猪圆环病毒2型感染猪肠上皮细胞对CD4+T细胞分化关键分子mRNA的影响

[目的]探讨猪圆环病毒2型感染的猪肠上皮细胞对CD4+T细胞分化关键分子mRNA的影响.[方法]利用免疫磁珠法分离猪外周血CD4+T细胞,与猪圆环病毒2型感染48 h的猪肠上皮细胞共培养,收集共培养后72 h的T细胞,荧光定量PCR检测CD4+T细胞分化关键分子变化.[结果]荧光定量PCR检测显示,细胞因子 IL-4、IL-17A、IFN-γ、TGF-β1 与转录因子 T-bet、GATA3、RORγt、Foxp3的 mRNA 水平均被显著下调,而IL-10的mRNA水平被显著上调.[结论]感染猪圆环病毒2型猪肠上皮细胞抑制CD4+T细胞分化关键分子mRNA水平,提示CD4+T细胞向Th1细胞、Th2细胞、Th17细胞、调节性T细胞分化被抑制,而IL-10的上调激活CD4+T细胞向高分泌IL-10的CD4+T细胞分化.     

鸡Mx蛋白在293T细胞中的表达及其抗病毒活性

[目的]研究鸡Mx蛋白在293T细胞中的表达并检测其抗病毒活性。[方法]将鸡的Mx基因克隆入pEGFP-C1载体,经筛选鉴定后磷酸钙法转染293T细胞,并对转染细胞进行荧光分析以及RT-PCR与Western blot鉴定,同时提取细胞蛋白质,微量细胞病变试验检测其抗新城疫病毒的活性。[结果]试验构建的pEGFP-C1-cMx真核表达载体转染293T细胞后,在胞浆中可检测到绿色荧光,RT-PCR与Western blot结果进一步证实,pEGFP-C1-cMx可在293T细胞中表达,微量细胞病变试验显示,表达的融合蛋白可保护鸡胚成纤维细胞免受新城疫病毒感染。[结论]试验构建的pEGFP-C1-cMx表达载体可在293T细胞中表达具有抗新城疫病毒活性的鸡Mx蛋白。     

公鸡生殖器官中T细胞分布密度研究

【目的】检测CD4+/CD8+T细胞在公鸡生殖器官中的分布密度。【方法】通过荧光定量PCR分析IL-8的表达水平,以及通过冰冻切片和免疫组化技术,定位健康公鸡的生殖系统中CD4+和CD8+T细胞。【结果】IL-8在附睾中表达水平最高,在睾丸中的表达水平最低;有少量的CD4+和CD8+T细胞被定位在睾丸间质细胞区。在附睾管和输精管的固有层和黏膜上皮中,均识别到两种类型的T细胞。而且CD4+和CD8+T细胞在公鸡生殖道中不同部位密度的变化趋势明显:CD4+和CD8+T细胞在公鸡生殖管道中段(输出管、附睾管)的密度高于两端(睾丸网、输精管)。【结论】T细胞介导的免疫防御主要发生在附睾,尤其附睾输出管和附睾管中。     

载脂蛋白A1对小鼠淋巴细胞频率及活性的影响

以载脂蛋白A1 (ApoA-Ⅰ)基因敲除及转基因小鼠为基础,利用流式细胞术检测小鼠体内T细胞、B细胞和NK细胞及其亚型频率、表面活化型分子表达情况,探讨ApoA-Ⅰ水平对小鼠淋巴细胞频率及活性的影响.结果 表明:相对于野生小鼠,生理状态下ApoA-Ⅰ基因敲除小鼠脾脏、外周血CD8+T细胞及效应型(CD8+ NKG2D+、CD8+ CD44+CD62L-、CD8+ CD44+ KLGR1+、CD8+ IFN-γ+)细胞频率显著下调,而以上指标在A poA-Ⅰ转基因小鼠中上调.CD4+T细胞、调节性T细胞(CD4+ CD25+ FoxP3+)、B细胞和NK细胞频率在3组小鼠中均无显著差异.该研究说明ApoA-Ⅰ参与调控CD8+T细胞活性,但对CD4+T细胞、B细胞及NK细胞无显著影响.     

子宫内膜异位症不孕患者手术前后CD4+CD29+T细胞变化与妊娠率的关系

目的 了解子宫内膜异位症(endometriosis,EMS)不孕患者手术前后CD4+CD29+T细胞变化及其与妊娠率的关系.方法 收集具有手术指征的EMS致不孕患者36例(手术组),无手术指征EMS患者38例(非手术组),同时选取38例健康女性作对照(健康组).观察EMS患者治疗前后外周血CD4+CD29+T细胞及其细胞因子白介素1(IL-1)、白介素4(IL-4)及CA125水平的变化,并将其与妊娠率进行相关性分析.结果 EMS不孕患者CD4+CD29+T细胞、IL-1、IL-4及CA125水平均显著高于健康女性(P＜0.01).治疗后,手术组和非手术组的CD4+CD29+T细胞、IL-1及CA125水平均显著低于治疗前,且手术组明显低于非手术组(P＜0.01);手术组的妊娠率明显高于非手术组(P＜0.05).CD4+CD29+T细胞、IL-1及IL-4与CA125均呈显著正相关,而与妊娠率呈显著负相关(P＜0.05或0.01).结论 CD4+CD29+T细胞升高是EMS不孕患者的重要免疫学表现,手术可显著降低该细胞及其细胞因子水平,提高妊娠率.     

c-Jun蛋白对A型流感病毒H1N1感染小鼠体内CD4~+和CD8~+T细胞增殖的调节作用

通过构建A型流感病毒H1N1感染小鼠模型,并使用c-Jun蛋白的特异性核酶Dz13抑制该蛋白的表达,探索c-Jun蛋白在流感病毒感染后对T淋巴细胞增殖和炎性反应的调节作用。蛋白质印迹法(Western-blotting)结果表明,核酶Dz13作用后c-Jun蛋白的表达量明显降低,且c-Jun蛋白的活化也随之降低。此外,用流式细胞术测定感染后3 d和6 d外周血内辅助性T细胞(CD4~+T)和杀伤性T细胞(CD8~+T)的增殖情况,同时用实时荧光定量聚合酶链式反应测定外周血内细胞因子IFN-γ、IL-6和IL-10表达。结果显示:小鼠感染病毒后,外周血CD4+和CD8+T细胞明显增多,相关细胞因子的表达量也明显上调;而抑制c-Jun蛋白表达后,CD4~+和CD8~+T细胞的增殖受到抑制,且相关细胞因子IFN-γ、IL-6和IL-10的表达量亦受到抑制。说明c-Jun蛋白参与调节A型流感病毒H1N1感染后引起的T淋巴细胞增殖和相关细胞因子的表达。     

人CD8~+T细胞基因调控网络研究

为研究CD8~+ T细胞的功能异常机制,对功能异常状态CD8~+ T细胞和3种正常CD8~+ T细胞亚型[中央记忆CD8~+ T细胞(HCM)、效应记忆CD8~+ T细胞(HEM)和na?ve CD8~+ T细胞(HW)]的ATAC-seq(Assay for transposase accessible chromatin with high-throughput sequencing)数据进行基因调控网络比较,结果表明,3种正常CD8~+ T细胞特异表达的基因参与维持相应T细胞正常功能的信号通路,功能异常CD8~+ T细胞特异的基因参与癌症相关通路。转录因子调控网络比较分析发现功能异常CD8~+ T细胞GMEB2转录因子表达关闭。细胞表面受体调控网络比较分析发现功能异常CD8~+ T细胞CD7和P4HB受体的表达关闭。采用基因调控网络的数据进行比较分析,发现CD8~+ T细胞功能异常机制可能与GMEB2转录因子、CD7和P4HB受体表达关闭有关,这为抗病育种寻找特定基因提供新思路。     

抗原浓度和DC数量对OT-I小鼠CD8+ T细胞分化与增殖的影响

【目的】探讨细胞毒性T细胞(CTL)表位肽SIINFEKL浓度和树突状细胞(DC)数量对CD8+T细胞活化和增殖的影响。【方法】用小鼠重组粒细胞集落刺激性生物因子(GM-CSF)和IL-4诱导骨髓细胞来源的DC增殖和分化,将所得成熟树突状细胞(maDC)和SIINFEKL抗原肽,与免疫磁珠法分离的OT-I小鼠CD8+T细胞共培养。用不同质量浓度的SIINFEKL(100,10,1,0.1,0.01和0.001 ng/mL)或不同数量的DC(DC/T比例分别为1/20和1/5)与CD8+T细胞共培养,刺激CD8+T细胞增殖分化。于共培养不同时间收集细胞,流式细胞术测定CD8+T细胞的数量、分裂速度、细胞表面活化分子的表达丰度,碘化丙叮(PI)染色测定细胞活力。【结果】在任一DC/T比例下,SIINFEKL浓度过低,均不能引起CD8+T细胞的充分增殖,而高于CD8+T细胞最佳增殖浓度的SIINFEKL则导致CD8+T细胞数量减少;在SIINFEKL质量浓度为0.001~0.1 ng/mL时,增加DC数量能促进CD8+T细胞的扩增;而SIINFEKL质量浓度为1~100 ng/mL时,提高DC数量反而导致CD8+T细胞数量减少;高浓度抗原和DC数量能有效诱导CD8+T细胞的活化和分裂增殖,但过量刺激会使细胞死亡,导致CD8+T细胞数量减少。【结论】CD8+T细胞的增殖受DC数量和抗原肽浓度的共同调节,要获得持久有效的CD8+T细胞免疫,必须保证CD8+T细胞得到足够的抗原信号,同时避免抗原信号过强导致的细胞活化后死亡。     

2种乳酸杆菌肠道黏膜免疫调节作用的比较

【目的】比较嗜酸乳酸杆菌(Lactobacillus acidophilus)和植物乳酸杆菌(Lactobacillus plantarum)肠道黏膜免疫调节作用的差异。【方法】以C57BL/6小鼠为受试动物,分别灌胃嗜酸乳酸杆菌和植物乳酸杆菌7d后取材,采用流式细胞术、ELISA等方法,对脾脏和肠系膜淋巴结中T细胞亚群、结肠PP结中TLR2+和TLR4+细胞、结肠组织中IFN-γ和IL-4水平及小肠sIgA水平进行检测,明确2种乳酸杆菌对肠道黏膜的免疫调节作用。【结果】2种乳酸杆菌对小鼠体质量无显著影响。2种乳酸杆菌均可显著提高小鼠肠系膜淋巴结中CD3+CD4+和CD3+CD8+T细胞比例,且与嗜酸乳酸杆菌相比,植物乳酸杆菌更倾向于提高CD3+CD8+T细胞比例;而与对照组相比,二者对脾脏中CD3+CD4+和CD3+CD8+T细胞比例均无显著影响。2种乳酸杆菌均可极显著提高结肠PP结中TLR2+细胞比例,而对TLR4+细胞比例无显著影响。2种乳酸杆菌均可极显著提高结肠组织中IFN-γ水平,嗜酸乳酸杆菌还可极显著提高IL-4水平。2种乳酸杆菌均能显著提高肠道黏膜sIgA水平。【结论】2种乳酸杆菌均可提高结肠PP结中TLR2+细胞比例,嗜酸乳酸杆菌上调Th1和Th2型免疫反应,植物乳酸杆菌上调Th1型免疫反应。     

泛素结合酶UFC1基因的荧光表达载体构建

利用pEGFP-C3作为载体,构建与绿色荧光蛋白基因相连的泛素结合酶UFC1(ubiquitin-fold modifier conjugating enzyme 1)序列的真核表达质粒,观察其在真核细胞中的表达和定位,为进一步研究UFC1基因功能打下了基础。采用RT-PCR方法从宫颈癌Hela细胞中扩增UFC1基因全长cDNA,双酶切后克隆到真核表达载体pEGFP-C3中,构建并鉴定pEGFP-C3-UFC1质粒。经脂质体介导转染Hela细胞后,在荧光显微镜下观察目的蛋白在真核细胞内的表达情况。双酶切鉴定和测序分析均证实,已成功构建pEGFP-C3-UFC1重组载体。重组载体转染Hela细胞,观察到绿色荧光蛋白表达,还可见绿色荧光呈点状分布于细胞质中;而对照pEGFP-C3质粒转染Hela细胞,绿色荧光蛋白则呈弥散状分布。成功克隆了UFC1基因,构建了pEGFP-C3-UFC1重组质粒。     

The reservoir for HIV-1 in human peripheral blood is a T cell that maintains expression of CD4

Human immunodeficiency virus type 1 (HIV-1) selectively infects cells expressing the CD4 molecule, resulting in substantial quantitative and qualitative defects in CD4+ T lymphocyte function in patients with acquired immunodeficiency syndrome (AIDS). However, only a very small number of cells in the peripheral blood of HIV-1-infected individuals are expressing virus at any given time. Previous studies have demonstrated that in vitro infection of CD4+ T cells with HIV-1 results in downregulation of CD4 expression such that CD4 protein is no longer detectable on the surface of the infected cells. In the present study, highly purified subpopulations of peripheral blood mononuclear cells (PBMCs) from AIDS patients were obtained and purified by fluorescence-automated cell sorting. They were examined with the methodologies of virus isolation by limiting dilution analysis, in situ hybridization, immunofluorescence, and gene amplification. Within PBMCs, HIV-1 was expressed in vivo predominantly in the T cell subpopulation which, in contrast to the in vitro observations, continued to express CD4. The precursor frequency of these HIV-1-expressing cells was about 1/1000 CD4+ T cells. The CD4+ T cell population contained HIV-1 DNA in all HIV-1-infected individuals studied and the frequency in AIDS patients was at least 1/100 cells. This high level of infection may be the primary cause for the progressive decline in number and function of CD4+ T cells in patients with AIDS.     

Demethylated CD8 gene in CD4+ T cells suggests that CD4+ cells develop from CD8+ precursors

Mature T cells and medullary thymocytes bear either the CD4 or CD8 differentiation antigen. Precursor cells in the thymus express neither CD4 nor CD8 (CD4-8-), but most cortical thymocytes are CD4+8+. Whether CD4+ and CD8+ mature T cells arise directly from CD4-8- precursors or from a CD4+8+ intermediate remains unresolved. In this study, methylation of the CD8 gene in murine T cells and thymocytes was examined. There was progressive demethylation of the CD8 gene in the thymus during the transition from CD4-8- to CD4+8+. A similar pattern of demethylation of the CD8 gene was seen in CD4+ mature T cells, suggesting previous expression of CD8 in the CD4+ lineage.     

A role for CD40 expression on CD8+ T cells in the generation of CD8+ T cell memory

The delivery of CD4 help to CD8+ T cell responses requires interactions between CD40 and CD40 ligand and is thought to occur through antigen-presenting cell (APC) activation. Here we show that generation of memory CD8+ T cells displaying an enhanced capacity for cell division and cytokine secretion required CD4 help but not CD40 expression by the APCs. Activated CD4+ and CD8+ T cells expressed CD40; and in the absence of this protein, CD8+ T cells were unable to differentiate into memory cells or receive CD4 help. These results suggest that, like B cells, CD8+ T cells receive CD4 help directly through CD40 and that this interaction is fundamental for CD8+ T cell memory generation.     

SLE患者CD4+CD25+调节性T细胞及Foxp3基因表达的研究

目的:研究系统性红斑狼疮(SLE)患者外周血CD 4 CD 25 调节性T细胞及Foxp3基因的表达水平,了解它们在SLE发病机制中的作用。方法:分别收集25例SLE患者(SLE组)及健康人(对照组)外周抗凝静脉血,分离纯化T淋巴细胞。PE标记抗CD 4单抗,F ITC标记的抗CD 25单抗,作双色流式细胞术,分析SLE患者外周血CD 4 CD 25 调节性T细胞百分率,RT-PCR检测T细胞Foxp3 mRNA表达。结果:SLE组外周血CD 4 T、CD 4 CD 25 T细胞百分率及T细胞Foxp3 mRNA水平均低于对照组(P<0.01),并且CD 4 CD 25 T细胞百分率与Foxp3mRNA水平呈依赖关系(P<0.01)。结论:SLE患者外周血存在细胞免疫功能失调,CD 4 CD 25 调节性T细胞数量减少和Foxp3mRNA表达下调可能与SLE的免疫学发病机制有关。     

Defective CD8 T cell memory following acute infection without CD4 T cell help

The CD8+ cytotoxic T cell response to pathogens is thought to be CD4+ helper T cell independent because infectious agents provide their own inflammatory signals. Mice that lack CD4+ T cells mount a primary CD8 response to Listeria monocytogenes equal to that of wild-type mice and rapidly clear the infection. However, protective memory to a challenge is gradually lost in the former animals. Memory CD8+ T cells from normal mice can respond rapidly, but memory CD8+ T cells that are generated without CD4 help are defective in their ability to respond to secondary encounters with antigen. The results highlight a previously undescribed role for CD4 help in promoting protective CD8 memory development.     

Expanded HIV-1 cellular tropism by phenotypic mixing with murine endogenous retroviruses

In view of the current interest in in vivo murine models for acquired immunodeficiency syndrome (AIDS), the interaction between human immunodeficiency virus type 1 (HIV-1) and endogenous murine leukemia virus (MuLV)-related retroviruses was investigated with a human leukemic T cell line (PF-382x) that acquired xenotropic MuLV (X-MuLV) after in vivo passage in immunosuppressed mice. Despite similar levels of membrane CD4 expression and HIV-1 125I-labeled gp 120 binding, a dramatic acceleration in the time course of HIV-1 infection was observed in PF-382x compared to its X-MuLV-negative counterpart (PF-382). Moreover, PF-382 cells coinfected by X-MuLV and HIV-1 generated a progeny of phenotypically mixed viral particles, enabling HIV-1 to productively infect a panel of CD4- human cells, including B lymphoid cells and purified normal peripheral blood CD4-/CD8+ T lymphocytes. Mixed viral phenotypes were also produced by human CD4+ T cells coinfected with an amphotropic MuLV-related retrovirus (A-MuLV) and HIV-1. These data show that endogenous MuLV acquired by human cells transplanted into mice can significantly interact with HIV-1, thereby inducing important alterations of HIV-1 biological properties.     

重组鸡α-干扰素对鸡外周血T淋巴细胞亚类的影响

重组鸡α-干扰素(rChIFN-α)静脉注射4~6周龄SPF鸡,24 h后采血分离淋巴细胞,通过流式细胞术测定不同时间外周血中CD4+和CD8+T淋巴细胞的百分率。结果显示,rChIFN-α可以在48~72 h内明显提高CD4+T淋巴细胞的百分率,并下调CD8+T淋巴细胞的百分率,证明rChIFN-α具有显著的免疫调节作用。     

Endogenous MHC class II processing of a viral nuclear antigen after autophagy

CD4+ T cells classically recognize antigens that are endocytosed and processed in lysosomes for presentation on major histocompatibility complex (MHC) class II molecules. Here, endogenous Epstein-Barr virus nuclear antigen 1 (EBNA1) was found to gain access to this pathway by autophagy. On inhibition of lysosomal acidification, EBNA1, the dominant CD4+ T cell antigen of latent Epstein-Barr virus infection, slowly accumulated in cytosolic autophagosomes. In addition, inhibition of autophagy decreased recognition by EBNA1-specific CD4+ T cell clones. Thus, lysosomal processing after autophagy may contribute to MHC class II-restricted surveillance of long-lived endogenous antigens including nuclear proteins relevant to disease.     

Induction of CD4+ human cytolytic T cells specific for HIV-infected cells by a gp160 subunit vaccine

Cytolytic T lymphocyte (CTL) responses were evaluated in humans immunized with recombinant human immunodeficiency virus type 1 (HIV) envelope glycoprotein gp160. Some vaccinees had gp160-specific CTLs that were shown by cloning to be CD4+. Although induced by exogenous antigen, most gp160-specific CTL clones also recognized gp160 synthesized endogenously in target cells. These clones lysed autologous CD4+ T lymphoblasts infected with HIV. Of particular interest were certain vaccine-induced clones that lysed HIV-infected cells, recognized gp160 from diverse HIV isolates, and did not participate in "innocent bystander" killing of noninfected CD4+ T cells that had bound gp120.