胸腺细胞在胸腺外凋亡的形态学证据

In vitro thymus explants culture designed in this paper can mimic the thymic microenvironment as it were in vivo. Theoretically, thymus explants are cut off free from blood stream. So if some developing or developed thymocytes had the inclination to migrate into the periphery, they would only be accumulated in the blood vessels within thymus explants. After 3-day's culture, under transmission electron microscope we observed the migrating thymocytes accumulated in the blood vessels of C57BL/6 mice thymus explants, and these thymocytes were occurring apoptosis at different stage. To our knowledge, this findings offers the first morphological evidence that thymocytes do not necessarily die inside the thymus in situ, and that having acquired the death signals thymocytes can migrate into the blood stream and die quickly outside the thymus. But this is not to say that we deny the intrathymic death hypothesis. On the contrary, we found the number of thymocytes occurring in situ apoptosis on the surfaces of stromal cells is far more than that of migrating into the blood vessels. So, our proposal is that there are two sites for thymocytes apoptosis, some die inside the thymus and the others die outside the thymus.     

IL-7和胸腺基质细胞系(MTEC5)诱导胸腺CD3- CD4- CD8- 细胞分化

胸腺细胞发育是细胞因子及胸腺细胞与胸腺基质细胞相互作用的结果.观察了IL-7及小鼠胸腺基质上皮细胞系MTEC5对成年小鼠CD3^- CD4^- CD8^- 胸腺细胞发育的影响.IL-7能促进TN细胞增殖.TN细胞经在IL-7条件下培养后表达CD3-TCR分子,其中20％～40％为TCRγδ⁺,60％～80％为TCRαβ⁺,但保持CD4^- CD8^- .该细胞获得对 Con A、抗CD3mAb及抗TCRmAb进行增殖应答的能力,抗CD28mAb能促进这种增殖应答,证明表达的CD3-TCR分子功能成熟.当在该系统中加入MTEC5,部分TN细胞转变为CD3⁺TCRβ⁺CD4⁺CD8^- 和CD3⁺TCRβ⁺CD4^- CD8⁺SP细胞,表明MTEC5可诱导TN细胞分化为表现型成熟的胸腺细胞.     

小鼠胸腺髓质上皮细胞系体内诱导胸腺细胞功能成熟

来源于BALB/c小鼠 (H-2^d)胸腺的髓质上皮细胞系MTEC1细胞表达H-2^d和I -A^d 分子 .为研究上皮细胞诱导胸腺细胞阳性选择及功能分化的能力 ,首先用γ 线照射C5 7BL/6J小鼠(H-2^d) ,再经静脉注射 (H-2^b×d)F1代骨髓细胞 ,制备 H-2^b×d→H-2^b)嵌合体 ,然后将MTEC1细胞注射到嵌合体胸腺内 .注射后 2个月 ,经HE染色显示 ,MTEC1细胞在嵌合体小鼠胸腺被膜下皮质区成簇存在 ,体外免疫学试验发现 ,嵌合体小鼠脾细胞含有抗原特异性H-2^d识别限制的杀伤T细胞 (CTL) ,IL -2产生T细胞及增殖应答T细胞 .在MLR试验中 ,嵌合体小鼠脾细胞显示对H-2^d 同种异型抗原的明显耐受 .从而证明MTEC1髓质上皮细胞能在胸腺微环境内诱导H-2^d识别限制的对特异抗原应答的T细胞发育及功能成熟 .由此提出在胸腺髓质区进行“2次胸腺选择”的假说 ,并讨论了其意义 .     

胸腺树突状细胞对胸腺细胞凋亡的促进作用

小鼠胸腺细胞在体外培养一定时间后自发出现凋亡,在与小鼠胸腺树突状细胞(MTSC4)共育后,其凋亡过程可被明显加速;与此相反,小鼠胸腺上皮细胞(MTECI)有抑制凋亡的作用.与MTSC4共育后的胸腺细胞中不仅CD4⁺CD8⁺双阳性细胞明显减小,而且CD4⁺CD8⁺单阳性细胞也减少.提示胸腺基质细胞可通过其对细胞凋亡的促进作用参与胸腺内的阴性选择,并提示阴性选择可能在胸腺髓质区仍在进行.     

EB病毒诱导胸腺恶性T细胞淋巴瘤的研究

为研究EB病毒在恶性T细胞淋巴瘤发生中的作用,将EB病毒感染的人胚胸腺细胞移植于Scid鼠皮下,于移植后第3日起在移植处对侧皮下注射TPA50ng／只,每周1次。于移植后第4周起,移植处皮下有结节状隆起形成,并逐渐增大。于6－15周内行病理学检查和免疫组织化学染色,证实为T细胞淋巴瘤8例。其中实验组胸腺细胞＋EBV的成瘤率为25％（1／4）,胸腺细胞＋EBV＋TPA组的成瘤率为53.8%（7／13）,对照组胸腺细胞＋TPA的成瘤率为0（0／5）。PCR和 闰杂交在诱导肿瘤可可检测到EB病毒的基因EBERs、LMP1和BARF1,并有病毒基因LMP1蛋白编码产物的表达。EB病毒可感染人胚胸腺细胞,并使其发生恶性转化,EB病毒可能在恶性T细胞淋巴瘤的发生中起病因作用。     

IL—7对早期胸腺细胞发育的作用

胸腺基质细胞分泌白细胞介素７（ＩＬ－７）。ＩＬ－７是早期Ｔ细胞的重要生长因子之一。它能选择性促进ＣＤ４＾－ＣＤ８＾－和ＣＤ４＾＋ＣＤ８＾－／ＣＤ４＾－ＣＤ８＾＋细胞增殖。ＩＬ－７主要维持早期前体Ｔ细胞存活,同时保持其Ｔ前体细胞潜能。ＩＬ－７结胸腺细胞分化的作用目前仍有争议。     

IL－7对早期胸腺细胞发育的作用

胸腺基质细胞分泌白细胞介素７（ＩＬ－７）。ＩＬ－７是早期Ｔ细胞的重要生长因子之一。它能选择性促进ＣＤ４－ＣＤ８－和ＣＤ４＋ＣＤ８－／ＣＤ４－ＣＤ８＋细胞增殖。ＩＬ－７主要维持早期前体Ｔ细胞存活,同时保持其Ｔ前体细胞潜能。ＩＬ－７对胸腺细胞分化的作用目前仍有争议。     

地噻咪松诱导乳鼠胸腺细胞凋亡的研究

本文用透射电镜的方法,研究了地噻咪松诱导乳鼠胸腺细胞的凋亡,并确立了乳腺胸腺细胞中凋亡细胞学形态的特征;含溴化乙锭的琼脂多糖电泳检测乳鼠胸腺细胞中凋亡细胞的断裂ＤＮＡ;末端标记法直接,持异的标记乳鼠胸腺细胞中凋亡细胞断裂ＤＮＡ。     

cAMP与小鼠胸腺细胞凋亡相关性的研究

本文采用Ｆｏｒｓｋｏｌｉｎ诱导小鼠胸腺细胞凋亡,探讨胞内ｃＡＭＰ浓度上升与细胞凋亡的相关性,采用透射电镜,ＦＣＭ和ＭＴＴ法,结果显示：Ｆｏｒｓｋｏｌｉｎ能诱导小鼠胸腺细胞凋亡,不同浓度其作用程度不同,已由ＦＣＭ证实,透射电镜显示细胞凋亡的形态学特征,Ｆｏｒｓｋｏｌｉｎ激活胞内腺苷酸环化酶,产生大量ｃＡＭＰ,其诱导细胞凋亡数目最多的浓度,ＭＴＴ法显示其光密度（ＯＤ５７０）值最高,表示细胞活性最强。我     

Effects of Chorionic Gonadotropin on Differentiation of Thymocytes in the Presence of Epithelial Thymus Cells

We studied the effects of the main placental hormone, chorionic gonadotropin, on differentiation of human thymocytes in vitro in the presence of thymic epithelial cells. It was shown that the hormone at a high dose (100 IU/ml) enhanced the epithelium-induced phenotypic maturation of thymocytes, which is registered by an increased expression of the membrane marker CD3 and transition of CD4⁺8⁺ thymocytes in the cells with CD4⁺8^– and CD4^–8⁺ phenotypes. In addition, gonadotropin enhanced the proliferative response of thymocytes to the mitogen during their cultivation with the epithelium. The stimulating effect of the hormone on the epithelium-induced differentiation of thymocytes is mediated by the humoral factors of epithelial cells. In addition, gonadotropin at this dose exerts its own differentiating activity with respect to thymocytes and stimulates their phenotypic and functional maturation in a monoculture.     

In vitro enhancing effects of thymic dendritic cells on apoptotic cell death of thymocytes

Using terminal deoxynucleotide transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay and propidium iodide-DNA staining flow cytometry assay, the effects of mouse thymic dendritic cells (MTSC4) on the process of programmed cell death of thymocytesin vitro were investigated. It was noticed that thymocytes bound to MTSC4 used in this study. That the percentages of apoptotic nuclei of the bound thymocytes on MTSC4 were much higher than those of medium-cultured thymocytes, while the bound thymocytes on mouse thymic epithelial cell (MTEC1) showed much lower percentages of apoptosis. FACS analysis quantitatively confirmed the observation. Phenotype analysis showed that MTSC4 induced the deletion of CD4 + CD8 + cells and CD4 + CD8-.cells in 18 h of coculture. The results suggest that the negative selection of medullary thymocytes may be achieved by thymic dendritic cells through their enhancing effects on apoptosis. Project supported by the National Natural Science Foundation of China (Grant No.39670685) and FokYin Tung Education Foundation.     

In vitro enhancing effects of thymic dendritic cells on apoptotic cell death of thymocytes

Ｔｈｅｍｉｃｒｏｅｎｖｉｒｏｎｍｅｎｔｃｏｎｓｔｉｔｕｔｅｄｂｙｔｈｙｍｉｃｓｔｒｏｍａｌｃｅｌｌｓｉｓａｎｉｍｐｏｒｔａｎｔｓｉｔｅｆｏｒｔｈｅｄｅｖｅｌｏｐｍｅｎｔｏｆｔｈｙｍｏｃｙｔｅｓ.95%ｏｆｔｈｙｍｏｃｙｔｅｓｄｉｅｉｎｔｈｅｔｈｙｍｕｓｅｖｅｒｙｄａｙ,ｉｎｔｈｅｗａｙｏｆａｐｏｐｔｏｓｉｓ[1].Ｔｈｅｃｅｌｌｄｅａｔｈｉｓｍａｉｎｌｙｃａｕｓｅｄｂｙｔｈｅｄｅｆａｕｌｔｏｆｐｏｓｉｔｉｖｅｓｅｌｅｃｔｉｏｎａｎｄｔｈｅａｃｔｉｏｎｏｆｎｅｇａｔｉｖｅｓｅｌｅｃｔｉｏｎｓｗｈｉｃｈａｒ…     

Molecular Mechanisms Underlying Differentiation of Thymocytes

A review of the main molecular events occurring during differentiation of T-lymphocytes in the thymus: T-cell specialization of early intrathymic precursors, formation and expression of antigen receptor, formation of antigen recognizing cell repertoire, and /- and CD4/CD8-commitment. The mechanisms of glucocorticoid-induced apoptosis of thymocytes and its blockade during antigen-dependent activation are considered. A special attention is paid to the analysis of intracellular signals underlying the clonal selection of thymocytes.     

Assembling the thymus medulla: Development and function of epithelial cell heterogeneity

The thymus is a unique primary lymphoid organ that supports the production of self-tolerant T-cells essential for adaptive immunity. Intrathymic microenvironments are microanatomically compartmentalised, forming defined cortical, and medullary regions each differentially supporting critical aspects of thymus-dependent T-cell maturation. Importantly, the specific functional properties of thymic cortical and medullary compartments are defined by highly specialised thymic epithelial cells (TEC). For example, in the medulla heterogenous medullary TEC (mTEC) contribute to the enforcement of central tolerance by supporting deletion of autoreactive T-cell clones, thereby counterbalancing the potential for random T-cell receptor generation to contribute to autoimmune disease. Recent advances have further shed light on the pathways and mechanisms that control heterogeneous mTEC development and how differential mTEC functionality contributes to control self-tolerant T-cell development. Here we discuss recent findings in relation to mTEC development and highlight examples of how mTEC diversity contribute to thymus medulla function.     

The thymic stromal cell line MTSC4 induced thymocyte apoptosis in a non-MHC-restricted manner

Mouse thymic stromal cell line 4 (MTSC4) is one of the stromal cell lines established in our laboratory. While losing the characteristics of epithelial cells, they express some surface markers shared with thymic dendritic cells (TDCs). To further study the biological functions of these cells, we compared the capability of MTSC4 with TDCs in the induction of thymocyte apoptosis, using thymic reaggregation culture system. Apoptosis of thymocytes induced by MTSC4 and TDCs was measured by Annexin V and PI staining and analyzed by flow cytometry. We found that MTSC4 selectively augmented the apoptosis of CD4^ 8^ (DP) thymocytes. This effect was Fas/FasL independent and could not be blocked by antibodies to MHC class I and class II molecules. In addition, MTSC4 enhanced the apoptosis of DP thymocytes from different strains of mice, which implies that MTSC4-induced thymocyte apoptosis is not mediated by the TCR recognition of self peptide/MHC molecules. In contrast to MTSC4, thymocyte apoptosis induced by TDCs was MHC-restricted. Thus, MHC-independent fashion of stromal-DP thymocyte interaction may be one of the ways to induce thymocyte apoptosis in thymus. Our study has also shown that the interaction of MTSC4 stromal cells and thymocytes is required for the induction of thymocyte apoptosis.     

Selective support of a mouse thymus epithelial cell line （MTEC1） to the viability and proliferation of CD4＋CD8—,CD4^—CD8^—,and CD4^＋CD8^＋thymocytes in vitro

The MTEC1 cell line,established in our laboratory,is a normal epithelial cell line derived from thymus medulla of Balb/c mice and these cells constituteively produce multiple cytokines.The selection of thymic microenvironment on developing T cells was investigated in an in vitro system.Unseparated fresh thymocytes from Balb/c mice were cocultured with MTEC1 cells or/and MTEC1-SN,then,the viability,proliferation and phenotypes of cultured thymocytes were assessed.Without any exogenous stimulus,both MTEC1 cells and MTEC1-SN were able to maintain the viability of thymocytes,while only the MTEC1 cells,not the MTEC1-SN,could directly activate thymocytes to exhibit moderate proliferation,indicating that the proliferative signal is delivered through cell surface interatcions of MTEC1 cells and thymocytes.Phenotype analysis on FACS of viable thymocytes after coculture revealed that MTEC1 cells preferentially activate the subsets of CD4^ CD8^-,CD4^ CD^8 and CD^4- CD^8- thymocytes;whereas MTEC1-SN preferentially maintained the viability of CD4^ CD^8- and CD4^-CD8^ thymocyte subsets.For the Con A-activated thymocytes.both MTEC1 cells and MTEC1-SN provided accessory signal(s) to significantly increase the number of viable cells and to markedly enhance the proliferation of thymocytes with virtually equal potency,phenotyped as CD4^ CD8^-,CD4^-CD8^ ,and CD^4-CD8^-subests,In summary,MTEC1 cells displayed Selection of thymic epithelial cells on thymocyte subsets. selective support to the different thymocyte subsets,and the selectivity is dependent on the status of thymocytes.     

Airway epithelial ITGB4 deficiency in early life mediates pulmonary spontaneous inflammation and enhanced allergic immune response

Lung immune responses to respiratory pathogens and allergens are initiated in early life which will further influence the later onset of asthma. The airway epithelia form the first mechanical physical barrier to allergic stimuli and environmental pollutants, which is also the key regulator in the initiation and development of lung immune response. However, the epithelial regulation mechanisms of early-life lung immune responses are far from clear. Our previous study found that integrin β4 (ITGB4) is decreased in the airway epithelium of asthma patients with specific variant site. ITGB4 deficiency in adult mice aggravated the lung Th2 immune responses and enhanced airway hyper-responsiveness (AHR) with a house dust mite (HDM)-induced asthma model. However, the contribution of ITGB4 to the postnatal lung immune response is still obscure. Here, we further demonstrated that ITGB4 deficiency following birth mediates spontaneous lung inflammation with ILC2 activation and increased infiltration of eosinophils and lymphocytes. Moreover, ITGB4 deficiency regulated thymic stromal lymphopoietin (TSLP) production in airway epithelial cells through EGFR pathways. Neutralization of TSLP inhibited the spontaneous inflammation significantly in ITGB4-deficient mice. Furthermore, we also found that ITGB4 deficiency led to exaggerated lung allergic inflammation response to HDM stress. In all, these findings indicate that ITGB4 deficiency in early life causes spontaneous lung inflammation and induces exaggerated lung inflammation response to HDM aeroallergen.     

Differentiation capacity of stromal fibroblast-like cells from human bone marrow, adipose tissue, hair follicle dermal papilla and derma

We compared the morphology and differentiation capacity of human stromal cells derived from bone marrow (BMSC), adipose tissue (ATSC), hair follicle dermal papilla (DPC) and dermal fibroblasts (DFb). All cells have fibroblast-like morphology. ATSC and DPC cells expressed stem cell the surface markers CD105, CD49d, and STRO-1, which were revealed immunocytochemically. CD49d was not found on BMSC. The low expression of CD49d and STRO-1 was registered in the DFb population. ATSC, BMSC, and DPC have similar capacities for adipo- and osteogenic differentiation. These cells, cultured in appropriate induction media, alter the phenotype and synthesize specific proteins. However, the expression of differentiation in the DPC population is lower than in ATSC and BMSC cultures. We propose that these cell populations have primitive progenitor cells with properties of mesenchymal stem cells.     

吸入维生素A和皮质激素对大鼠哮喘性肺炎的疗效及TSLP表达的影响

目的:观察胸腺与肺的胸腺基质淋巴细胞生成素(TSLP)和Th因子在病程发展中变化,分析吸入维生素A(VA)与皮质激素对哮喘性肺炎疗效。方法:卵蛋白激发大鼠哮喘4周后休息1周,VA与皮质激素吸入治疗1周,吸入90%乙醇液作为哮喘组。通过免疫荧光染色、组织化学染色等法检查胸腺与肺。结果:激发第4～5周后,哮喘组胸腺与肺TSLP阳性细胞、肺泡巨噬细胞(AM)较多,胸腺和脾增生,血Th2因子升高,肺部炎症逐渐加重;VA组第4周胸腺与肺TSLP和Th2因子表达均较低;第5周TSLP轻度增加,胸腺、脾T细胞区增生由强转弱,AM明显增多,肺炎症逐渐消退。激素组第4～5周胸腺和肺TSLP、Th2因子表达低,胸腺、脾增生渐强。AM数量少,肺炎症渐重。结论:激发哮喘期间TSLP表达增强伴有Th2类反应,VA和皮质激素均抑制TSLP与Th2因子表达,但VA促进T淋巴细胞增生与AM清除抗原功能,停药后肺炎症逐渐消退;皮质激素停药后炎症加重。