【作者】 李红梅；

【导师】 孔宪涛； 陈慰峰；

【作者基本信息】 第二军医大学 ， 临床检验诊断学， 2003， 博士

【摘要】 胸腺是T细胞发育的主要场所，胸腺前体细胞由骨髓迁入胸腺，经历一系列克隆选择，最终产生成熟的功能性T细胞，迁出胸腺，形成外周T细胞库。胸腺内按照细胞表面标志的变化，α β T细胞的发育过程可分为以下步骤：CD4~-CD8~-→CD4~+CD8~+→CD4~+CD8~-或CD4~-CD8~+。一旦CD4~+CD8~+胸腺细胞表达克隆特异的TCR α β，这些细胞就经历阳性、阴性选择，上调TCR分子表达，下调CD4或CD8分子。由胸腺皮质迁入髓质细胞并非过去认为的是处于一种静止状态，而是要经历表型乃至功能的进一步成熟，才能成为功能成熟的T细胞。抗TCR单克隆抗体是有效的免疫耐受诱导剂，能减少T细胞数量和阻断T细胞功能，明显造成机体的免疫抑制状态。本文根据抗TCR单克隆抗体对正常BALB／C及自身免疫性糖尿病模型NOD小鼠胸腺细胞阴性选择的诱导研究，得出以下结论。 1．抗TCR抗体、PMA、Con A诱导胸腺及外周T细胞的活化分裂，我们用CFSE标记细胞、流式细胞仪分析法检测，证明此法不仅可以测定单细胞水平上细胞的分裂，还可根据荧光强度判断细胞的分裂次数。 2．正常BALB／C小鼠胸腺不同亚群T细胞对抗TCR抗体诱导敏感性有差异，CD3~-CD4~-CD8~-(三阴TN)、CD8~+CD3~-、CD3~+CD4~+CD8~-(单阳SP)、CD3~+CD4~-CD8~+ SP细胞出现不同变化，TN细胞相对耐受凋亡，而CD8~+CD3~-、DP细胞对凋亡诱导敏感。 3．胸腺和脾脏表面CD3、CD4、CD8的表达与性别无明显关系，6-9周小鼠脾脏CD3~+细胞明显多于3-4周小鼠；胸腺细胞的增殖与年龄、性别无关，而3周雌性小鼠脾细胞对PMA+IONO刺激后的增殖应答比雄性明显，4-6周雄性小鼠脾细胞的增殖能力强于雌性小鼠，7-8周雌性小鼠脾细胞对抗TCR抗体的应答能力明显减小。表明免疫系统的雌雄异型可能早于青春期。第二军医大学博士论文中文摘要4.雌、雄不同性别BALB/C小鼠对相同TCR受体诱导凋亡的敏感性有差异，雄性小鼠胸腺细胞的凋亡明显于雌性。且随着大量皮质区CD4+CDS+胸腺细胞的凋亡，CD4一CDS一细胞、成熟的髓质CD4+CDS一细胞增加，CD4一cDS+细胞水平无明显改变。5.anti.CD28”2月b刺激能明显增强anti矛C尺阴月b诱导胸腺细胞的凋亡。胸腺T细胞对antiJ℃尺阴月b或anti~7’C R mAb+CD28阴Ab刺激的不同应答与细胞的成熟阶段密切相关。胸腺皮、髓质中的胸腺细胞亚群发生凋亡时，经CD28传导的胞内途径均受到影响。6.抗TCR抗体处置后，小鼠成熟髓质区高表达T细胞受体的单阳性细胞数目成倍增加，皮质区低表达T细胞受体的不成熟双阳性细胞数目减少。成熟的单阳性胸腺细胞高表达归巢受体L一selectin，表型分析(TCRQ日、CD69、HSA、V p7一integrin、Qa一2)显示增加的这群细胞为胸腺的新迁出细胞，同时小鼠外周淋巴结及脾脏CD4+、CDS‘新迁出细胞数量减少，表明抗TCR抗体能抑制胸腺T细胞向外周迁移。7.与BALB/c小鼠胸腺细胞结果相比较:抗TCR抗体诱导的增殖应答较弱，此种缺乏与年龄及NOD胸腺CD4+CDS一和CD4一CDS十SP细胞有关，rIL一2能部分恢复对TCR抗体应答的缺乏。NOD小鼠对PMA+I ONO和PMA+anti一TCR卫IAb应答正常，但对anti一TCRlllAb+I ONO应答缺乏，推测与年龄有关的NOD小鼠胸腺细胞对TCR抗体应答的缺乏与T细胞激活时上游PKC信号通路的缺乏有关。8.与BALB/c小鼠胸腺细胞结果相比较:1)双信号刺激对NOD和BALB/cCD4’CD8十亚群的明显促凋亡作用相似;而NOD小鼠髓质6C 10+HSAh‘CD4+CDS一SP细胞对anri矛C天mAb+CD28mAb介导凋亡的敏感性明显弱于BALB/c小鼠。2)体内抗 TCR抗体处置后，N OD髓质6C 10+HSA，，iCD4SP不成熟单阳细胞的凋亡耐受明显。表明NOD小鼠胸腺髓质不成熟单阳性细胞亚群的阴性选择缺乏，反映了NOD小鼠疾病的发作可能不仅与外周耐受有关、同时与胸腺细胞的中枢耐受有关。更多还原

【Abstract】 Thymus is the site for T cell differentiation, maturation, and selection. These three interrelated processes lead to the production of mature function T cells. Following selection through the pre-T-cell receptor(pre-TCR), early CD4-CD8- cells(2-3% of adult thymocytes, many of which are dividing ) acquire CD4 and CD8 on the cell surface, and become ’double-positive’(DP) thymocytes. The early, large DP cells (10-15% of all thymocytes) divide several times before they stop proliferating and become small DP cells (70% of all thymocytes). DP thymocytes make up the bulk of cells in the thymic cortex. Most DP cells die, either by defult(no positive selection) or by negative selection.A small proportion of DP cells survive after thymic selection and become CD4+CD8- or CD4-CD8+ ’single-positive’ (SP) thymocytes, the majority of which are located in the thymic medulla and express higher levels of TCR.In contrast to the cortex, the thymic medulla is generally considered to be a relatively inactive compartment, where little cell divisions occurs, cell death is minimal and cells predominantly await export to the periphery. However, this point of view has been challenged recently. The average medullery residence time of SP thymocytes is almost 14 days. During this period, final maturation process may take place. Functional maturation process of medullary-type CD4+CD8 and CD4"CD8+ thymocytes remains largely unclear. To this end, it is necessary to define the subgroups among these cells. Anti-TCR monoclonal antibodies are potent apoptosis-inducing agents at pharmacological and physiological doses on Tlymphocytes. The activation of peripheral T lymphocytes could be stimulated by anti-TCRαβAb , ConA and PMA+Ionomycin, the latter activated both T and B lymphocytes. The effects of different stimuli on the division of T lymphocytes were different, as shown in the order of: PMA+Ionomycin> ConA > anti-TCRapmAb. The same rule was also applicable to thymocyte proliferation. However, the frequency and dividing times of thymocytes were far less than those of peripheral T lymphocytes due to their different degree of cell maturity.Analysis the susceptibility of different subsets of immature mice thymocytes to undergo apoptosis. In Vitro apoptosis was induced in unfractionated mice thymocytes by anti-TCR mAbs.In Vivo apoptosis was induced in BALB/c mice by anti-TCR mAbs ,and thymocytes were examined for it by FACS. Results showed that CD4+CD8+DP thymocytes and CD4-CD8+CD3- thymocytes were equally sensitive to apoptosis after treatment with the anti-TCR mAbs. In sharp contrast, the early migrants or precursor-containing thymocytes which are CD4-CD8-CD3- TN have a lower spontaneous apoptosis rate and were relativel resistant to the anti-TCR mAbs. The findings showed a breakpoint in thymocyte sensitivity to apoptosis which occurs after the onset of CD8 expression, suggesting that susceptibility of thymocytes to apoptosis is developmentally regulated.With regard to distribution of immune cells, no significant sex-related changes were seen in thymocyte expression of CD3, CD8, CD4 or splenocyte expression of CD3 , CD8, CD4.For splenocytes, significantly more cells were positive for CD3 in 6-9 week old compared with 3-4 weekold mice. Thymocyte proliferation was not related to age or sex of the mice. For splenocytes of the 3 weeks old mice, the response to a cell of PMA and ionomycin induced a significantly greater then that by cells from females. For mice 7-8 weeks of age, splenocytes from female mice responded significantly less to stimulation by antibody to TCR. Conclusion Sexual dimorphism in the immune system may be demonstrated prior to puberty.Investigate a link between anti-TCRmAb-induced apoptosis in mice thymocytes and sex. Apoptosis of thymocytes was studied in male and female mice by in intraperitionealy injection with anti-TCRmAb and cell culture with anti-TCR raAb added, and a possible link between apoptosis sensitivity and cell cycle was also analyzed. The mice showed a slight increase in the total number of m更多还原