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(TCR
Q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。
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 T
lymphocytes. 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 week
old 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
引文
1． Wyllie AH, Kerr JFR, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol, 1980,68:251
2． McPhee D, Pye J, Shortman K, et al. The differentiation of thymocytes. V. Evidence of intrathymic death of most thymocytes. Thumus, 1991, 1:151
3． T Hunig R, Mitnacht, et al. T cell receptor-mediated selection of functional rat CD8 T cells from defined immature thymocyte precursors in short-term suspension culture. J Exp Med, 1991, 173:561-568
4． 张洪涛，王蕙芬，等.抗TCR/CD3单克隆抗体研究应用的现状与展望.国外医学免疫学分册,1996,5:239-243
5． Kishimoto HJ, Sprent. The thymus and negative selection. Immunol Rev, 2001, 21(2) : 315-323
6． Deborah J, Theresa L, Jeffrey A, et al. CD28/B7 System of T Cell Costimulation. Annu Rev Immunol, 1996, 14: 33-258．
7． Andjelic S, Jain N, J Nikolic-Zugic. Immuture thymocytes become sensitive to calcuim-madiated apoptosis with the onset of CD8, CD4, and the T cell receptor expression: a role for bcl-2? J Exp Med, 178(1993) 1745-1751
8． Strasser AW, Harris S, Cory, bcl-2 transgene inhibits T cell death and perturbs thymic self-censorship. Cell ,1991,67:889-899
9． Shinkai Y, Ma A, Cheng HL. CD3 epsilon and CD3 zeta cytoplasmic domains can independ-ently generate signals for T cell development and function. Immunity, 1995, 2(4) : 401-11
10． Freedman BD, Lin QH, Gaulton G, et al. ATP-evoked Ca2+ transients and currents in marine thymocytes possible role for p2x receptors in death by neglect. Eur J Immmunol,1999, 29(5) :1653-46
11． Michel F, Attal-Bonnefoy G, Mangino G. CD28 as a molecular amplifier extending TCR ligation and signaling capabilities. Immunity, 2001, 15(6) : 935-45
12． Sprent J, Kishimoto H. The thymus and central tolerance. Philo Trans R Sol Lond B Biol Sci, 2001, 356(1409) : 609-16
13． Wulfing C, Sumen C, Sjaastad MD, et al. Constimulation and endogenous MHC ligands contribute to T cell recognition. Nat Immunol, 2002, 3(1) : 42-7
14． Del Terra E, Francesconi A, Meli A, et al. Radiation dependent apoptosis on cultured thyroid cells .Phys Med, 2001, 1: 261-3
15． Nishzuka Y. The role of protein kinase in cell surface signal transduction and tumour promotion. Nature, 1984, 308(5961) : 693-698
16． Knight DE, Scrutton MC. Cyclic nucleotides control a system which regulates Ca2+sensitivity of platelet secretion. Nature, 1984, 309(5963) : 66-68
17． Havran WL, Poenie M, Kimura J, et al. Expression and function of the CD3-antigen recorptor on marine CD4+8+thymocytes. Nature, 1987, 330 (6144) : 171-173
18． Finkel TH, McDuffie M, Kappler JW, et al. Both immature and mature T cells mobilize Ca2+ in response to antigen receptor crosslinking. Nature, 1987,330(6144) : 179-180
19． Ali A, Garrovillo M, Oluwole OO, et al. Mechanisms of acquired thymic tolerance: induction of transplant tolerance by adoptive transfer of in vivo allomhc peptide activated syngeneic T cells. Transplantation, 2001, 71(10) : 1442-8
20． Mckean DJ, Huntoon CJ, Bell MP, et al. Maturation versus death of developing double-positive thymocytes reflects competing effects on Bcl-2
expression and can be regulated by the intensity of CD28 costimulation. J Immunol, 2001, 166(5) : 3468-3475
21． Turka LA, Ledbetter JA, Lee K, et al. CD28 is an inducibles a proliferative signal in CD3+ mature thymocytes. J Immunol, 1990, 144:1646-1653
22． Burkhardt AL, Costa T. Ig a and Ig B are functionally homologous to the signaling proteins of the T cell receptor. Mol Cell Biol, 1994, 14:1095-1103
23． Kishimoto H, Sprent J. The thymus and negative selection. Immunol Rev, 2000, 21(2) : 315-323
24． Mariathason S, Jones RG, Ohashi PS, et al. Signals involved in thymocyte positive and negative selection. Semin Immunol, 1999, 11: 263-272
25． Deborah J, Theresa L, Jeffrey A, et al. CD28/B7 System of T Cell Costimulation. Annu Rev Immunol, 1996, 14:233-258
26． Noel PJ, Alegre ML, Reiner SL, et al. Impaired negative selection in CD28-deficient mice. Cell Immunity, 1998, 187:131-138
27． Burr JS, Savage ND, Messah GE, et al. Cutting edge: distinct motifs within CD28 regulate T cell proliferation and induction of Bcl-XL. J Immunol, 2000, 166(4) : 5331-5335
28． Kishimoto H, Surh CD, Sprent J, et al. A role for Fas in negative selection of thymocytes in vivo. J Exp Med, 1998, 187:1427-1438．
29． Debatin KM, Sess D, Krammer PH, et al. Differential expression of APO-1 on human thmocytes inplication for negative selection? Eur J Immunol. 1994,24:753-758．
30． Kishimoto H, Charles D S, Sprent J, et al. Upregulation of surface markers on dying thymocytes. J Exp Med, 1995,181: 649-655．
31． 邱玉华,张学光,季玉红,等.鼠抗人CD28分子单克隆抗体的研制及生物学特性研究.细胞与分子免疫学杂志,2001,17(4) : 368-370．
32． Papadaki HA, Kritikes HD, Gemetzi C, et al. Bone marrow progenitor
rheumatoid arthritis: evidence for a tumor necrosis factor alpha-mediated effect. Blood, 2002, 99(5) : 1610-9
33． Soory M.Hormone mediation of immune responses in the progression of diabetes, rheumatoid arthritis and periodoutal diseases. Curr Drug Targets. Immune Endocr Metabol Disord, 2002, 2(1) 13-25
34． Utsuyama M, Kanno J, Inoue T, et al. Age/sex dependent and non-monotonous dose-response effect of diethylstilbestrol on the immune functions in mice. Toxicol Lett, 2002,135(1-2) : 145-53
35． Dulos GJ, Bagchus WM. Androgens indivectly accelerate thymocyte apoptosis. Int Immunopharmacol, 2001, l(2) :321-8
36． Fried J, Perez AG, Clarkson BD, et al. Rapid hypotonic method for flow cytofluorimetry of monolayer cell cultures. J Nutr, 1977, 107:1889-1895
37． 张洪涛,王蕙芬,等.抗TCR/CD3单克隆抗体研究应用的现状与展望.国外医学免疫学分册,1996, 5: 239-243
38． Kishimoto H, Sprent J. The thymus and negative selection. Immunol Rev, 2001,21:315-323
39． Cutolo M, Seriolo B, Villaggio B, et al. Androgens and estrogens modulate the immune and inflammatory responses in rheumatoid arthritis. Ann N Y Acad Sci, 2002, 966:131-42
40． Viselli SM, Olsen NJ, Olson G, et al. Androgen receptors in thymic epithelium modulate thymus size and thymocyte development. Endocrinology, 2001, 142(3) : 1278-83
41． Erlandsson MC, Ohlsson C, Gustafsson JA, et al. Role of oestrogen receptors alpha and beta in immune organ development and in oestrogen mediated effects on thymus. Immunology, 2001, 103(1) : 17-25
42． Asselin-Paturel C, Megherat S, Vergnon I, et al. Differential effect of high doses versus low doses of interleukin-12 on the adoptive transfer of human
specific cytotoxic T lymphocyte in autologous lung tumors engrafted into severe combined immunodeficiency disease-nonobese diabetic mice: relation with interleukin-10 induction. Cancer, 2001, 91(1) : 113-22
43． Chiu PP, Jevnikar AM, Danska JS. Genetic control of T and B lymphocyte activation in nonobese diabetic mice. J Immunol, 2001, 167(12) :7169-79
44． Nelson BH. Interleukin-2 signaling and the maintenance of self-tolerance. Curr Dir Autoimmun, 2002, 5:92-112．
45． Arreaza GA, Cameron MJ, Jaramillo A, et al. Neonatal activation of CD28 signaling overcomes T cell anergy and prevents autoimmune diabetes by an IL-4-dependent mechanism. J Clin Invest, 1997, 100(9) :2243-53
46． Bodey B, Body B Jr, Kaiser HE. Apoptosis in the mammalian thymus during normal histogenesis and under various in vitro and in vivo experimental conditions. In Vivo, 1998, 12(1) :123-33
47． Ramsdell R, Fowkles BJ. Clonal deletion versus clonal anergy: the role of the thymus in inducing self-tolerance. Science,1990,248:1335
48． Nelson BH.Interleukin-2 signaling and the maintenance of self-tolerance. Curr Dir Autoimmun, 2002, 5:92-112
49． Irvine RF. Nuclear lipid signaling. Sci STKE, 2002, 2002(150) :RE13． Review
50． Simon VR, Moran MF. SERCA activity is required for timely progression through G1/S. Cell Prolif, 2001, 34(1) : 15-30．
51． Galron D, Tamir A, Altman A, et al. Inhibition of PMA-induced human T cell proliferation by bryostatin is associated with enhanced degradation of conventional protein kinase C (cPKC): Ca2+ signals restore mitogenic activity without abrogating enhanced cPKC degradation. Cell Immunol, 1994, 158(1) : 195-207．
52． Nieland JD, Kruisbeek AM. A T cell lymphoma can provide potent co-
stimulatory effects to T cells that are not mediated by B7-1, B7-2, CD40, HSA or CD70． Int Immunol, 1995, 7(11) : 1827-38．
53． Rose NR. Mechanisms of autoimmunity. Semin Liver Dis, 2002, 22(4) :387-94
54． Buschard K, Hanspers K, Fredman P, et al. Treatment with sulfatide or its precursor, galactosylceramide, prevents diabetes in NOD mice. Auto-immunity, 2001,34(1) : 9-17
55． Delovitch TL, Singh B. The nonobese diabetic mouse as a model of autoimmune diabetes: immune dysregulation gets the NOD. Immunity, 1997,7:727-738
56． Hugues S, Mougneau E, Ferlin W, et al. Tolerance to islet antigens and prevention from diabetes induced by limited apoptosis of pancreatic beta cells. Immunity, 2002, 16(2) : 169-181
57． Chiu PP, Jevniker AM, Danska JS, et al. Genetic control of T and B lymphocyte activation in nonobese diabetic mice. J Immunol, 2001, 167(2) : 7169-79
58． Ridgway WM, Fasso M, Lanctot A, et al. Braking self-tolerance in nonobese diabetic mice. J Exp Med, 1996, 183: 1657-1662
59． Serreze DV, Jognson EA, Chapman HD, et al. Autoreactive diabetagenic T cells in NOD mice can efficiently expand from a greatly reduced precursor pool. Diabetes, 2001, 50(9) : 1992-2000
60． , 2002, 5(12) : 533-537
61． Kishimoto H, Sprent J. A defect in central tolerance in NOD mice. Nat Immunol, 2001, 11 (2) : 1027-1031
62． Kishimoto H, Sprent J. The thymus and negative selection. Immuno Rev, 2000, 21(2) : 315-323
63． Hayakawa K, Hardy RR. Marine CD4+T cell subsets. Immunol Rev, 1991, 123: 145-168
64． Lucas B, Vasseur F, Penit C. Production, selection, and maturation of thymocytes with high surface density of TCR. J Immunol, 1994, 153:53-62
65． Kishimoto H, Surh CD, Sprent J, et al. A role for Fas in negative selection of thymocytes in vivo. J Exp Med, 1998,187:1427-1438．
66． Mariathason S, Jones RG, Ohashi PS, et al. Signals involved in thymocyte positive and negative selection. Semin Immunol, 1999, 11: 263-272．
67． 周汝麟,薛昭华.见:毕爱华,主编,医学免疫学.人民军医出版社,1996,275-323
68． Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods, 1983,65:55
69． Claude Penit and Florence Vasseur. Sequential events in thymocyte differen-tiation and thymus regenreton revealed by a combination of bromodeoxyuridine DNA labeling and antimitic drug treatment. J Immunol, 1998,15:3315-3323
70． Abbas AK.Cellular and Molecular Immunology. 3rd ed. Philadelphia: W.B. Saunders Company, 1997, 139-169
71． Ramsdell F, et al. The majority of CD4+8-thymocytes are functionally immature. J Immunol, 1991, 147:1779．
72． Nitta Y, Nelson K, An drewa RG, et al. CFSE dye dilution mixed lymphocyte react ions quantify donor-specific alloreactive precursors in non-human primate cardiac graft rejection. Transplant Proc, 2001, 33(1-2) :326-329
73． Kanez R, SW, Nunez R, Geimm F. Flow cytometry analysis of the effect of allopurinol and dinitroaniline compound(Chloralin) on the viability and
proliferation of Leishmania infantum promastigotes. BMC Pharmacol, 2001,1(1) : 1
74． Patel HR. Analysis of protein phosphorylation patterns reveals unanticipated complexity in T lymphocyte activation pathways. J Immunol, 1991,146:3332
75． 韩作宁,刘白,马树俊,等.PMA对人外周T细胞和CD4+细胞表型影响的研究.中华微生物和免疫学杂志,1994, 14: 179-179
76． Hirst M. Transitions to informal care in Great Britain during the 1990s. J Epidemiol Community Health, 2002, 56(8) : 579-87
77． Dulos GJ, Bagchus WM. Audrogens indirectly accelerate thymocytes apoptosis. Int Immunopharmacol, 2001, 1(2) : 321-81．
78． Sakabe K, Kawashima J, Seibi K, et al. Hormone and immune response, with special reference to steroid hormone.2． Sex steroid receptors in rat thymus. J Exp Clin Med, 1998, 15(2) : 201-11
79． L Dominguez-Gerpe, M.Rey-Mendez. Age-related change in primary and secondary immune organs of the mouse. Immunol Invest, 1998, 27:153-165
80． Aspinall R, Andrew D. Gender-related differences in the rats of age associated thymic atrophy. Dev Immunol, 2001, 8(2) : 95-106
81． Cavallott D, Artico M, Lannetti G, et al. Occurrence of adrenergic nerve fibers in human thymus during immune response. Neurochem Int, 2002, 40(3) : 211-21
82． Mc Carty MF. Upregulation of lymphocyte apoptosis as a strategy for preventing and treating autoimmune disorders a role for whole-food vegan diets, fish oil and dopamine agonists. Med Hypotheses, 2001, 57(2) : 258-75
83． Oner H, Ozan E. Effect of gonadal hormones on thymus gland after bilateral ovariectomy and orchidectomy in rats. Arch Androl, 2002, 48(2) : 115-26
84． Nakayama M, Otsuka K, Sato K, et al. Activation by estrogen of the number and function of forbidden T-cell clones in intermediate T-cell receptor cells. Cell Immunol, 1996, 172(2) : 163-71
85． Kalra R, Singh SP, Kracko D, et al. Chronic self-administration of nicrotine in rats impairs T cell responsiveness. J Pharmacol Exp Ther, 2002, 302(3) : 935-9
86． Westerink RH, Klompmakers AA, Westenberg HG, et al. Signaling pathways involved in Ca (2+)-and Pb(2+)-induced vesicular catecholamine release from rat PC12 cells. Brain Res, 2002, 57(1) : 25-36