p56^lck与T细胞发育

ｐ５６~（ｌｃｋ）与Ｔ细胞发育田兰,陈慰峰（北京医科大学免疫系１０００８３）来源于骨髓的多能干细胞在胸腺内经过一系列复杂的发育过程成为功能成熟的Ｔ细胞。根据细胞表面ＣＤ４、ＣＤ８分子的表达,可将胸腺细胞分为４个不同的亚群,其发育顺序为ＣＤ４－ＣＤＳ－...     

抗凝血酶因子Ⅲ的分子生物学研究进展

ＡＴ－Ⅲ是人血浆中重要的丝氨酸蛋白酶剂,由９个α－螺旋结构,３个β－折叠,一个反应中心环组成。天然ＡＴ－Ⅲ与肝素结合使半掩埋的ＲＣＬ排出分子表面,ＡＴ－Ⅲ分子处于高抑制活性构象。ＡＴ－Ⅲ基因位于１ｑ２３－２５,长度为１９ｋｂ。外显子Ⅰ前后的ＤＮＡ序列调控ＡＴ－Ⅲ基因的表达。基因的缺失、变异将导致ＡＴ－Ⅲ分子在血浆中的浓度低下或功能异常,引发血栓性疾病。     

推进早期胚胎发育正常调控过渡的输卵管细胞因子的初步研究

本研究对勉输卵管上皮细胞分泌蛋白的特征及功能能作了初步研究和分析。ＲＯＳｓ分子量介于１３５－４４ｋＤ,可分为３组,其中第２,３组为酸性糖蛋白。经糖苷酶消化,第３组ＲＯＰＳ显著地降解为分子量接近３０ｋＤ的蛋白。利用制备的抗总ＲＯＰｓ多抗以及针对不同分子量ＲＯＰ的抗血清,分别进行功能分析发现,Ａｒｏｐ－Ｔ和Ａｒｏｐ－Ⅰ能使受精卵发育完全阻断．     

CD3单抗诱导小鼠胸腺细胞凋亡的流式细胞仪检测

采用不同浓度抗小鼠ＣＤ３ 复合物单抗刺激幼龄小鼠胸腺细胞,培养后分别在不同时间用流式细胞仪检测胸腺细胞的凋亡情况。结果表明,在ＣＤ３ 单抗诱导幼龄小鼠胸腺细胞４ 小时后,流式细胞仪即可测出凋亡细胞特有的ＡＰ峰。本项研究提示,用ＣＤ３ 单抗刺激未成熟胸腺细胞可以通过内源性的凋亡途径引起细胞死亡。未成熟Ｔ 细胞通过ＴＣＲ－ＣＤ３ 复合物与自身抗原接合激活上述过程可能与克隆清除的形成机制及自我耐受有关。     

钙调素对细胞周期的调节

ＲＣ３细胞是一种用真核表达载体Ｐ＾ｃａｍ转染ＮＩＨ３Ｔ３细胞建成的可调钙调素（Ｃａｌｍｏｄｕｌｉｎ,ＣａＭ）高表达细胞模型。通过分子杂交及蛋白免疫印迹方法证实在地塞米松（Ｄｅｘａｍｅｔｈａｓｏｍｅ,ＤＸＭ）作用下,ＲＣ３细胞可高表达ＣａＭ．ＣａＭ拮抗剂三氟拉嗪（ｔｒｉｆｌｕｏｐｒａｚｉｎｅ,ＴＦＰ）则使Ｇ１期细胞增加,Ｓ期细胞减少。高表达ＣａＭ使细胞分裂指数提高,Ｇ２期细胞减少,有丝分裂前期细胞     

反义RNA抑制人黑色素瘤细胞表面粘附分子CD44的表达及抑瘤效应

将细胞表面粘附分子ＣＤ４４Ｓ的ｃＤＮＡ反向插入到真核细胞表达载体ｐＭＡＭｎｅｏ－ＣＡＴ和ＭＭＴＶ－ＬＴＲ启动子下游,构成ＣＤ４４Ｓ的反义ＲＮＡ载体．将其用电击法导入ＣＤ４４＋的人黑色素瘤细胞系ＨＭＭ２３９,转录出的反义ＲＮＡ能不同程度地抑制ＨＭＭ２３９表面ＣＤ４４的表达．ＣＤ４４的表达被抑制后,瘤细胞与透明质酸的结合力下降,细胞的体外生长速率不受影响．将其接种裸鼠皮下,发现其致瘤性明显降低     

正常的和转化的C3H小鼠胚胎成纤维细胞neu基因结构的初步研究

应用ＰＣＲ－ＳＳＣＰ技术并结合Ｓｏｕｔｈｅｒｎ印迹杂交从基因水平对正常和￣３Ｈ－ＴｄＲ恶性转化小鼠胚胎成纤维细胞ＮＣ３Ｈ１０和ＴＣ３Ｈ１０中ｎｅｕ基因进行研究。Ｓｏｕｔｈｅｒｎ印迹杂交结果表明恶性转化的ＴＣ３Ｈ１０细胞ｎｅｕ基因出现重排和扩增,ＳＳＣＰ分析未发现ＴＣ３Ｈ１０细胞ｎｅｕ基因跨膜区突变。上述结果说明ＴＣ３Ｈ１０细胞ｎｅｕ基因结构异常可能在跨膜区外,ｎｅｕ基因异常在￣３Ｈ－ＴｄＲ诱导的细胞恶性转化过程中可能有重要的作用,ＥＧＦ可促进ｎｅｕ基因表达增高,研究发现在ＥＧＦ持续作用下,ＮＣ３Ｈ１０细胞ｎｅｕ基因甲基化水平无显著变化,说明ＥＧＦ可能是通过其它途径调控ｎｅｕ基因表达增高的,排除了ＥＧＦ通过改变ｎｅｕ基因甲基化水平而调控ｎｅｕ基因表达的可能性。     

反义RNA抑制人黑色素瘤细胞表面粘附分子CD44的表达及抑瘤效应

将细胞表面粘附分子ＣＤ４４Ｓ的ＣＤＮＡ反向插入到真核细胞表达载体ＰＭＡＭｎｅｏ－ＣＡＴ和ＭＭＴＶ－ＬＴＲ启动下游,构成ＣＤ４４Ｓ的反应ＲＮＡ载体,将其用电击法导入ＣＤ４４的人黑色素瘤细胞系ＨＭＭ２３９,转录出的反义ＲＮＡ能不同程度地换制ＨＭＭ２３９表面ＣＤ４４的表达,ＣＤ４４的表达被抑制后,瘤细胞与透明质酸的结合力下降,细胞的体外生长速率不受影响,将其接种裸鼠皮下,发现其致瘤性明显降低。     

用PT－PCR法从U937细胞系克隆CD44H的cDNA

用ＰＴ－ＰＣＲ法从Ｕ９３７细胞系克隆ＣＤ４４Ｈ的ｃＤＮＡ罗振革,高杰英,孔祥英,苏新,朱锡华（军事医学科学院微生物流行病研究所,北京１００８５０）（第三军医大学免疫教研室,重庆６３００３８）ＣＤ４４是分布于多种细胞表面的糖蛋白分子,属粘附分子的成员,...     

人胎脾交错突细胞对T,B淋巴细胞发育影响的免疫组织化学研究

用免疫酶单重和双重染色研究人胎儿脾连续切片中交错突细胞（ＩＤＣ）与Ｔ,Ｂ淋巴细胞的定位关系及ＨＬＡ－ＤＲ表达。结果表明,Ｓ－１００阳性树突状细胞为ＩＤＣ,多数表达ＨＬＡ－ＤＲ。９－１２周的胎脾中就可见到散在分布的ＩＤＣ。１３－１６周胎脾中ＩＤＣ开始定位于白髓的Ｔ细胞集落内和周缘,及Ｂ细胞集落的周边。在上述区域ＩＤＣ常与Ｔ细胞形成ＩＤＣ－Ｔ细胞聚合体。在脾的发育过程中,ＩＤＣ不仅与Ｔ,Ｂ淋巴细胞在分布上关系密切,而且可与这两类细胞形成突起－胞体、胞体－胞体的连接。提示,胎儿脾中ＩＤＣ与Ｔ,Ｂ细胞的迁移,定位及功能成熟过程有密切联系。     

The CD3gamma chain is essential for development of both the TCRalphabeta and TCRgammadelta lineages.

CD3gamma and CD3delta are the most closely related CD3 components, both of which participate in the TCRalphabeta-CD3 complex expressed on mature T cells. Interestingly, however, CD3delta does not appear to participate functionally in the pre-T-cell receptor (TCR) complex that is expressed on immature T cells: disruption of CD3delta gene expression has no effect on the developmental steps controlled by the pre-TCR. Here we report that in contrast with CD3delta, CD3gamma is an essential component of the pre-TCR. We generated mice selectively lacking expression of CD3gamma, in which expression of CD3delta, CD3epsilon, CD3zeta, pTalpha and TCRbeta remained undisturbed. Thus, all components for composing a pre-TCR are available, with the exception of CD3gamma. Nevertheless, T-cell development is severely inhibited in CD3gamma-deficient mice. The number of cells in the thymus is reduced to <1% of that in normal mice, and the large majority of thymocytes lack CD4 and CD8 and are arrested at the CD44-CD25+ double negative (DN) stage of development. Peripheral lymphoid organs are also practically devoid of T cells, with absolute numbers of peripheral T cells reduced to only 2-5% of those in normal mice. Both TCRalphabeta and TCRgammadelta lineages fail to develop effectively in CD3gamma-deficient mice, although absence of CD3gamma has no effect on gene rearrangements of the TCRbeta, delta and gamma loci. Furthermore, absence of CD3gamma results in a severe reduction in the level of TCR and CD3epsilon expression at the cell surface of thymocytes and peripheral T cells. The defect in the DN to double positive transition in mice lacking CD3gamma can be overcome by anti-CD3epsilon-mediated cross-linking. CD3gamma is thus essential for pre-TCR function.     

CD1d-independent developmental acquisition of prompt IL-4 gene inducibility in thymus CD161(NK1)-CD44lowCD4+CD8- T cells is associated with complementarity determining region 3-diverse and biased Vbeta2/Vbeta7/Vbeta8/Valpha3.2 T cell receptor usage

Among Ag-inexperienced naive T cells, the CD1d-restricted NKT cell that uses invariant TCR-alpha-chain is the most widely studied cell capable of prompt IL-4 inducibility. We show in this study that thymus CD161-CD44lowCD4+CD8- T cells promptly produce IL-4 upon TCR stimulation, a response that displays biased Vbeta(2/7/8) and Valpha3.2 TCR usage. The association of Vbeta family bias and IL-4 inducibility in thymus CD161-CD44lowCD4+CD8- T cells is found for B6, B10, BALB/c, CBA, B10.A(4R), and ICR mouse strains. Despite reduced IL-4 inducibility, there is a similarly biased Vbeta(2/7/8) TCR usage by IL-4 inducibility+ spleen CD161-CD44lowCD4+CD8- T cells. Removal of alpha-galacotosylceramide/CD1d-binding cells from CD161-CD44lowCD4+CD8- thymocytes does not significantly affect their IL-4 inducibility. The development of thymus CD161-CD44lowCD4+CD8- T cells endowed with IL-4 inducibility and their associated use of Vbeta(2/7/8) are beta2-microglobulin-, CD1d-, and p59fyn-independent. Thymus CD161-CD44lowCD4+CD8- T cells produce low and no IFN-gamma inducibility in response to TCR stimulation and to IL-12 + IL-18, respectively, and they express diverse complementarity determining region 3 sequences for both TCR-alpha- and -beta-chains. Taken together, these results demonstrate the existence of a NKT cell distinct, TCR-repertoire diverse naive CD4+ T cell subset capable of prompt IL-4 inducibility. This subset has the potential to participate in immune response to a relatively large number of Ags. The more prevalent nature of this unique T cell subset in the thymus than the periphery implies roles it might play in intrathymic T cell development and may provide a framework upon which mechanisms of developmentally regulated IL-4 gene inducibility can be studied.     

Cytokine production by mature and immature CD4-CD8- T cells. Alpha beta-T cell receptor+ CD4-CD8- T cells produce IL-4.

This study follows our previous investigation describing the production of four cytokines (IL-2, IL-4, IFN-gamma, and TNF-alpha) by subsets of thymocytes defined by the expression of CD3, 4, 8, and 25. Here we investigate in greater detail subpopulations of CD4-CD8- double negative (DN) thymocytes. First we divided immature CD25-CD4-CD8-CD3- (CD25- triple negative) (TN) thymocytes into CD44+ and CD44- subsets. The CD44+ population includes very immature precursor T cells and produced high titers of IL-2, TNF-alpha, and IFN-gamma upon activation with calcium ionophore and phorbol ester. In contrast, the CD44- subset of CD25- TN thymocytes did not produce any of the cytokines studied under similar activation conditions. This observation indicates that the latter subset, which differentiates spontaneously in vitro into CD4+CD8+, already resembles CD4+CD8+ thymocytes (which do not produce any of the tested cytokines). We also subdivided the more mature CD3+ DN thymocytes into TCR-alpha beta- and TCR-gamma delta-bearing subsets. These cells produced cytokines upon activation with solid phase anti-CD3 mAb. gamma delta TCR+ DN thymocytes produced IL-2, IFN-gamma and TNF-alpha, whereas alpha beta TCR+ DN thymocytes produced IL-4, IFN-gamma, and TNF-alpha but not IL-2. We then studied alpha beta TCR+ DN T cells isolated from the spleen and found a similar cytokine production profile. Furthermore, splenic alpha beta TCR+ DN cells showed a TCR V beta gene expression profile reminiscent of alpha beta TCR+ DN thymocytes (predominant use of V beta 8.2). These observations suggest that at least some alpha beta TCR+ DN splenocytes are derived from alpha beta TCR+ DN thymocytes and also raises the possibility that these cells may play a role in the development of Th2 responses through their production of IL-4.     

The requirement for pre-TCR during thymic differentiation enforces a developmental pause that is essential for V-DJβ rearrangement

T cell development occurs in the thymus and is critically dependent on productive TCRβ rearrangement and pre-TCR expression in DN3 cells. The requirement for pre-TCR expression results in the arrest of thymocytes at the DN3 stage (β checkpoint), which is uniquely permissive for V-DJβ recombination; only cells expressing pre-TCR survive and develop beyond the DN3 stage. In addition, the requirement for TCRβ rearrangement and pre-TCR expression enforces suppression of TCRβ rearrangement on a second allele, allelic exclusion, thus ensuring that each T cell expresses only a single TCRβ product. However, it is not known whether pre-TCR expression is essential for allelic exclusion or alternatively if allelic exclusion is enforced by developmental changes that can occur in the absence of pre-TCR. We asked if thymocytes that were differentiated without pre-TCR expression, and therefore without pause at the β checkpoint, would suppress all V-DJβ rearrangement. We previously reported that premature CD28 signaling in murine CD4(-)CD8(-) (DN) thymocytes supports differentiation of CD4(+)CD8(+) (DP) cells in the absence of pre-TCR expression. The present study uses this model to define requirements for TCRβ rearrangement and allelic exclusion. We demonstrate that if cells exit the DN3 developmental stage before TCRβ rearrangement occurs, V-DJβ rearrangement never occurs, even in DP cells that are permissive for D-Jβ and TCRα rearrangement. These results demonstrate that pre-TCR expression is not essential for thymic differentiation to DP cells or for V-DJβ suppression. However, the requirement for pre-TCR signals and the exclusion of alternative stimuli such as CD28 enforce a developmental "pause" in early DN3 cells that is essential for productive TCRβ rearrangement to occur.     

B7-CD28 interaction promotes proliferation and survival but suppresses differentiation of CD4-CD8- T cells in the thymus

Costimulatory molecules play critical roles in the induction and effector function of T cells. More recent studies reveal that costimulatory molecules enhance clonal deletion of autoreactive T cells as well as generation and homeostasis of the CD25(+)CD4(+) regulatory T cells. However, it is unclear whether the costimulatory molecules play any role in the proliferation and differentiation of T cells before they acquire MHC-restricted TCR. In this study, we report that targeted mutations of B7-1 and B7-2 substantially reduce the proliferation and survival of CD4(-)CD8(-) (double-negative (DN)) T cells in the thymus. Perhaps as a result of reduced proliferation, the accumulation of RAG-2 protein in the DN thymocytes is increased in B7-deficient mice, which may explain the increased expression of TCR gene and accelerated transition of CD25(+)CD44(-) (DN3) to CD25(-)CD44(-) (DN4) stage. Qualitatively similar, but quantitatively less striking effects were observed in mice with a targeted mutation of CD28, but not CTLA4. Taken together, our results demonstrate that the development of DN in the thymus is subject to modulation by the B7-CD28 costimulatory pathway.     

Characterization of TCR gene rearrangements during adult murine T cell development

Development of the alphabeta and gammadelta T cell lineages is dependent upon the rearrangement and expression of the TCRalpha and beta or gamma and delta genes, respectively. Although the timing and sequence of rearrangements of the TCRalpha and TCRbeta loci in adult murine thymic precursors has been characterized, no similar information is available for the TCRgamma and TCRdelta loci. In this report, we show that approximately half of the total TCRdelta alleles initiate rearrangements at the CD44highCD25+ stage, whereas the TCRbeta locus is mainly in germline configuration. In the subsequent CD44lowCD25+ stage, most TCRdelta alleles are fully recombined, whereas TCRbeta rearrangements are only complete on 10-30% of alleles. These results indicate that rearrangement at the TCRdelta locus can precede that of TCRbeta locus recombination by one developmental stage. In addition, we find a bias toward productive rearrangements of both TCRdelta and TCRgamma genes among CD44highCD25+ thymocytes, suggesting that functional gammadelta TCR complexes can be formed before the rearrangement of TCRbeta. These data support a model of lineage commitment in which sequential TCR gene rearrangements may influence alphabeta/gammadelta lineage decisions. Further, because TCR gene rearrangements are generally limited to T lineage cells, these analyses provide molecular evidence that irreversible commitment to the T lineage can occur as early as the CD44highCD25+ stage of development.