止哮平喘方对哮喘豚鼠T淋巴细胞免疫功能影响的实验研究
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摘要
目的：哮喘是以嗜酸细胞，淋巴细胞及肥大细胞浸润为主的气道慢性变态反应性炎症，其中淋巴细胞尤其是T淋巴细胞活化起关键作用。CD_4~+辅助性T细胞-2(TH_2)的激活为主要发病环节，TH_2细胞占优势的反应导致一系列由IgE介导的变应性炎症。迄今为止，对哮喘仍然首选糖皮质激素全身或局部用药，长期应用副作用明显。因此探讨中医中药防治哮喘具有广阔前景。本实验研究探讨中药复方止哮平喘方对致敏哮喘豚鼠T淋巴细胞免疫功能及相关细胞因子的作用机理，为指导临床应用提供理论基础和实验依据。
方法：复制卵蛋白致敏哮喘模型。实验动物分组：分正常对照组、哮喘模型组、地塞米松阳性对照组(西药对照组)、中药小剂量组、中药大剂量组和中西结合组，共六组。第一次诱喘24小时后开始给药，共治疗8天。采集标本。采用光镜、电镜观察支气管及肺脏的组织形态学改变；测量全血及肺泡灌洗液(BALF)中血细胞计数，以反映哮喘的炎症情况；免疫组织化学法测定外周单个核细胞(PBMC)中CD_4~+、CD_8~+T细胞的含量；原位末端标记法(TUNEL)法测定PBMC细胞中T淋巴细胞凋亡情况；原位杂交法观察IL-4、IFN－γmRNA的表达及变化情况，进一步探讨止哮平喘方对TH_1、TH_2型细胞因子的影响。
结果：1 常规病理研究显示，哮喘模型组豚鼠气道周围大量炎性细胞浸润，以嗜酸细胞和淋巴细胞为主；BALF中炎性细胞增加，而正常对照组气道基本上无炎症反应，BALF中炎性细胞数无明显变化。
2 哮喘模型组全血及BALF中炎性细胞数显著高于正常对照组(P＜0.01)；中药小剂量组与哮喘模型组比较无明显差异(P＞0.05)；中药大剂量组、中西结合组和西药对照组炎性细胞数均显著降低(P＜0.05或P＜0.01)；其中中西结合组优于中药大剂量组、西药对照组(P＜0.05)。
3 哮喘模型组TH_2细胞(CD_4~+T细胞)高于正常对照组(P＜0.05)、CD_8~+T细胞无明显差异(P＞0.05)；中药小剂量组与哮喘模型组比较无明显差异(P＞0.05)；中药大剂量组、西药对照组和中西结合组CD_4~+T细胞均显著降低(P＜0.05)、CD_8~+T细胞无明显差异(P＞0.05)。其中中西结合组优于中药大剂量组、西药对照组(P＜0.05)。
4 哮喘模型组T淋巴细胞凋亡率显著低于正常对照组(P＜0.01)；中药小剂量组与哮喘模型组比较无明显差异(P＞0.05)；中药大剂量组、西药对照组和中西结合组T淋巴细胞凋亡率均显著降低(P＜0.05)。
5 哮喘模型组IL-4 mRNA表达显著高于正常对照组(P＜0.05)、IFN-γmRNA无明显差异(P＞0.05)；中药小剂量组与哮喘模型组比较无明显差异(P＞0.05)；中药大剂量组、西药对照组和中西结合组IL-4 mRNA均显著降低(P＜0.05)、IFN-γmRNA无明显差异(P＞0.05)；其中中西结合组优于中药大剂量组、西药对照组(P＜0.05)。
6 病理研究显示：哮喘模型组气道炎症明显、管腔狭窄、肺泡隔明显增宽、平滑肌增厚；中药大剂量组和西药对照组、中西结合组均能显著减轻大、小气道及肺组织炎症情况，支气管肺组织大致正常；而中药小剂量组则变化较小。
结论：以卵蛋白致敏复制豚鼠哮喘模型是成功的。大剂量中药止哮平喘方通过诱导T淋巴细胞凋亡；降低TH_2细胞(CD_4~+T细胞)数量、相对增强CD_8~+T细胞；抑制IL-4mRNA表达，相对增加IL-4mRNA表达，调节TH_1／TH_2型细胞因子失衡；进而降低体内IgE水平。
达到调节免疫功能、减轻气道局部炎症。中西结合组疗效明显优于大剂量中药止哮平喘方。
Objective Asthma is a chronic airway allergic inflammation with eosinophilics and lymphocytes infiltration.Among them the activation of T lymphocytes, especially CD/ helper cells (Th2) play the most important role in the inducement and maintenance of asthmatic airway inflammation.It's the prevailing reaction of Th-, cells that result in a course of inflammation changes mediated by IgE.Up to now, dexamethasone (DM) is still the first choice to the treatment of asthma with partial or total used,but its side -effects are obvious.TCM has got many useful theories and experiences to the treatment of asthma, so the therapy of TCM should be paid more. The study is to investigate the effects of zhixiaopingchuanfang on immune function of T lymphocyte and related cytokines in guinea-pigs asthma model, and to study the immune regulating and controlling mechanism of zhixiaopingchuanfang in the treatment of bronchial asthma.
Methods The guinea-pigs asthma model was established with ovalbumin challenge method The guinea-pigs were divided into 6 groups randomly:normal control group , asthma group, DM-treated group, small-dose group of TCM,high-dose group of TCM and integrative group The medicines were gived from the next day of the first inducement to the 8th day.
1. The pathologic change of the bronchioles and lung tissues were observed under optical and electron microscope.
2 The total and differential white blood cells counts of blood and bronchoalveolar lavage fluid (BALF) were carried out.
3 CD/. CD/T cells counts were detected with immunocyutochemistry (ICC) in the peripheral blood mononuclear cells (PBMC).
4 Apoptosis of T lymphocyte was detected by TdT-mediated dUTP nick and labeling in PBMC
5 Expression of IL-4. IFN- v mRNA was detected by in situ hybridization staining Results 1 Significant infiltration of eosinphils and lymphocytes was observed in
airways and lung tissues in asthma group, and the total cell counts especially eosinphils and lymphocytes in BALF were significantly increased. No inflammatory changes were observed in airway in normal control group ,there was no remarkable changes in the inflammatory cells in BALF
2 The inflammatory cells in blood and BALF in asthma group were significantly increased than in normal group(p<0.01) There was no remarkable difference in inflammatory cells between the small-dose group and the asthma group(p>0.05) The inflammatory cells were significantly decreased in the high-dose group. DM-treated group and the integrative group than in the asthma group(p<0 05 or p<0.01).The effect of the integrative group was better than the high-dose group and DM-treated group(p<0 05)
3 The number of CD/ cells (Th2) was significantly increased in asthma group than in the normal group (p<0.05). but there was no remarkable difference in the number of CD/ cells (Thl) between the asthma group and the normal group (p>0 05) .The number of CD/ cells was significantly decreased in the high-dose group. DM-treated group and the integrative group than
in the asthma group(p<0.05), but there was no remarkable difference in CDS+ cells between them(p>0.05) .The effect of the integrative group was better than that of the high-dose group and the DM-treated group(p<0.05).
4.The percentages of apoptosis cells in T lymphocytes were significantly decreased in the asthma group than in the normal group(p<0.01) ,but there was no remarkable difference between the small-dose group and the asthma group (p>0.05).The percentages of apoptosis cells in T lymphocytes were significantly increased in the high-dose group > DM-treated group and integrative group(p<0.05).
5.The IL-4 mRNA expression was significantly increased in the asthma group than in the normal group(p<0.05), but there was no remarkable difference in the expression of IFN- Y mRNA between them (p>0.05). There was no remarkable difference between the small-dose group and the asthma group (p>0.05).The IL-4mRNA expression were significantly decreased in the high-dose group > DM-treated group and the i
方法：复制卵蛋白致敏哮喘模型。实验动物分组：分正常对照组、哮喘模型组、地塞米松阳性对照组(西药对照组)、中药小剂量组、中药大剂量组和中西结合组，共六组。第一次诱喘24小时后开始给药，共治疗8天。采集标本。采用光镜、电镜观察支气管及肺脏的组织形态学改变；测量全血及肺泡灌洗液(BALF)中血细胞计数，以反映哮喘的炎症情况；免疫组织化学法测定外周单个核细胞(PBMC)中CD_4~+、CD_8~+T细胞的含量；原位末端标记法(TUNEL)法测定PBMC细胞中T淋巴细胞凋亡情况；原位杂交法观察IL-4、IFN－γmRNA的表达及变化情况，进一步探讨止哮平喘方对TH_1、TH_2型细胞因子的影响。
结果：1 常规病理研究显示，哮喘模型组豚鼠气道周围大量炎性细胞浸润，以嗜酸细胞和淋巴细胞为主；BALF中炎性细胞增加，而正常对照组气道基本上无炎症反应，BALF中炎性细胞数无明显变化。
2 哮喘模型组全血及BALF中炎性细胞数显著高于正常对照组(P＜0.01)；中药小剂量组与哮喘模型组比较无明显差异(P＞0.05)；中药大剂量组、中西结合组和西药对照组炎性细胞数均显著降低(P＜0.05或P＜0.01)；其中中西结合组优于中药大剂量组、西药对照组(P＜0.05)。
3 哮喘模型组TH_2细胞(CD_4~+T细胞)高于正常对照组(P＜0.05)、CD_8~+T细胞无明显差异(P＞0.05)；中药小剂量组与哮喘模型组比较无明显差异(P＞0.05)；中药大剂量组、西药对照组和中西结合组CD_4~+T细胞均显著降低(P＜0.05)、CD_8~+T细胞无明显差异(P＞0.05)。其中中西结合组优于中药大剂量组、西药对照组(P＜0.05)。
4 哮喘模型组T淋巴细胞凋亡率显著低于正常对照组(P＜0.01)；中药小剂量组与哮喘模型组比较无明显差异(P＞0.05)；中药大剂量组、西药对照组和中西结合组T淋巴细胞凋亡率均显著降低(P＜0.05)。
5 哮喘模型组IL-4 mRNA表达显著高于正常对照组(P＜0.05)、IFN-γmRNA无明显差异(P＞0.05)；中药小剂量组与哮喘模型组比较无明显差异(P＞0.05)；中药大剂量组、西药对照组和中西结合组IL-4 mRNA均显著降低(P＜0.05)、IFN-γmRNA无明显差异(P＞0.05)；其中中西结合组优于中药大剂量组、西药对照组(P＜0.05)。
6 病理研究显示：哮喘模型组气道炎症明显、管腔狭窄、肺泡隔明显增宽、平滑肌增厚；中药大剂量组和西药对照组、中西结合组均能显著减轻大、小气道及肺组织炎症情况，支气管肺组织大致正常；而中药小剂量组则变化较小。
结论：以卵蛋白致敏复制豚鼠哮喘模型是成功的。大剂量中药止哮平喘方通过诱导T淋巴细胞凋亡；降低TH_2细胞(CD_4~+T细胞)数量、相对增强CD_8~+T细胞；抑制IL-4mRNA表达，相对增加IL-4mRNA表达，调节TH_1／TH_2型细胞因子失衡；进而降低体内IgE水平。
达到调节免疫功能、减轻气道局部炎症。中西结合组疗效明显优于大剂量中药止哮平喘方。
Objective Asthma is a chronic airway allergic inflammation with eosinophilics and lymphocytes infiltration.Among them the activation of T lymphocytes, especially CD/ helper cells (Th2) play the most important role in the inducement and maintenance of asthmatic airway inflammation.It's the prevailing reaction of Th-, cells that result in a course of inflammation changes mediated by IgE.Up to now, dexamethasone (DM) is still the first choice to the treatment of asthma with partial or total used,but its side -effects are obvious.TCM has got many useful theories and experiences to the treatment of asthma, so the therapy of TCM should be paid more. The study is to investigate the effects of zhixiaopingchuanfang on immune function of T lymphocyte and related cytokines in guinea-pigs asthma model, and to study the immune regulating and controlling mechanism of zhixiaopingchuanfang in the treatment of bronchial asthma.
Methods The guinea-pigs asthma model was established with ovalbumin challenge method The guinea-pigs were divided into 6 groups randomly:normal control group , asthma group, DM-treated group, small-dose group of TCM,high-dose group of TCM and integrative group The medicines were gived from the next day of the first inducement to the 8th day.
1. The pathologic change of the bronchioles and lung tissues were observed under optical and electron microscope.
2 The total and differential white blood cells counts of blood and bronchoalveolar lavage fluid (BALF) were carried out.
3 CD/. CD/T cells counts were detected with immunocyutochemistry (ICC) in the peripheral blood mononuclear cells (PBMC).
4 Apoptosis of T lymphocyte was detected by TdT-mediated dUTP nick and labeling in PBMC
5 Expression of IL-4. IFN- v mRNA was detected by in situ hybridization staining Results 1 Significant infiltration of eosinphils and lymphocytes was observed in
airways and lung tissues in asthma group, and the total cell counts especially eosinphils and lymphocytes in BALF were significantly increased. No inflammatory changes were observed in airway in normal control group ,there was no remarkable changes in the inflammatory cells in BALF
2 The inflammatory cells in blood and BALF in asthma group were significantly increased than in normal group(p<0.01) There was no remarkable difference in inflammatory cells between the small-dose group and the asthma group(p>0.05) The inflammatory cells were significantly decreased in the high-dose group. DM-treated group and the integrative group than in the asthma group(p<0 05 or p<0.01).The effect of the integrative group was better than the high-dose group and DM-treated group(p<0 05)
3 The number of CD/ cells (Th2) was significantly increased in asthma group than in the normal group (p<0.05). but there was no remarkable difference in the number of CD/ cells (Thl) between the asthma group and the normal group (p>0 05) .The number of CD/ cells was significantly decreased in the high-dose group. DM-treated group and the integrative group than
in the asthma group(p<0.05), but there was no remarkable difference in CDS+ cells between them(p>0.05) .The effect of the integrative group was better than that of the high-dose group and the DM-treated group(p<0.05).
4.The percentages of apoptosis cells in T lymphocytes were significantly decreased in the asthma group than in the normal group(p<0.01) ,but there was no remarkable difference between the small-dose group and the asthma group (p>0.05).The percentages of apoptosis cells in T lymphocytes were significantly increased in the high-dose group > DM-treated group and integrative group(p<0.05).
5.The IL-4 mRNA expression was significantly increased in the asthma group than in the normal group(p<0.05), but there was no remarkable difference in the expression of IFN- Y mRNA between them (p>0.05). There was no remarkable difference between the small-dose group and the asthma group (p>0.05).The IL-4mRNA expression were significantly decreased in the high-dose group > DM-treated group and the i
引文
1． Mosmann TR.Chewinski H.Bond MW.et al.Two types of murine T helper cell clone: a) Definition according to profiles of lymphokine activities and secreted protiens.J Immunol.1986: 136: 2348-2357
2． Maggi E.Biswas p.Del-Prete G.et al.Accumulation of TH2-like helper T cells in the conjunctive of patients with conjunctivities. J Immunel.1991: 146: 1169-
1174
3. Ying S,Durham SR,Corrigan CJ,et al. Phenotype of cells expressing mRNA for TH_2-type and TH_1-type cytokines in bronchoalveolar lavage and bronchial biopsies from atopic asthmatic and normal control subjects. Am J Respir Cell MolBiol,1995; 12: 477-487
4．薛建敏，徐永健，张珍祥，等．内源性一氧化氮对哮喘患者白细胞介素-4、5及γ干扰素表达的影响．中华内科杂志，1999；11：772-773
5．王长征，何彬，汪泽厚，等．致敏豚鼠淋巴细胞白细胞介素-5mRNA表达等活性检测。中华结核和呼吸杂志，1997；1：41
6．王群，林江涛，孙洪涛，等．哮喘患者TH亚群的失衡及与相细胞因子、肺通气功能改变的相关性研究．中华结核和呼吸杂志，2000；3：147-150
7. Kopt M. Le-Gios G,Bachmann M,et al.Disruption of murine IL-4 gene blocks TH_2 cytokine responses. Nature, 1993; 362: 245-247
8. Umetus DT, Dekruyff RH.TH_1 and TH_2 CD4+ cell in human allergic diseases.J Allergy Clin Immunol,1997; 100: 1-6
9. Gajewski TF. M Pinnas. J Wong, et al.Murine TH_1 and TH_2 clones proliferate optimally in response to distinct antigenpresenting cell populations. J Immunol,1991; 146: 1750-1758
10. Swain SL.IL-4 directs the development of TH_2 like helper effectors.J Immunol,1991; 145(11): 3796-3800
11. Anderson GP. Resolution of chronic inflammation-by therapeutic induction of apoptosis. Trends Pharmacol Sci,1996; 17: 438-442
12. Spinozzi F. Fizzotti M.Agea E,et al. Defective expression of Fas messenger RNA and Fas receptor on pulmonary T cells from patients with asthma. Annal Intern Med. 1998; 128: 363-369
13．薛建敏，徐永健，张珍祥，等．抗CD_4+人-鼠嵌合抗体对哮喘患者淋巴增殖和凋亡的影响．中华结核和呼吸杂志，1999；8：457-460
14. Benbernou N. Esnault S,Shin HC,et al. Differential regulation of IFN-gamma. IL-10 and inducible nitric oxide synthase in human T cells by cyclic AMP-dependent signal transduction pathway. Immunology, 1997; 91: 361-368
15. Nagai H.Maeda Y. Tanaka H.The effect of anti-IL-4 monoclonal antibody.rapamycin and Interferor-γ on airway hyperreactivity to acetylcholine in mice. Clin Exp Allergy. 1997; 27: 218-224
16. Nimmagadda SE.Spahn JD.Surs W. et al.Allerger exposure decreases glucocorticoid receptor binding affffinity and steroid responsiveness in atopic asthmatics. Am J Respir Cell Mol Biol, 1997; 155: 87-93
17. Upham JW. Athwahl D,Chou CC,et al.Retinoic acid selectively inhibit IL-5 receptor expression during differentiation of hemopoitic progenitor cells.J Allergy Clin Immunol.1998; 101 suppl: 214
18. Boguniewicz M.Martin RJ,Martin D,et al. The effects of nebulized recombinant interferon-gamma in asthmatic airways. J Allergy Clin Immunol. 1995; 95: 33-135
19. Fahy JV, Fleming HE.Wong HH,et al.The effect of an anti-IgE monoclonal antibody on the early and late phase response to allergen inhalation in asthmatic subjects. Am J Respir Crit Care Med, 1997; 155: 1828-1834
2． Maggi E.Biswas p.Del-Prete G.et al.Accumulation of TH2-like helper T cells in the conjunctive of patients with conjunctivities. J Immunel.1991: 146: 1169-
1174
3. Ying S,Durham SR,Corrigan CJ,et al. Phenotype of cells expressing mRNA for TH_2-type and TH_1-type cytokines in bronchoalveolar lavage and bronchial biopsies from atopic asthmatic and normal control subjects. Am J Respir Cell MolBiol,1995; 12: 477-487
4．薛建敏，徐永健，张珍祥，等．内源性一氧化氮对哮喘患者白细胞介素-4、5及γ干扰素表达的影响．中华内科杂志，1999；11：772-773
5．王长征，何彬，汪泽厚，等．致敏豚鼠淋巴细胞白细胞介素-5mRNA表达等活性检测。中华结核和呼吸杂志，1997；1：41
6．王群，林江涛，孙洪涛，等．哮喘患者TH亚群的失衡及与相细胞因子、肺通气功能改变的相关性研究．中华结核和呼吸杂志，2000；3：147-150
7. Kopt M. Le-Gios G,Bachmann M,et al.Disruption of murine IL-4 gene blocks TH_2 cytokine responses. Nature, 1993; 362: 245-247
8. Umetus DT, Dekruyff RH.TH_1 and TH_2 CD4+ cell in human allergic diseases.J Allergy Clin Immunol,1997; 100: 1-6
9. Gajewski TF. M Pinnas. J Wong, et al.Murine TH_1 and TH_2 clones proliferate optimally in response to distinct antigenpresenting cell populations. J Immunol,1991; 146: 1750-1758
10. Swain SL.IL-4 directs the development of TH_2 like helper effectors.J Immunol,1991; 145(11): 3796-3800
11. Anderson GP. Resolution of chronic inflammation-by therapeutic induction of apoptosis. Trends Pharmacol Sci,1996; 17: 438-442
12. Spinozzi F. Fizzotti M.Agea E,et al. Defective expression of Fas messenger RNA and Fas receptor on pulmonary T cells from patients with asthma. Annal Intern Med. 1998; 128: 363-369
13．薛建敏，徐永健，张珍祥，等．抗CD_4+人-鼠嵌合抗体对哮喘患者淋巴增殖和凋亡的影响．中华结核和呼吸杂志，1999；8：457-460
14. Benbernou N. Esnault S,Shin HC,et al. Differential regulation of IFN-gamma. IL-10 and inducible nitric oxide synthase in human T cells by cyclic AMP-dependent signal transduction pathway. Immunology, 1997; 91: 361-368
15. Nagai H.Maeda Y. Tanaka H.The effect of anti-IL-4 monoclonal antibody.rapamycin and Interferor-γ on airway hyperreactivity to acetylcholine in mice. Clin Exp Allergy. 1997; 27: 218-224
16. Nimmagadda SE.Spahn JD.Surs W. et al.Allerger exposure decreases glucocorticoid receptor binding affffinity and steroid responsiveness in atopic asthmatics. Am J Respir Cell Mol Biol, 1997; 155: 87-93
17. Upham JW. Athwahl D,Chou CC,et al.Retinoic acid selectively inhibit IL-5 receptor expression during differentiation of hemopoitic progenitor cells.J Allergy Clin Immunol.1998; 101 suppl: 214
18. Boguniewicz M.Martin RJ,Martin D,et al. The effects of nebulized recombinant interferon-gamma in asthmatic airways. J Allergy Clin Immunol. 1995; 95: 33-135
19. Fahy JV, Fleming HE.Wong HH,et al.The effect of an anti-IgE monoclonal antibody on the early and late phase response to allergen inhalation in asthmatic subjects. Am J Respir Crit Care Med, 1997; 155: 1828-1834