Smad4的条件性缺失对T细胞活化的影响

发布时间：2018-01-02 00:16

本文关键词：Smad4的条件性缺失对T细胞活化的影响　出处：《中国人民解放军军事医学科学院》2012年硕士论文　论文类型：学位论文

【摘要】：转化生长因子-β（Transforming growth factor-β，TGF-β)超家族是一类多功能的多肽，在维持T细胞免疫的过程中发挥重要的作用，能够影响T细胞的发育、分化和增殖等各个环节。在哺乳动物体内，TGF-β亚类包括3种亚型，即TGF-β1、TGF-β2和TGF-β3。其中，TGF-β1在机体免疫系统中广泛存在并且发挥着重要的调节作用，能够调节免疫系统内淋巴细胞及非淋巴细胞的活化及效应细胞的功能。TGF-β1发挥其在细胞水平的功能是通过与其异质性的复合物组成的TGF-β受体Ⅰ及受体Ⅱ介导的受体偶联丝氨酸/苏氨酸激酶进行信号的转导。Smad（Smaand Mad Homologue）蛋白家族是TGF-β受体的底物之一，目前认为Smad蛋白家族包括八个成员：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；两个I-Smads包括Smad6和Smad7。 Smad4蛋白是TGF-β/Smad信号通路在细胞内传导过程中最重要的中介分子之一。在TGF-β/Smad信号转导过程中，Smad2和Smad3能够被其C端受体介导的磷酸化所激活。Smad2和Smad3经过受体诱导的磷酸化活化之后，，能够与Smad4形成杂聚体，转导进入细胞核内。在细胞核内该聚合体能够与转录因子、转录因子活化蛋白及阻遏蛋白相结合，从而调节靶基因的转录。在过去的几年里有大量的证据表明，接受TGF-β信号刺激后Smad4能够与Smad2和Smad3形成Smad2/Smad2/Smad4、Smad3/Smad3/Smad4以及Smad2/Smad3/Smad4聚合体，并且认为Smad4是TGF-β信号通路中唯一的Co-Smad而发挥着协助Smad2和Smad3进入细胞核发挥调节基因转录的蛋白分子。然而，在2006年He等科学家发现了另外一种蛋白分子——转录调节因子1γ（Transcriptional IntermediaryFactor1γ，TIF1γ），能够与Smad4竞争结合磷酸化活化的Smad2、Smad3，介导该聚合体进入细胞内调节基因的转录。除了TGF-β/Smad信号通路，TGF-β还能够激活非Smad信号通路，包括细胞外调节蛋白激酶(extracellular regulated proteinkinase, ERK)、c-Jun氨基端激酶（c-Jun N-terminal kinase，JNK）和p38激活的蛋白激酶信号通路。尽管Smad4蛋白是TGF-β/Smad信号通路中一个重要的分子，而TGF-β在维持T细胞免疫过程中起着重要的作用，目前关于Smad4蛋白缺失对T细胞免疫影响的研究却很少。我们的研究主要的目的就是利用Smad4条件性缺失小鼠模型，探索Smad4对于T细胞增殖、活化和效应T细胞的分化过程是否有着与TGF-β同样重要的作用。鉴于Smad4完全敲除的小鼠由于外胚层形成的缺陷在胚胎早期就会死亡，我们引进了利用Cre-loxP系统的Smad4基因T细胞条件性敲除的小鼠（Smad4~(Cre/Co/Co)）作为动物模型，Smad4条件基因打靶小鼠（Smad4Co/Co）作为对照。 1.研究目的和内容 1) Smad4条件性缺失对T细胞发育和增殖的影响 TGF-β最早被发现的功能是它能够将细胞周期阻滞在G1期，从而发挥它对各种细胞的抑制作用，其中也包括了免疫系统的T细胞。而有研究认为，Smad4蛋白在TGF-β发挥对细胞周期的阻滞功能过程中处于中枢地位，Smad4通过与R-Smad聚合体在细胞核内抑制有丝分裂信号的转导，调节细胞周期。因此，我们的研究目的之一就是探讨Smad4对T细胞发育和增殖的影响，以及Smad4条件性缺失小鼠模型是否会造成T细胞发育不完全和对T增殖抑制作用的消失。 2) Smad4条件性缺失对T细胞活化和分化的影响研究表明，TGF-β通过促进CD4~+CD25~+Treg细胞的发育和分化来发挥其抑制其他T细胞的活化及分化的功能，CD4~+CD25~+Treg能够分泌TGF-β1或表达膜结合型的TGF-β1继而抑制其它免疫细胞的活化和分化。因此，我们的研究内容之一是观察Smad4条件性缺失小鼠模型T细胞活化和分化的过程是否与对照组小鼠有着显著性的差异。 3) Smad4条件性缺失对CD8~+T细胞活化和增殖的影响在研究过程中我们发现，年老的Smad4条件性缺失小鼠CD8~+T细胞的活化能力有缺陷，而其增殖能力与对照组小鼠相比没有明显的差别。在接下来的工和作中我们利用尾静脉注射李斯特菌的方法建立动物模型，在用李斯特菌免疫0天、5天、7天后研究活化的CD8~+CD44~(hi)T细胞比例Smad4~(Cre/Co/Co)和Smad4~（Co/Co）小鼠之间有无显著性差别。 4) Smad4条件性缺失对CD8~+效应CTL的影响目前，关于Smad4与抗原特异性CD8~+T细胞的研究还未见报道，而CD8~+T细胞在早期李斯特菌感染的清除过程中起着重要的作用。因此，这部分我们的研究内容主要是Smad4条件性缺失对CD8~+效应CTL的影响及其机制。 2.研究方法 1) PCR鉴定Smad4基因型小鼠尾部组织提取基因组DNA作为模板，PCR反应条件：94°C,1分钟；68°C,1分钟20秒；72°C,30秒；94°C,2分钟；94°C,30秒，60°C,30秒，72°C,1分钟，30个循环；72°C,8分钟；4°C，保存。 1) Immunoblotting Analysis M2buffer裂解，蛋白样品经SDS-PAGE蛋白电泳、蛋白转印、封闭、一抗孵育、二抗孵育和辣根过氧化物酶-ECL法检测其中Smad4蛋白含量。 2)流式细胞术用荧光标记的单抗对待检测表面Marker进行标记，1%多聚甲醛固定，上机FACS检测其表达水平。对细胞内细胞因子检测，则需要将待检细胞用PMA/Ino/BFA处理4h后，先对表面Marker进行标记，用透化液处理后，用荧光抗体标记胞内因子，上机FACS检测其表达水平。 3)统计组间差异比较使用t检验,以P0.05为差异有统计学意义。 4)其它本实验使用了细胞计数、FITC-标记的Annexin V、CFSE以及BrdU掺入等方法检测细胞的增殖能力。 3.研究结果和结论 1)基因型鉴定我们用DNA鉴定、Western-Blot和荧光抗体染色的方法证实了引进的确实是Smad4基因T细胞条件性敲除小鼠。 2) Smad4条件性缺失对T细胞发育和分化的影响我们的实验没有发现Smad4基因的T细胞条件性敲除对Treg细胞、CD4~+T细胞、CD8~+T细胞发育有显著的影响，同样的，我们发现Smad4基因条件性缺失对Th1/Th2/Th17细胞的分化也没有显著性的影响。 3) Smad4条件性缺失对T细胞增殖的影响 Smad4条件性缺失对幼年小鼠CD4~+和CD8~+T细胞数目没有影响，对年老小鼠脾细胞总数和CD4~+T细胞数目也没有影响，而我们发现Smad4~(Cre/Co/Co)与Smad4Co/Co相比CD8~+T细胞数却有着显著性的差别，产生这一现象的原因尚不明确。 4) Smad4条件性缺失对T细胞活化的影响对新生小鼠脾细胞和年老（52周龄）小鼠的脾、淋巴结细胞的分析，我们发现Smad4Co/Co与Smad4~(Cre/Co/Co)相比CD4~+CD44hi细胞比例均没有显著性差异。而在对年老小鼠脾细胞进行分析时发现，Smad4~(Cre/Co/Co)小鼠脾细胞中CD8~+CD44hi细胞低于对照组小鼠，说明Smad4条件性缺失对CD8~+T细胞的活化有一定影响。 5) Smad4条件性缺失对胃肠道上皮稳态的影响曾有研究发现，用Lck-Cre的方法T细胞条件性敲除Smad4基因小鼠在年老时会出现胃肠道多发性的肿瘤；而另有研究表明，以Lck-Cre或CD4-Cre技术T细胞特异性Smad4条件性敲除小鼠在年老时只会在十二指肠部位出现上皮细胞性肿瘤。在我们的研究中，对52周龄的Smad4条件性敲除小鼠肝、十二指肠和结肠病理切片，HE染色分析，并未发现有胃肠道肿瘤的发生。我们推测，Smad4基因T细胞条件性缺失不一定会造成胃肠道的肿瘤，Smad4基因T细胞条件性缺失造成胃肠道的肿瘤的发生与否可能与基因型及饲养环境有着密切的关系。 6) Smad4条件性缺失对CD8~+效应CTL的影响我们使用李斯特菌感染小鼠模型诱导抗原特异性CD8~+T细胞免疫应答，探讨Smad4基因T细胞条件性敲除对CD8~+T细胞活化的影响。在研究过程中我们发现，Smad4基因条件性缺失会导致CD8~+效应CTL细胞活化明显滞后，李斯特菌尾静脉注射5天时Smad4~(Cre/Co/Co)小鼠脾细胞中CD8~+CD44hi细胞低于对照组小鼠，而在7天时这种显著性差异消失。我们在研究中，发现李斯特菌感染Smad4~(Cre/Co/Co)小鼠脾细胞中CD8~+T细胞的Gran B、IFN-γ等杀伤Marker水平要远远低于对照组小鼠，同时Smad4~(Cre/Co/Co)小鼠脾细胞中短期效应细胞CD127(low)/KLRG1(hi) CD8~+T细胞的比例也低于对照组小鼠，说明Smad4基因条件性缺失对CD8~+效应CTL细胞的杀伤功能有一定的影响。 7) Smad4条件性缺失小鼠影响CD8~+CTL活化和杀伤效应的机制我们在实验中发现，Smad4基因的T细胞条件性缺失对抗原特异性CD8~+T细胞表面的IL-2Rα链、β链、γ链以及IL-15Rα链表达并没有显著的影响，因此，李斯特菌诱导的Smad4基因T细胞条件性敲除小鼠(Smad4~(Cre/Co/Co))脾细胞中CD8~+T细胞活化滞后并不是由于其细胞表面IL-2R及IL-15R的异常表达所造成的。研究中发现Smad4~(Cre/Co/Co)小鼠脾细胞中CD43~+CD27~+细胞的比例明显低于对照组小鼠，而CD43~+CD27~+细胞对CD8~+效应CTL活化和功能维持有重要的作用，我们推测这是造成其CD8~+CTL活化滞后和杀伤效应缺陷的原因之一。在体外实验中，我们用体外增殖分化实验排除了Smad4条件性缺失造成的抗原特异性CD8~+T细胞之间的差别是由于CD8~+T细胞内在的缺陷的影响。而接下来的实验显示，Smad4~(Cre/Co/Co)小鼠脾细胞中CD4~+T细胞表面的CD40L表达低于对照组小鼠，CD40与CD40L的相互作用能够诱导CD80/CD86共刺激信号的表达，继而活化pre-CTL。因此，我们认为Smad4~(Cre/Co/Co)小鼠脾细胞中CD4~+T细胞表面的CD40L低表达是造成CD8~+CTL活化滞后的另外一个原因。 4.本文创新点关于Smad4对CTL细胞分化有何影响的研究目前还未见报道，本研究利用Cre重组酶系统Smad4基因T细胞特异性条件性敲除小鼠对Smad4与CTL细胞分化的关系进行了探讨，发现Smad4条件性缺失影响CD8~+T细胞的活化；同时，我们用李斯特菌感染的方式开展了Smad4条件性缺失对影响CD8~+CTL活化及杀伤功能影响的研究，并初步探讨了造成这种影响可能的机制。
[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.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

【共引文献】