)小鼠脾細(xì)胞中短期效應(yīng)細(xì)胞CD127(low)/KLRG1(hi) CD8~+T細(xì)胞的比例也低于對(duì)照組小鼠，說明Smad4基因條件性缺失對(duì)CD8~+效應(yīng)CTL細(xì)胞的殺傷功能有一定的影響。 7) Smad4條件性缺失小鼠影響CD8~+CTL活化和殺傷效應(yīng)的機(jī)制 我們?cè)趯?shí)驗(yàn)中發(fā)現(xiàn)，Smad4基因的T細(xì)胞條件性缺失對(duì)抗原特異性CD8~+T細(xì)胞表面的IL-2Rα鏈、β鏈、γ鏈以及IL-15Rα鏈表達(dá)并沒有顯著的影響，因此，李斯特菌誘導(dǎo)的Smad4基因T細(xì)胞條件性敲除小鼠(Smad4~(Cre/Co/Co))脾細(xì)胞中CD8~+T細(xì)胞活化滯后并不是由于其細(xì)胞表面IL-2R及IL-15R的異常表達(dá)所造成的。研究中發(fā)現(xiàn)Smad4~(Cre/Co/Co)小鼠脾細(xì)胞中CD43~+CD27~+細(xì)胞的比例明顯低于對(duì)照組小鼠，而CD43~+CD27~+細(xì)胞對(duì)CD8~+效應(yīng)CTL活化和功能維持有重要的作用，我們推測(cè)這是造成其CD8~+CTL活化滯后和殺傷效應(yīng)缺陷的原因之一。在體外實(shí)驗(yàn)中，我們用體外增殖分化實(shí)驗(yàn)排除了Smad4條件性缺失造成的抗原特異性CD8~+T細(xì)胞之間的差別是由于CD8~+T細(xì)胞內(nèi)在的缺陷的影響。而接下來的實(shí)驗(yàn)顯示，Smad4~(Cre/Co/Co)小鼠脾細(xì)胞中CD4~+T細(xì)胞表面的CD40L表達(dá)低于對(duì)照組小鼠，CD40與CD40L的相互作用能夠誘導(dǎo)CD80/CD86共刺激信號(hào)的表達(dá)，繼而活化pre-CTL。因此，我們認(rèn)為Smad4~(Cre/Co/Co)小鼠脾細(xì)胞中CD4~+T細(xì)胞表面的CD40L低表達(dá)是造成CD8~+CTL活化滯后的另外一個(gè)原因。 4.本文創(chuàng)新點(diǎn) 關(guān)于Smad4對(duì)CTL細(xì)胞分化有何影響的研究目前還未見報(bào)道，本研究利用Cre重組酶系統(tǒng)Smad4基因T細(xì)胞特異性條件性敲除小鼠對(duì)Smad4與CTL細(xì)胞分化的關(guān)系進(jìn)行了探討，發(fā)現(xiàn)Smad4條件性缺失影響CD8~+T細(xì)胞的活化；同時(shí)，我們用李斯特菌感染的方式開展了Smad4條件性缺失對(duì)影響CD8~+CTL活化及殺傷功能影響的研究，并初步探討了造成這種影響可能的機(jī)制。
[Abstract]:Transforming growth factor beta (Transforming growth factor- TGF- beta, beta) superfamily is a kind of multifunctional polypeptide, play an important role in maintaining T cell immunity, can affect T cell development, differentiation and proliferation of each link. In mammals, TGF- beta subtype includes 3 subtypes TGF- TGF-, beta 1, beta 2 and beta 3. TGF-, TGF- beta 1 in the immune system widely exists and plays an important role in the immune system, can regulate lymphocyte activation and effector cells and non lymphocyte function of.TGF- beta 1 play at the cellular level is the function of serine / receptor coupling TGF- beta receptor 1 and receptor mediated composition of the composites through its heterogeneity by threonine kinase signal transduction of.Smad (Smaand Mad Homologue) protein family is one of the substrates of TGF- receptor, the Smad protein family It includes eight members: R-Smad in the TGF- beta pathway, including Smad2 and Smad3; R-Smad in the BMP pathway includes Smad1, Smad5 and Smad8; Co-Smad is Smad4; the two one includes "he he".
Smad4 protein is one of the most important intermediary molecules of TGF- beta signaling pathway of /Smad in cells in the process. In the TGF- beta /Smad signal transduction process, Smad2 and Smad3 can be the end of the C receptor mediated phosphorylation and activation of.Smad2 receptor induced phosphorylation of Smad3 after activation, can form heteromers with Smad4 transduced into the cell nucleus. The nucleus in the polymer with transcription factor, transcription factor activation and protein repressor protein combination, thereby regulating the transcription of target genes. In the past few years there is plenty of evidence that TGF- beta signal after stimulation with Smad2 and Smad4 can form Smad2/Smad2/Smad4 Smad3, and Smad3/Smad3/Smad4 Smad2/Smad3/Smad4 polymer, and that the Smad4 is the only Co-Smad TGF- beta signaling pathway and plays to assist Smad2 and Smad3 to enter the nucleus play in regulating gene transcription protein . however, another protein molecule transcription factor 1 gamma was found in 2006 He (Transcriptional scientist IntermediaryFactor1 y, TIF1 y), to phosphorylation and activation of Smad2, combined with the Smad4 Smad3 competition, the polymer mediated transcription into cells regulated gene. In addition to TGF- beta /Smad signaling pathway. TGF- can also activate non beta Smad signaling pathway, including extracellular regulated protein kinase (extracellular regulated, proteinkinase, ERK), c-Jun N-terminal kinase (c-Jun N-terminal, kinase, JNK) and p38 activated protein kinase signaling pathway.
Although the Smad4 protein is an important molecule of TGF- beta /Smad signaling pathway, and TGF- beta in maintaining T cell immunity plays an important role in the process, the current research on the effect of Smad4 deficiency on T cell immunity is few. The main purpose of our research is to use a Smad4 deficiency mouse model, explore Smad4 T for cell proliferation, differentiation and activation of effector T cells is to play the same important role. In view of TGF- beta Smad4 complete knockout mice because the ectoderm will defect in early embryonic death, we introduce the Cre-loxP system using Smad4 cell T gene conditional knockout mice (Smad4~ (Cre/Co/Co)) as the animal model, the Smad4 conditional gene targeting mice (Smad4Co/Co) as control.
1. the purpose and content of the study
1) the effect of Smad4 conditional deletion on the development and proliferation of T cells
The first discovery of TGF- beta function is capable of cell cycle arrest in G1 phase, in order to exert its inhibitory effect on various cells, including T cells of the immune system. One study suggests that Smad4 proteins play in the central position of block function on cell cycle in TGF- beta, with Smad4 R-Smad polymer in the nucleus inhibits mitogenic signal transduction, regulation of cell cycle. Therefore, one of the purpose of our research is to investigate the effect of Smad4 on T cell growth and proliferation, and the disappearance of Smad4 conditional mice lacking T cells will cause incomplete growth and inhibit the proliferation of T.
2) the effect of Smad4 conditional deletion on the activation and differentiation of T cells
Research shows that TGF- beta by promoting CD4~+CD25~+Treg cell development and differentiation to inhibit other T cell activation and differentiation, CD4~+CD25~+Treg can secrete TGF- beta 1 expression or membrane-bound TGF- beta 1 and inhibit other immune cell activation and differentiation. Therefore, one of our research content is to observe the conditional deletion of Smad4 a mouse model of T cell activation and differentiation process and the mice of control group have significant differences.
3) the effect of Smad4 conditional deletion on the activation and proliferation of CD8~+T cells
In the course of the study we found that the activation ability of old Smad4 conditional deletion of mouse CD8~+T cells are defective, and the ability of proliferation compared with control mice had no significant difference. In the next work and we use the method of intravenous injection of bacteria Lester animal model, the Lester immunized 0 days. 5 days, 7 days after the activation of CD8~+CD44~ (HI) T cell ratio (Cre/Co/Co) and Smad4~ Smad4~ (Co/Co) had no significant difference between the mice.
4) the effect of Smad4 conditional deletion on the CD8~+ effect CTL
At present, the research on Smad4 and antigen specific CD8~+T cells has not been reported, and CD8~+T cells in the elimination process of early Lester infection plays an important role. Therefore, we study the content of this part is Smad4 conditional deletion of CD8~+ effect of CTL and its mechanism.
2. research methods
1) identification of Smad4 genotypes by PCR
Mice tail tissue extract genomic DNA as template, PCR reaction conditions: 94 degrees C, 1 minutes; 68 degrees C, 1 minutes 20 seconds; 72 degrees C, 30 seconds; 94 degrees C, 2 minutes; 94 degrees C, 30 seconds, 30 degrees C, second second, 94 degrees, C, minutes, cycle, C, C, minutes, C.
1) Immunoblotting Analysis
M2buffer lysis and protein samples were detected by SDS-PAGE protein electrophoresis, protein transfer, blocking, one antibody incubation, two anti incubation and horseradish peroxidase -ECL method to detect the content of Smad4 protein.
2) flow cytometry
The fluorescent labeled monoclonal antibody was used to detect the surface Marker, 1% polyoxymethylene was fixed, and the expression level was detected by FACS.
The detection of cytokines in cells requires the cells to be treated with PMA/Ino/BFA after 4H. Then the surface Marker is labeled, and then the cytokines are labeled with fluorescent antibody. The expression level is detected by FACS.
3) statistics
The difference between the groups was compared with the t test, and the difference was statistically significant with the difference of P0.05.
4) other
In this experiment, cell counts, FITC- labeled Annexin V, CFSE and BrdU incorporation were used to detect cell proliferation.
3. the results and conclusions of the study
1) genotyping
We identified by DNA, Western-Blot and fluorescent antibody staining confirmed that the imported Smad4 gene T cell conditioned knockout mice were indeed introduced.
2) the effect of Smad4 conditional deletion on the development and differentiation of T cells
Our experiments did not find that conditional knockout of Smad4 gene T cells had significant effects on the development of Treg cells, CD4~+T cells and CD8~+T cells. Similarly, we found that conditional deletion of Smad4 gene had no significant effect on Th1/Th2/Th17 cell differentiation.
3) the effect of Smad4 conditional deletion on the proliferation of T cells
Conditional deletion of Smad4 had no effect on CD4~+ and CD8~+T cell number in juvenile mice, spleen cells had no effect on the total number and the number of CD4~+T cells in old mice, we found that Smad4~ (Cre/Co/Co) compared with Smad4Co/Co CD8~+T cells had no significant difference, the reason of this phenomenon is not clear.
4) the effect of Smad4 conditional deletion on the activation of T cells
The spleen cells of newborn mice (52 weeks old) and old mice spleen cells of lymph node, we found that Smad4Co/Co and Smad4~ (Cre/Co/Co) compared to the proportion of CD4~+CD44hi cells were not significantly different. And found in the analysis of the old mice spleen cells, Smad4~ (Cre /Co/Co) of CD8~+CD44hi cells was lower than the control group in mice spleen cells, indicating that Smad4 conditional deletion of CD8~+T cell activation has certain effect.
5) the effect of Smad4 conditionality loss on the homeostasis of gastrointestinal epithelium
Studies have found that by using the method of Lck-Cre T cells in Smad4 conditional knockout mice appear gastrointestinal multiple tumors in old age; and another study showed that Lck-Cre or CD4-Cre T cell specific Smad4 knockout mice in old age will only appear in the epithelial cells of the tumor in the duodenum. In our study, 52 week old Smad4 knockout mice liver, duodenum and colon pathological sections, HE staining analysis found no gastrointestinal tumor. We speculate that the Smad4 gene of T cell conditional deletion does not necessarily cause gastrointestinal tumors, the Smad4 gene of T cells caused by conditional deletion of the gastrointestinal tract the tumor may close relationship with genotype and feeding environment there.
6) the effect of Smad4 conditional deletion on the CD8~+ effect CTL
We use induction of antigen specific CD8~+T cell immune response Lester bacteria infection mouse model of Smad4 gene conditional knockout T cell activation in CD8~+T cells. During the study, we found that Smad4 gene conditional deletion causes activation of CD8~+ effector CTL cells obviously lags behind, Lester bacteria 5 days intravenous injection of Smad4~ (Cre/Co/Co) CD8~+CD44hi cells were lower than the control group of mice spleen cells of mice, and that on the 7 day difference disappeared. In our study, Lester found Smad4~ infection (Cre/Co/Co) in CD8~+T cells of mouse spleen cells in Gran B, IFN- y killer Marker levels are much lower than the control mice, while Smad4~ (Cre/Co/Co) short term the effect of CD127 cells in mouse spleen cells (low) /KLRG1 (HI) of CD8~+T cells was lower than in control mice, indicating that Smad4 gene conditional missing on CD8~+ effect CTL The killing function of the cell has a certain influence.
7) the mechanism of Smad4 conditioned loss of mice affects the activation and killing effect of CD8~+CTL
In our experiments, the IL-2R alpha chain, Smad4 gene deletion of T cells conditioned antigen-specific CD8~+T cell surface beta chain, gamma chain and IL-15R alpha chain expression and no significant effect, therefore, Lester bacteria induced Smad4 cell T gene conditional knockout mice (Smad4 ~ (Cre/Co/Co)) of spleen cells in the activation of CD8~+T cells is not lag due to abnormal cell surface expression of IL-2R and IL-15R caused by Smad4~. The study found (Cre/Co/Co) CD43~+CD27~+ cells of mouse spleen cells was significantly lower than the control mice, and the effect of CTL on CD8~+ CD43~+CD27~+ cell activation and function maintenance plays an important role, we speculate that this is caused by one of the the activation of CD8~+CTL lag and the killing effect of defects. In vitro experiment, we exclude the Smad4 conditional deletion caused by antigen specific CD8~+T cell proliferation and differentiation in vitro experiment The difference between the two is due to the effect of the intrinsic defects of the CD8~+T cells. And the subsequent experiments showed that Smad4~ (Cre/Co/Co) lower than that of the control group of mice CD4~+T cell surface expression of CD40L in mouse spleen cells, the interaction between CD40 and CD40L can induce CD80/CD86 costimulatory signal expression and activation of pre-CTL., therefore, we believe that the Smad4~ (Cre/Co/Co) in mice spleen cell surface CD4~+T low expression of CD40L is caused by another reason CD8~+CTL activation lag.
4. the innovation point of this article
There is no report on the effect of Smad4 on the differentiation of CTL cells, this study uses Cre recombinase system Smad4 gene T cell specific knockout of Smad4 and the differentiation of CTL cells in mice was discussed and found Smad4 conditional deletion affect CD8~+T cell activation; at the same time, we carried out the conditional deletion of Smad4 influence of CD8~+CTL effect on the activation and cytotoxicity of Lester with bacteria infection, and to explore the mechanism of this effect may be caused.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

【共引文獻(xiàn)】

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