T淋巴细胞亚群在哮喘发病机制中的作用
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摘要
目的：探讨Th1／Th2、TC1／TC2失衡在哮喘发病机制中的作用，了解IFN-γ和糖皮质激素对其的影响。方法：采用Ficoll-Hypague密度梯度离心及贴壁培养法对18例哮喘患者和10例健康对照者外周血单个核细胞进行分离，纯化后，加入地塞米松及IFH-γ培养24小时，加入PMA，莫能菌素，艾罗霉素培养4.5小时，促使细胞内细胞因子聚集于高尔基体，取培养上清中的淋巴细胞经流式细胞仪检测分析CD4+T、CD8+T淋巴细胞及其细胞内细胞因子IL-2、IL-10、TGF-β表达率。结果：(1)CD4+T细胞亚群TH1、TH2、TH3及TH1／TH2检测结果的比较。哮喘组TH1，TH2与正常对照组比较，均显著增加(P＜0.05)，TH1／TH2显著降低(P＜0.05)，TH3显著增加(P＜0.05)。加入地塞米松培养的哮喘组TH1，TH2均显著降低(P＜0.05)，TH1／TH2无显著差异(P＞0.05)，TH3无显著差异(P＞0.05)。加入IFN-γ培养的哮喘组TH1显著增加(P＜0.05)，TH2差异无显著性(P＞0.05)，TH1／TH2显著增加(P＜0.05)，TH3显著降低(P＜0.05)。(2)CD8+T细胞亚群TC1、TC2、TC1／TC2检测结果的比较。哮喘组TC1，TC2与正常对照组比较，均显著增加
山西省医科大学硕士毕业论文
(P(0.05)，TClffCZ显著增加(P(0.05)。加人地塞米松培养
的哮喘组TCI，TCZ均显著降低(P<0 .05)，TCI汀C2无显著差
异(P>0.05)。加人IFN一y培养的哮喘组TCI显著增加
(P<0.05)，TCZ差异无显著性(P>0.05)，TCI厅C2显著增加
(P<0.05)。(3)CDs+T叮一乒及CE吟、CDS细胞内总TGF一乒检
测结果的比较。哮喘组CDS十TGF一月与正常对照组比较，显著
增高(P<0.05);加人地塞米松培养的哮喘组CDS+TGF一日显著
降低(P<0.05)，加人IFN培养的哮喘组CDS+TGF一尽的变化显
著降低，(P<0.05)，CD4、CDS细胞内总TGF一p哮喘比正常组显
著增加(P<0.05)，加地塞米松后无显著变化(P>0.05)，加
IFN一y后显著降低(P<0 .05)。结论:(1)哮喘急性发作期有
THI汀H2、TCI汀C2的比例失衡。(2)地塞米松可通过抑制哮喘
T淋巴细胞产生细胞因子缓解哮喘的炎症反应，但并不能纠正
THI汀H2、TCI/TC2的比例失衡，而且也不能降低气道重塑。
(3)IFN一y可通过纠正THI/TH2、TCI汀C2的比例失衡及抑制
TGF一日的分泌治疗哮喘，降低哮喘气道重塑。
Objective: To investigate the imbalance of TH1/TH2,TC1/ TC2 in peripheral blood of bronchial asthma and their variation under dexamethasone or IFN-7. Methods: Peripheral blood mononuclear cells from 18 asthmatic patients and 10 normal donors were isolated by density centrifugation with Ficoll - Hypague and cultivated with or without dexamethasone,
IFN - γ respectively for 24 hours. Untill the 19. 5th hours,PMA,Monesin and Ionomycin were added into the cultive palate for continuing 4.5 hours. Then,CD4+T,CD8+T and their intracellular cytokine such as IL2,IL10,TGF-β were analysed by Flow cytometery. Results: (1) The frequencies of TH1 and TH2 in asthmatic group were significantly increased than that in normal control group, TH1/TH2 lower, TH3 higher. The percentages of TH1 and TH2 in asthmatic group stimulated with dexamethasone were significantly decreased than those in asthmatic control group, but TH1 /TH2 and TH3 no significant difference. The frequencies of TH1 in asthmatic group stimulated with IFN-γ were significantly increased than that in asthmatic control group, TH2 no significant difference, TH1/TH2 significantly increased, and TH3 significantly decreased. (2) The frequencies of Tc1 and Tc2 in asthmatic group were significantly increased than those in normal control group,
Tc1/Tc2 higher. The percentages of Tc1 and Tc2 in asthmatic
group stimulated with dexamethasone were significantly decreased than those in asthmatic control group, but Tc1/Tc2 no significant difference. The frequency of Tc1 in asthmatic group stimulated with IFN-γ was significantly increased than that in asthmatic control group, Tc2 no significant difference, Tc1/Tc2 significantly increased . (3) The frequencies of CD8 + TGF-β in asthmatic group were significantly increased than that in normal control group. The percentages of CD8 + TGF -β in asthmatic group stimulated with either dexamethasone or IFN -γ were all significantly decreased than that in asthmatic control group, while in asthmatic group, TGF -β stimulated with dexmethasone in both CD4 + T and CD8 + T was not significant difference. The frequency of CD8 + TGF - β in asthmatic group stimulated with IFN -γ has no significant difference than that in asthmatic control group. Conclusion: (1)There are the imbalances of TH1/TH2 and TC1/TC2 in peripheral blood of acute asthmatic patients. (2)The production of cytokine by CD4 + T can be suppressed by dexamethasone Which can neither reverse the imbalance ofTh1/Th2 and Tc1/Tc2 nor alleviate the airway remodeling. (3) IFN -γ may play a therapic role on asthma by reversing the imbalance of Th1/Th2 and Tc1/Tc2 and reducing the production of TGF - βin Lymph-ocyte which was thought to being related to asthmatic airway remodeling.
山西省医科大学硕士毕业论文
(P(0.05)，TClffCZ显著增加(P(0.05)。加人地塞米松培养
的哮喘组TCI，TCZ均显著降低(P<0 .05)，TCI汀C2无显著差
异(P>0.05)。加人IFN一y培养的哮喘组TCI显著增加
(P<0.05)，TCZ差异无显著性(P>0.05)，TCI厅C2显著增加
(P<0.05)。(3)CDs+T叮一乒及CE吟、CDS细胞内总TGF一乒检
测结果的比较。哮喘组CDS十TGF一月与正常对照组比较，显著
增高(P<0.05);加人地塞米松培养的哮喘组CDS+TGF一日显著
降低(P<0.05)，加人IFN培养的哮喘组CDS+TGF一尽的变化显
著降低，(P<0.05)，CD4、CDS细胞内总TGF一p哮喘比正常组显
著增加(P<0.05)，加地塞米松后无显著变化(P>0.05)，加
IFN一y后显著降低(P<0 .05)。结论:(1)哮喘急性发作期有
THI汀H2、TCI汀C2的比例失衡。(2)地塞米松可通过抑制哮喘
T淋巴细胞产生细胞因子缓解哮喘的炎症反应，但并不能纠正
THI汀H2、TCI/TC2的比例失衡，而且也不能降低气道重塑。
(3)IFN一y可通过纠正THI/TH2、TCI汀C2的比例失衡及抑制
TGF一日的分泌治疗哮喘，降低哮喘气道重塑。
Objective: To investigate the imbalance of TH1/TH2,TC1/ TC2 in peripheral blood of bronchial asthma and their variation under dexamethasone or IFN-7. Methods: Peripheral blood mononuclear cells from 18 asthmatic patients and 10 normal donors were isolated by density centrifugation with Ficoll - Hypague and cultivated with or without dexamethasone,
IFN - γ respectively for 24 hours. Untill the 19. 5th hours,PMA,Monesin and Ionomycin were added into the cultive palate for continuing 4.5 hours. Then,CD4+T,CD8+T and their intracellular cytokine such as IL2,IL10,TGF-β were analysed by Flow cytometery. Results: (1) The frequencies of TH1 and TH2 in asthmatic group were significantly increased than that in normal control group, TH1/TH2 lower, TH3 higher. The percentages of TH1 and TH2 in asthmatic group stimulated with dexamethasone were significantly decreased than those in asthmatic control group, but TH1 /TH2 and TH3 no significant difference. The frequencies of TH1 in asthmatic group stimulated with IFN-γ were significantly increased than that in asthmatic control group, TH2 no significant difference, TH1/TH2 significantly increased, and TH3 significantly decreased. (2) The frequencies of Tc1 and Tc2 in asthmatic group were significantly increased than those in normal control group,
Tc1/Tc2 higher. The percentages of Tc1 and Tc2 in asthmatic
group stimulated with dexamethasone were significantly decreased than those in asthmatic control group, but Tc1/Tc2 no significant difference. The frequency of Tc1 in asthmatic group stimulated with IFN-γ was significantly increased than that in asthmatic control group, Tc2 no significant difference, Tc1/Tc2 significantly increased . (3) The frequencies of CD8 + TGF-β in asthmatic group were significantly increased than that in normal control group. The percentages of CD8 + TGF -β in asthmatic group stimulated with either dexamethasone or IFN -γ were all significantly decreased than that in asthmatic control group, while in asthmatic group, TGF -β stimulated with dexmethasone in both CD4 + T and CD8 + T was not significant difference. The frequency of CD8 + TGF - β in asthmatic group stimulated with IFN -γ has no significant difference than that in asthmatic control group. Conclusion: (1)There are the imbalances of TH1/TH2 and TC1/TC2 in peripheral blood of acute asthmatic patients. (2)The production of cytokine by CD4 + T can be suppressed by dexamethasone Which can neither reverse the imbalance ofTh1/Th2 and Tc1/Tc2 nor alleviate the airway remodeling. (3) IFN -γ may play a therapic role on asthma by reversing the imbalance of Th1/Th2 and Tc1/Tc2 and reducing the production of TGF - βin Lymph-ocyte which was thought to being related to asthmatic airway remodeling.
引文
1. Dale T, Umetsu, Jennifer J. et al. Asthma: an epidemic of dysregulated immunity. Nature immunology 2002; Aug 3(8): 715.
2．中华医学会呼吸病学分会哮喘学组。支气管哮喘防治指南(支气哮喘的定义，诊断，治疗疗效判断标准及教育和管理方案)。中华结核和呼吸杂志。1997；20：261。
3. Farrell AM, Antrobus P, Simpson D, et al. A rapid flow cytometric assay to detect CD4 + and CD8 + T helper (Th) 0, Th1 and Th2 cells in whole blood and its application to study cytokine levels in atopic dermatitis before and after cyclosporine therapy. British Journal of derm. 2001; 144 (1):24-73.
4. Sad S, Marcotte R, Mosmann TR. Cytokine-induced differentiation of precursor mouse CD8 + T cells into cytotoxic CD8 + Tcells secreting Th1 or Th2 cytokines. Immunity 1995; 2:271-279.
5. Hansen G, et al. CD4 + Th cells engineered to produce latent TGF, reverse allergen induced airway hyperreactivity and inflammation. J Clin Invest 2000; 11: 89-96.
6. Kimura M, Tsuruta S, Yoshida T. Differences in cytokine production by peripheral blood mononuclear cells between patients with atopic dermatitis and bronchial asthma. Clin Exp Immunol 1999;118:192-6.
7. David Broide, MD, Better insights into the molecular mechanisms of TH1/TH2 may lead to novel new treatments. 96th International Conference of the American Thoracic Society.
8. Adachi M, et al. Int Arch Allergy Immunol. 1999; 118:391-394.
9. Xian chang li, Gulcin Demirci et al. IL15 and IL2: a matter of life and death for T cells in vivo. Nature Medcine. 2001; 7(1): 114-118.
10. O lesur, A kokis, C hermans. Interleukine-2 involvement in early acute respiratory distress syndrome: relationship with polymorphonuclear nutrophil apoptpsis and patient survival. Critical Care Med. 2001; 28(12):3814-3822.
11. Chosh, Peripheral blood CD4 + T and CD8 + T cell type 1 and type 2 cytokine production in atopic asthmatic and normal subjects. Clin and Experi Allergy. 2002; 32 (3): 427-433.
12. Tanaka H, Masuda J, Tokuoka S, et al, The effects of allergeninduced airway inflammation on airway remodeling in a murine model of allergic asthma.. Inflam Res, 2001; 50 (12): 616-24.
13. Kenyon NJ, Kelly EA,Jarjoun NN, Enhanced cytokine generation by peripheral blood monocular cells in allergic and asthma subjects. Ann Allergy Asthma Immunol 2000 Aug:85(2): 115-20.
14. Prescott sl, macaubas c, holt bj, et al. Transplacental priming of the human immune system to environmental allergens:Universal skewing of initial T cell responses toward the TH2 cytokine profile J Immunol 1998 ; 160:4730 - 4737.
15. Maddox L,Schwartz DA, The pathophysiology of asthmas Annu Rev Med, 2002: 53: 477-98.
16. Viqnola Am, Gagliardo R, Siena A, etc, Airway remodeling in the pathogenesis of asthma, Cur Allergy Asthma Rep, 2001: 1 (2): 108-15.
17. Kraft, M, Lewis C, Rham D, et al, IL-4, IL-13 and dexamethasone augment fibroblast proliferation in asthma. Journal of Allergy and clinic Imuuno,2001; 107(4):602-606.
18. Sato Y, Kasukawa R, DNA vaccines against allergy. Nippon Rinsho. 2000; Jun 58(6): 1307-14.
19. Szabo SJ, et al. Distinct effects of T- bet in TH1 lineage commitment and IFN production in CD4 and CD8 + T cells. Science 2002; 295: 338-342.
20. Boorish LC, Nelson HS, Lanx MJ, st al. Interleukin-4 receptor in moderate atopic asthma: a phase Ⅰ/Ⅱ randoixed, placebocontrolled trial. Am J Respir crit care Med 1999; 160:1816-1823.
21. Finotto S, Neurath MF, Glickman JN, et al. Development of spontaneous airway changes consistent with human asthma in mice lacking T-bet. Science 2002; 295:336-338.
2．中华医学会呼吸病学分会哮喘学组。支气管哮喘防治指南(支气哮喘的定义，诊断，治疗疗效判断标准及教育和管理方案)。中华结核和呼吸杂志。1997；20：261。
3. Farrell AM, Antrobus P, Simpson D, et al. A rapid flow cytometric assay to detect CD4 + and CD8 + T helper (Th) 0, Th1 and Th2 cells in whole blood and its application to study cytokine levels in atopic dermatitis before and after cyclosporine therapy. British Journal of derm. 2001; 144 (1):24-73.
4. Sad S, Marcotte R, Mosmann TR. Cytokine-induced differentiation of precursor mouse CD8 + T cells into cytotoxic CD8 + Tcells secreting Th1 or Th2 cytokines. Immunity 1995; 2:271-279.
5. Hansen G, et al. CD4 + Th cells engineered to produce latent TGF, reverse allergen induced airway hyperreactivity and inflammation. J Clin Invest 2000; 11: 89-96.
6. Kimura M, Tsuruta S, Yoshida T. Differences in cytokine production by peripheral blood mononuclear cells between patients with atopic dermatitis and bronchial asthma. Clin Exp Immunol 1999;118:192-6.
7. David Broide, MD, Better insights into the molecular mechanisms of TH1/TH2 may lead to novel new treatments. 96th International Conference of the American Thoracic Society.
8. Adachi M, et al. Int Arch Allergy Immunol. 1999; 118:391-394.
9. Xian chang li, Gulcin Demirci et al. IL15 and IL2: a matter of life and death for T cells in vivo. Nature Medcine. 2001; 7(1): 114-118.
10. O lesur, A kokis, C hermans. Interleukine-2 involvement in early acute respiratory distress syndrome: relationship with polymorphonuclear nutrophil apoptpsis and patient survival. Critical Care Med. 2001; 28(12):3814-3822.
11. Chosh, Peripheral blood CD4 + T and CD8 + T cell type 1 and type 2 cytokine production in atopic asthmatic and normal subjects. Clin and Experi Allergy. 2002; 32 (3): 427-433.
12. Tanaka H, Masuda J, Tokuoka S, et al, The effects of allergeninduced airway inflammation on airway remodeling in a murine model of allergic asthma.. Inflam Res, 2001; 50 (12): 616-24.
13. Kenyon NJ, Kelly EA,Jarjoun NN, Enhanced cytokine generation by peripheral blood monocular cells in allergic and asthma subjects. Ann Allergy Asthma Immunol 2000 Aug:85(2): 115-20.
14. Prescott sl, macaubas c, holt bj, et al. Transplacental priming of the human immune system to environmental allergens:Universal skewing of initial T cell responses toward the TH2 cytokine profile J Immunol 1998 ; 160:4730 - 4737.
15. Maddox L,Schwartz DA, The pathophysiology of asthmas Annu Rev Med, 2002: 53: 477-98.
16. Viqnola Am, Gagliardo R, Siena A, etc, Airway remodeling in the pathogenesis of asthma, Cur Allergy Asthma Rep, 2001: 1 (2): 108-15.
17. Kraft, M, Lewis C, Rham D, et al, IL-4, IL-13 and dexamethasone augment fibroblast proliferation in asthma. Journal of Allergy and clinic Imuuno,2001; 107(4):602-606.
18. Sato Y, Kasukawa R, DNA vaccines against allergy. Nippon Rinsho. 2000; Jun 58(6): 1307-14.
19. Szabo SJ, et al. Distinct effects of T- bet in TH1 lineage commitment and IFN production in CD4 and CD8 + T cells. Science 2002; 295: 338-342.
20. Boorish LC, Nelson HS, Lanx MJ, st al. Interleukin-4 receptor in moderate atopic asthma: a phase Ⅰ/Ⅱ randoixed, placebocontrolled trial. Am J Respir crit care Med 1999; 160:1816-1823.
21. Finotto S, Neurath MF, Glickman JN, et al. Development of spontaneous airway changes consistent with human asthma in mice lacking T-bet. Science 2002; 295:336-338.