Flt3L对小鼠胸腺树突状细胞在FTOC体系中分化发育的作用

为了借助FTOC体系探讨Flt3L对小鼠胸腺树突状细胞分化发育的影响。摘取15～16d龄胎鼠胸腺进行体外器官培养(胎鼠胸腺器官培养-FTOC),根据所使用培养基的不同实验分为两组:对照组(基础培养基)和Flt3L组(培养基中含有细胞因子Flt3L),在体外进行FTOC常规培养,12d后分别收集两实验组的胸腺细胞,流式细胞仪检测细胞表面分子CD4、CD8、CD11c、Ia等的表达,通过光学显微镜观察细胞形态。骨髓来源的c-kit+造血干细胞通过悬滴培养方法种植入2-脱氧鸟苷处理过的胸腺,随后将胸腺放置于组织器官培养皿中所使用的培养基为基础培养基或加入细胞因子Flt3L的培养基,进行为期12d的FTOC常规培养。12d后收集不同条件下FTOC培养的胸腺细胞,通过流式细胞仪对胸腺细胞的表型进行分析;将在不同条件下FTOC培养获得的胸腺细胞进行MACS分选,从而获得胸腺树突状细胞(CD11c+DC),再与异源的CD4+T细胞进行混合淋巴细胞反应,通过MTT法检测T细胞的增殖情况。结果:在正常FTOC体系中,流式细胞仪检测结果和细胞形态学结果显示:Flt3L组胸腺DC有明显的增加,且FTOC联合悬滴培养体系中Flt3L组胸腺DC的生成率明显高于对照组;MTT检测结果也显示:没有CpG2006刺激时,胸腺DC刺激T细胞增殖的能力比较弱,但添加CpG2006刺激后,胸腺DC趋向于成熟表型,刺激T细胞增殖的能力有所增强。提示,Flt3L在小鼠胸腺DC的分化发育中发挥重要的调节作用,明显促进小鼠骨髓来源的c-kit+造血干细胞向胸腺DC的分化。     

CCL20在胸腺CD4+CD25+T细胞发育过程中的作用及其意义

目的:研究趋化因子CCL20对小鼠胸腺CD4 CD25 双阳性T细胞发育的影响,为天然调节性T细胞调控的研究提供基础数据.方法:采用14.5 d龄胚鼠胸腺进行体外培养,以流式细胞术(FCM)检测不同时间点胸腺CD4 CD25 细胞的变化,同时计数每个胸腺小叶的细胞数变化.结果:体外胸腺培养的第1 ～6天胸腺细胞比例,细胞数量趋势变化与胸腺体内发育的第14.5、15、16、17、18、19天CD4 CD25 双阳性T细胞的比例,细胞数量的趋势变化相似;在胸腺体外培养的第1 ～6天,CD4 CD25 T细胞占CD4 T细胞的比例分别为58.29%、12.14%、6.08%、17.78%、9.06%、4.04%,占CD25 T细胞的比例分别为3.75%、10.81%、17.20%、51.93%、61.64%、80.06%,这一发育趋势与体内结果具有一致性.在4 mg/L的CCL20干预下,胸腺体外培养的第3、6天CD4 CD25 胸腺细胞分别从3.24±0.18、3.96±0.24下降至1.27±0.11、1.76±0.22(P<0.001).结论:体外培养的CD4 CD25 双阳性胸腺细胞数量和比例变化与体内发育变化趋势一致,CCL20明显下调胸腺CD4 CD25 的表达,这将为天然调节性T细胞的调控研究提供有效的参考依据.     

IL-7对胸腺T细胞及树突状细胞体外分化发育的影响

目的:探讨IL-7对胸腺T细胞及胸腺树突状细胞分化的影响。方法:摘取15～16日龄胎鼠胸腺进行体外器官培养(胚胎胸腺器官培养-FTOC),分别将细胞因子IL-7和培养基滴加在胸腺小叶上,12天后收集不同条件下经FTOC培养获得的胸腺细胞,流式细胞仪检测细胞表面分子CD4、CD8、CD11c、B220、Ia等的表达,通过光学显微镜观察细胞形态,通过细胞计数检测细胞数目的变化。再将经FTOC培养获得的胸腺细胞和异源的T细胞进行混合淋巴细胞反应,通过MTT法检测T细胞的增殖情况。结果:细胞计数结果表明添加外源性IL-7组的胸腺细胞数目明显减少,流式细胞仪检测结果显示其中胸腺CD4-CD8-双阴性细胞及CD8+单阳性细胞比例有所增加,而CD4+CD8+双阳性细胞比例显著下降,CD4+单阳性细胞比例没有明显变化;此外,B细胞和树突状细胞、NK细胞数量均有不同程度的增加。结论:IL-7在胸腺T细胞及胸腺树突状细胞的分化发育中发挥重要的调节作用。     

OP9-DL1细胞共培养系统在T细胞体外定向诱导分化中的应用

T细胞是一类在机体免疫应答中起核心作用的免疫活性细胞。T细胞的分化发育指来自骨髓的淋巴样祖细胞(lymph-oid pregnitors)进入胸腺增殖、分化,并从胸腺皮质迁移至髓质,成为成熟T细胞的过程。长期以来,T细胞发育的体内外研究一直依赖于胸腺。但人或鼠的胸腺组织来源有限且取材困难,加大了T细胞发育的研究难度。近年来,随着T细胞发育过程中一些关键分子的发现及其研究的不断深入,一种可高效诱导造血干/祖细胞在体外向T细胞分化的简单模型骨髓基质细胞OP9-DL1体外培养系统得以建立,这种系统的出现大大简化了T细胞分化发育的研究,本文将就其在T细胞体外定向诱导分化中的应用作一综述。     

胚胎胸腺细胞在体外胚胎胸腺器官培养中的发育研究

体外可调控的Ｔ细胞培养系统的建立 ,为研究Ｔ细胞发育及Ｔ细胞和基质细胞的作用提供了很好的实验模型 ,如胸腺或基质细胞的分离培养和胚胎胸腺器官培养 (ＦＴＯＣ) 〔1〕。近年来研究表明 ,长期体外培养的胸腺基质细胞 ,由于表型的变化如ＭＨＣⅡ类分子丢失 ,而失去了支持Ｔ细胞发育的能力 ,其应用受到限制。而ＦＴＯＣ保持了体内胸腺的三维空间结构、恒定的细胞表型、适宜的细胞 细胞间的相互作用、基质细胞分泌的多种细胞因子 ,为研究Ｔ细胞的发育提供了更符合生理的胸腺微环境。研究表明 ,ＦＴＯＣ是目前唯一的能支持前体Ｔ细胞完成整…     

小鼠胚胎肝祖细胞的分离和扩增培养

目的从胎龄14.5 d的C57小鼠胚胎肝脏中分离、培养小鼠胚胎肝祖细胞(m HPCs),并将其诱导分化为胆管细胞。方法用荧光激活细胞筛选法(FACS)分选DLK1表面抗原阳性的小鼠胚胎肝细胞,并和小鼠胚胎成纤维细胞(MEFs)Transwell共培养或者单独培养。细胞免疫荧光检测刚分选和共培养4和6 d的DLK1~+细胞的甲胎蛋白(AFP)、白蛋白(ALB)及细胞角蛋白19(CK19)抗原的表达。结果体外共培养时,大部分DLK1~+细胞分裂增殖明显,呈葡萄状聚集生长。第4天,部分细胞开始贴壁增殖,形态开始变成梭形。结果显示,分选的DLK1~+细胞表达AFP和少量ALB,但不表达CK19;在共培养的第4天其开始表达CK19,和微弱表达ALB;第6天,其高表达CK19,而几乎不表达的ALB。结论应用FACS技术成功从E14.5胎肝细胞中分选出DLK1~+细胞并鉴定其大部分为mHPCs,并可在体外与MEFs Transwell共培养的条件下,诱导培养其分化为胆管细胞。     

大鼠胚胎脑组织神经干细胞的培养和鉴定

目的探讨从不同胎龄的大鼠脑组织中分离,培养神经干细胞(NSC)并对其鉴定,了解生物特性。方法通过采用机械分离和消化分离相结合的方法分离不同胎龄大鼠脑NSC。在无血清DMEM/F12(含20ng/m lbFGF,20ng/m lEGF及B27辅助培养液)中培养、传代和鉴定。诱导分化后采用SABC法对分化的细胞进行神经元特异烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)检测作细胞鉴定。结果从不同胎龄的胎鼠脑组织中成功培养出神经干细胞,胎龄为12.5天的胎鼠提取的神经干细胞集落最多,在上述条件下培养及传代的细胞不断分裂增殖,形成悬浮生长的呈巢素蛋白(nestin)阳性的神经球;用血清诱导分化为大量表达NSE阳性的神经元和GFAP阳性的星形胶质细胞。结论胎龄为12.5天胎鼠大脑皮质培养出的神经干细胞数量最多,可分化为神经元、神经胶质细胞及少突胶质细胞。     

链球菌蛋白对RAW264.7小鼠巨噬细胞免疫学活性的调节作用

目的:探讨链球菌蛋白对RAW264.7小鼠巨噬细胞免疫活性调节及其相关作用机制.方法:不同浓度的链球菌蛋白与RAW264.7小鼠巨噬细胞共同作用后,采用MTT 法检测细胞的增殖活化;吞噬中性红试验观察巨噬细胞的吞噬功能;生物化学法检测巨噬细胞上清液中TNF-α和IL-6 的含量;RT-PCR法检测细胞中TNF-α、IL-6和Toll样受体(Toll-like receptors,TLRs)的mRNA表达情况;流式细胞术检测细胞表面TLR2和TLR4的表达强度.结果:链球菌蛋白对巨噬细胞的生长增殖和吞噬功能有较强的刺激作用(P<0.05),促进TNF-α和IL-6的表达和分泌(P<0.05),并可上调巨噬细胞表面模式识别受体TLR2和TLR4的表达(P<0.05).结论:链球菌蛋白通过刺激RAW264.7小鼠巨噬细胞的增殖,增强细胞吞噬活性以及诱导细胞因子的产生等发挥其免疫调节作用.     

Sombati细胞模型中Toll样受体4的表达及意义

 目的：研究Sombati细胞模型中神经元Toll样受体4（TLR4）表达变化,探讨TLR4在Sombati癫痫细胞发病过程中的意义。方法：将新生SD乳鼠海马神经元进行体外培养至第9天,随机分为对照组和Sombati细胞模型组,分别检测TLR4蛋白及TLR4 mRNA的表达。结果：免疫组化结果显示Sombati细胞TLR4蛋白表达增强,荧光定量PCR显示Sombati细胞组TLR4 mRNA表达增加(P<0. 05),且随时间的延长而增强。结论：Sombati细胞模型中TLR4 mRNA与蛋白表达均增加,可能与癫痫的发病有关。     

小鼠胚胎脊髓Draxin蛋白表达水平的动态变化研究

目的:探讨正常小鼠胚胎脊髓不同发育时期背侧抑制性轴突导向蛋白(Draxin)的表达部位和表达水平的动态变化过程。方法:应用免疫组织化学、Western Blot观察不同发育阶段胎鼠脊髓内Draxin的分布及表达变化特点;应用体外培养结合免疫荧光双染标记,观察不同类型神经细胞内Draxin的表达情况。结果:免疫组织化学染色结果显示,不同发育时期胎鼠脊髓内部及其周边组织内,Draxin的表达呈现明显的动态变化趋势; Western Blot检测结果显示,E11. 5开始Draxin表达量逐渐增加,至E14. 5达到高峰,此后下降并逐渐消失;体外培养结合免疫荧光双标的结果显示,部分Tuj-1阳性的未成熟神经元、Nestin阳性的神经干细胞以及GFAP阳性的星形胶质细胞均表达Draxin。结论:Draxin在胎鼠脊髓发育过程中的表达,具有明显的动态变化趋势,多种类型的神经元和星形胶质细胞表达Draxin。     

Enhancement of IL-7 following irradiation of fetal thymus

The effect of ionizing radiation on intra-thymic T cell development was investigated using a fetal thymic organ culture (FTOC) method in vitro. When double-negative (DN) fetal (day 15) thymocytes were co-cultured with an irradiated (25 Gy) fetal (day 15) thymus in the absence of direct contact or mitogenic stimulation, the induction of TCRgammadelta+ T cells was observed. About 50% of the TCRgammadelta+ T cells developed after 4-day-co-culture with the irradiated fetal thymus, whereas only a few TCRgammadelta+ T cells developed after co-culture with the non-irradiated fetal thymus. About 50% of the TCRgammadelta+ T cells were CD8+ cells with alphabeta heterodimeric chains. Cultured supernatants of the irradiated fetal thymi also induced the differentiation from DN thymocytes to CD8+ TCRgammadelta+ T cells after 3-day-culture. To clarify the factor in the cultured supernatants, several neutralizing antibodies (Abs) were used. Only anti-IL-7-Ab inhibited the differentiation from DN thymocytes to CD8+ TCRgammadelta+ T cells. RT-PCR revealed the increased expression of IL-7 mRNA in the fetal thymus 24 hours after radiation. Electron microscope studies demonstrated proliferative epithelial cells in the irradiated fetal thymus. These findings strongly suggest that fetal thymic epithelial cells affected by irradiation proliferate and enhance the production of IL-7, which induces the differentiation of CD8+ TCRgammadelta+ T cells from DN thymocytes.     

Developmental changes predispose the fetal thymus to positive selection of CD4+CD8- T cells.

Selection of a competent T-cell repertoire is dependent on complex interactions between immature thymocytes and components of the thymic stroma. These events may be preserved in vitro by excising developing thymus rudiments and maintaining them under carefully controlled conditions in fetal thymus organ cultures (FTOC). Using this approach, we have shown that the ability of C57B1/6 thymi to sustain positive selection of mature CD4+CD8- cells is profoundly influenced by the day of gestation on which they are excised: while thymocytes from day 14 rudiments fail to progress beyond the CD4+CD8+ stage of the developmental pathway, day 15 and day 16 thymi support the differentiation of CD4+CD8- thymocytes. Importantly, day 16 thymocytes transferred to day 14 deoxyguanosine-treated rudiments are likewise arrested at the CD4+CD8+ stage, suggesting that the thymic microenvironment of day 14 rudiments, rather than the state of differentiation of the thymocytes they contain, is responsible for the block in positive selection. Our studies of the stromal elements of day 14 rudiments have, however, revealed no obvious deficiencies in the cell types represented, or their expression of class II major histocompatibility complex (MHC) determinants. Furthermore, we have been unable to circumvent the blockage in positive selection by the addition of certain cytokines expressed late during gestation. These results suggest that subtle changes occurring at day 15 of ontogeny render the thymic microenvironment capable of positive selection.     

Differentiation and maturation of macrophages into interdigitating cells and their multicellular complex formation in the fetal and postnatal rat thymus

Three mouse anti-rat macrophage monoclonal antibodies, TRPM-1, TRPM-2, and TRPM-3, as well as anti-rat Ia monoclonal antibody, were used to study the emergence, differentiation, and maturation of macrophages in the fetal and postnatal rat thymus immunohistochemically and immunoelectron microscopically. At 14 days of gestation, primitive/fetal macrophages entered the thymic primordium and showed Ia expression, where afterwards the epithelial cells also expressed Ia antigens prominently at 15 days of gestation. After 16 days of gestation, differentiation of a subpopulation of primitive/fetal macrophages into interdigitating cells (IDCs) is suggested. From 19 days of gestation, TRPM-1-positive dendritic cells including IDCs started forming multicellular complexes with thymocytes and the epithelial cells also formed similar complexes with thymocytes. One day after birth, TRPM-1 positive IDC-thymocyte complexes distributed throughout the thymic medulla. The number of TRPM-1- and Ia-positive IDCs increased by day, and Langerhans cells (LCs) appeared in the thymic medulla within a few days after birth. By two weeks after birth, the distribution pattern of Ia- and TRPM-1-positive cells became similar to that of adult rats. In ontogeny, intimate cell membrane appositions were frequently observed between thymocytes and Ia-positive epithelial cells or IDCs in the thymic multicellular complexes. These complexes were discriminated into two types; epithelial cell-thymocyte complexes and IDC- or LC-thymocyte ones. In vitro, two types of the thymic nurse cells (TNCs) were identified: epithelial cells and IDCs or LCs. Besides epithelial cells, IDCs or macrophages formed rosettes with thymocytes. These TNCs and rosettes in vitro seem to correspond to the thymic multicellular complexes in vivo.     

Involvement of CCR9 at multiple stages of adult T lymphopoiesis

The chemokine CCL25 is constitutively expressed in the thymus, and its receptor CCR9 is expressed on subsets of developing thymocytes. Nevertheless, the function of CCL25/CCR9 in adult thymopoiesis remains unclear. Here, we demonstrate that purified CCR9(-/-) hematopoietic stem cells are deficient in their ability to generate all major thymocyte subsets including double-negative 1 (DN1) cells in competitive transfers. CCR9(-/-) bone marrow contained normal numbers of lineage(-) Sca-1+c-kit+, common lymphoid progenitors, and lymphoid-primed multipotent progenitors (LMPP), and CCR9(-/-) LMPP showed similar T cell potential as their wild-type (WT) counterparts when cultured on OP9-delta-like 1 stromal cells. In contrast, early thymic progenitor and DN2 thymocyte numbers were reduced in the thymus of adult CCR9(-/-) mice. In fetal thymic organ cultures (FTOC), CCR9(-/-) DN1 cells were as efficient as WT DN1 cells in generating double-positive (DP) thymocytes; however, under competitive FTOC, CCR9(-/-) DP cell numbers were reduced significantly. Similarly, following intrathymic injection into sublethally irradiated recipients, CCR9(-/-) DN cells were out-competed by WT DN cells in generating DP thymocytes. Finally, in competitive reaggregation thymic organ cultures, CCR9(-/-) preselection DP thymocytes were disadvantaged significantly in their ability to generate CD4 single-positive (SP) thymocytes, a finding that correlated with a reduced ability to form TCR-MHC-dependent conjugates with thymic epithelial cells. Together, these results highlight a role for CCR9 at several stages of adult thymopoiesis: in hematopoietic progenitor seeding of the thymus, in the DN-DP thymocyte transition, and in the generation of CD4 SP thymocytes.     

Onset of promiscuous gene expression in murine fetal thymus organ culture

T-cell differentiation and induction of tolerance to self-antigens occurs mainly in the thymus. Thymic stromal cells, specifically medullary thymic epithelial cells, express a diverse set of genes encoding parenchymal organ-specific proteins. This phenomenon has been termed promiscuous gene expression (PGE) and has been implicated in preventing organ-specific autoimmunity by inducing T-cell tolerance to self antigens. Early thymopoiesis and the critical factors involved in T-cell differentiation can be reproduced in vitro by murine fetal thymus organ culture (FTOC), which mimics the natural thymic microenvironment. To evaluate the occurrence of PGE in FTOC, gene expression profiling during in vitro thymic development in BALB/c mice was performed using a set of nylon cDNA microarrays containing 9216 sequences. The statistical analysis of the microarray data (sam program) revealed the temporal repression and induction of 57 parenchymal and seven lymphoid organ-specific genes. Most of the genes analysed are repressed during early thymic development (15-17 days post-coitum). The expression of the autoimmune regulator (AIRE) gene at 16 days post-coitum marks the onset of PGE. This precedes the induction of parenchymal organ genes during the late developmental phase at 20 days post-coitum. The mechanism of T-cell tolerance induction begins during fetal development and continues into adulthood. Our findings are significant because they show a fine demarcation of PGE onset, which plays a central role in induction of T-cell tolerance.     

Role of IL-2 in rat fetal thymocyte development

Early during rat thymus ontogeny, an important proportion of thymocytes expresses IL-2R and contains IL-2 mRNA. To investigate the role of the IL-2-IL-2R complex in rat T cell maturation, we supplied either recombinant rat IL-2 or blocking anti-CD25 mAb to rat fetal thymus organ cultures (FTOC) under several experimental conditions. The IL-2 treatment initially stimulated the growth of thymocytes and, as a result, induced T cell differentiation, but the continuous addition of IL-2 to rat FTOC, as well as the anti-CD25 administration, resulted in cell number decrease and inhibition of thymocyte maturation. These results indicate that immature rat thymocytes bear functional high- affinity IL-2R and that IL-2 promotes T cell differentiation as a consequence of its capacity to stimulate cell proliferation. Modifications in TCR alpha beta repertoire and increased numbers of NKR- P1+ cells, largely NK cells, were also observed in IL-2-treated FTOC. Furthermore, IL-2-responsiveness of different thymocyte subsets changed throughout thymic ontogeny. Immature CD4-CD8-cells responded to IL-2 in two stages, early in thymus development and around birth, in correlation with the maturation of two distinct waves of thymic cell progenitors. Mature CD8+ thymocytes maximally responded to IL-2 around birth, supporting a role for IL-2 in the increased proliferation of mature thymocytes observed in vivo in the perinatal period. Taken together, these findings support a role for IL-2 in rat T cell development.      

Tetrachlorodibenzo-p-Dioxin (TCDD) Inhibits Differentiation and Increases Apoptotic Cell Death of Precursor T-Cells in the Fetal Mouse Thymus

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes thymic atrophy as well as alterations in thymocyte maturity in mice. Multiple mechanisms for thymic hypocellularity have been suggested, and include an increase in thymocyte apoptosis, a maturation arrest of thymocyte development, inhibited thymocyte proliferation, and a diminution of seeding of the thymus by the hematopoietic progenitors in the fetal liver or adult bone marrow. Fetal mice are highly sensitive to hypocellularity induction by TCDD when the chemical is administered during the window of thymic development, between days 10 and 18 of gestation. Treatment of pregnant C57Bl/6 mice in the present experiments with doses of 5 or 10 mu g/kg TCDD by oral gavage on gestation days 14 and 16 severely depressed day 18 thymic cellularity. Histopathologic evaluation of day 18 fetal thymi showed disruption of the normal organ architecture with loss of clear distinction between cortical and medullary regions after TCDD. A decrease in thymocyte density was noted in all regions, and was most dramatic in the cortical zones where pyknotic cells were increased by TCDD treatment. Using day 18 thymocyte suspensions and flow cytometry, the marker 7-AAD showed a decrease in viable thymocytes from TCDD-treated fetal mice, and a concomitant and dose-related increase of thymocytes in early apoptosis. Specifically, relative to control, thymocytes from the 5 and 10 mug/kg TCDD exposure groups displayed 1.9% and 5.3% respective increases in early apoptotic cells. When thymocytes were co-identified by CD4 and CD8 cell surface antigen expression, the enhanced apoptosis occurred in the CD4(+)CD8(+) phenotype with no significant apoptosis seen in the CD4(-)CD8(-), CD4(+)CD8(-), or CD4(-)CD8(+) thymocytes. Given the rapid clearance of apoptotic cells from the thymus, these histopathologic and cytometric data suggest increased thymocyte apoptosis contributes to fetal thymic atrophy after TCDD exposure.     

Human intrathymic T cell differentiation

The human thymus develops early on in fetal gestation with morphologic maturity reached by the beginning of the second trimester. Endodermal epithelial tissue from the third pharyngeal pouch gives rise to TE3+ cortical thymic epithelium while ectodermal epithelial tissue from the third pharyngeal cleft invaginates and splits during development to give rise to A2B5/TE4+ medullary and subcapsular cortical thymic epithelium. Fetal liver CD7+ T cell precursors begin to colonize the thymus between 7 and 8 weeks of fetal gestation, followed by rapid expression on thymocytes of other T lineage surface molecules. Human thymic epithelial cells grown in vitro bind to mature and immature thymocytes via CD2 and CD11a/CD18 (LFA-1) molecules on thymocytes and by CD58 (LFA-3) and CD54 (ICAM-1) molecules on thymic epithelial cells. Thymic epithelial cells produce numerous cytokines including IL1, IL6, G-CSF, M-CSF, and GM-CSF--molecules that likely are important in various stages of thymocyte activation and differentiation. Thymocytes can be activated via several cell surface molecules including CD2, CD3/TCR, and CD28 molecules. Finally, CD7+ CD4-CD8- CD3- thymocytes give rise to T cells of both the TCRab+ and TCR gd+ lineages.