哮喘缓解期患者外周血T淋巴细胞亚群的增殖及其分子机制研究

Proliferation of T lymphocyte subsets in asthma patients in clinical remission and molecular mechanism thereof

目的探讨哮喘缓解期患者外周血 T 淋巴细胞亚群增殖规律及其分子机制。方法收集45例哮喘急性发作期患者(哮喘急发组)、哮喘缓解期患者(哮喘缓解组)和正常对照(对照组)的外周血标本(各15例),体外分离获得并培养3组患者的外周血 CD^+_4和 CD^+_8 T 淋巴细胞,用流式细胞术检测细胞周期时相分布、细胞周期调节蛋白[P27^(kipl)、细胞周期素(Cyclin)D、E]、PI_3K 和 STAT_6的表达水平。结果 (1)3组 CD^+_4 T 淋巴细胞的 G_0/G_1期分布比例、S 期分布比例,G_2/M 期和(S+G_2)/M 期比例差异有统计学意义(Z 值分别为12.35、8.05、9.16、12.80,均 P<0.05),3组 CD^+_8 T 淋巴细胞的 G_0/G_1期分布比例、S 期分布比例和(S+G_2)/M 期比例差异有统计学意义(Z 值分别为21.60、25.22和21.52,均 P<0.01);(2)3组 CD^+_4 T 淋巴细胞的 P27^(kipl)、Cyclin D 和 Cyclin E 表达水平差异有统计学意义(Z 值分别为10.63、10.66和6.64,均 P<0.05),3组 CD^+_8 T 淋巴细胞的 P27^(kipl)表达水平差异有统计学意义(Z 值为9.30,P<0.05);(3)3组 CD^+_4 T 淋巴细胞的 PI_3K、STAT_6蛋白表达水平差异有统计学意义(Z 值分别为9.04和18.08,P 值分别<0.05和0.01),哮喘缓解期患者处于增殖状态的 CD^+_4 T 细胞较正常对照者明显增多,与哮喘发作期患者相比处于增殖状态的 CD^+_4、CD^+_8 T 细胞显著减少。PI_3K 信号转导途径可能通过上调 CyclinD 和/或 CyclinE 影响 CD^+_4、CD^+_8 T 细胞增殖活化,而 JAK1-STAT_6信号转导途径可能通过下调 P27^(kipl)发挥作用。结论哮喘缓解期患者体内 CD^+_4 T 细胞存在过度增殖,可能与细胞周期蛋白和信号转导蛋白异常有密切关系。

Objective To explore the rule of proliferation of T lymphocyte subsets in patients with clinical asthma remission and the molecular mechanism. Methods Peripheral blood samples were collected from 15 asthmatic patients, 15 asthmatic patients in clinical remission, and 15 healthy control subjects, all sex-, and age-matched. CD4^＋ T and CD8^＋ T lymphocytes were isolated. Flow cytometry was used to examine the cell cycles of CD4^＋ T and CD8^＋ T lymphocytes. Fluorescence immunohistochemistry was used to detect the expression levels of cell cycle regulatory proteins （ CCRPs）, including cyclin D, cyclin E, and P27^kipl , PI3K, and STAT6. Results （ 1 ） The percentage of G0/G1 phase of the CD4^＋ T lymphocytes of the asthmatic patients was 82.00%, significantly lower than those of the asthmatic patients in clinical remission and healthy controls （92. 50% and 99. 00%, Z= 12.35, P 〈0. 01 ）. The percentage of S phase of the CD^4＋ T lymphocytes of the asthmatic patients was 18.00% , significantly higher than those of the asthmatic patients in clinical remission and healthy controls （ 6. 10% and 0. 20% respectively, Z = 8.05, P 〈 0. 05 ）. The percentage of G2/M phase of the CD4^＋ T lymphocytes of the asthmatic patients was 2. 80%, significantly higher than those of the asthmatic patients in clinical remission and healthy controls （0.40% and 0 respectively, Z = 9. 16, P 〈 0. 05 ）. The S ＋ G2/M phase of the CD4^＋ T lymphocytes of the asthmatic patients was 18.00%, significantly higher than those of the asthmatic patients in clinical remission and healthy controls（7. 50% and 0. 20% respectively, Z = 12. 80, P 〈 0. 05 ）. The distribution of Go/Gl phase of CD8^＋T lymphocyte of the asthmatic patients was 44.60% , significantly lower than those of the asthmatic patients in clinical remission and healthy controls（95.90% and 100. 00% respectively, Z = 21.60, P 〈 0. 01 ）. The distribution of S phase of CD8^＋ T lymphocytes of the asthmatic patients was 51.70%, significantly lower than those of the asthmatic patients in clinical remission and healthy controls （0. 80% and 0 respectively, Z = 25. 22, P 〈0. 01 ）. The distribution of S ＋ G2/M phase of the CD8^＋ T lymphocytes of the asthmatic patients was 55.40% , significantly higher than those of the asthmatic patients in clinical remission and healthy controls（4. 10% and 0 respectively,Z =21.52, P 〈0. 01 ）. （2） The expression level of P27kipl of the CD4^＋ T lymphocytes of the asthmatic patients was 13. 20%, significant lower than those of the asthmatic patients in clinical remission and healthy controls （38. 80% and 47. 20% respectively, Z = 10. 63, P 〈 0. 01 ）. The expression level of cyclin D of the CD4^＋ T lymphocyte of the asthmatic patients was 35.00%, significant higher than those of the asthmatic patients in clinical remission and healthy controls （28. 20% and 13.10% respectively, Z = 10. 66, P 〈 0. 01 ）. The expression level of cyclin E of the CD4^＋ T lymphocytes of the asthmatic patients was 7. 90%, significant higher than those of the asthmatic patients in clinical remission and healthy controls （6. 30% and 3. 70% respectively, Z = 6. 64% , P 〈 0. 05 ）. The expression level of P27kipl of the CD8^＋ T lymphocyte of the asthmatic patients was 4. 50%, significant lower than those of the asthmatic patients in clinical remission and healthy controls （33.80% and 46. 30% respectively, Z =9. 30, P 〈 0. 05 ）. The expression level of cyclin D of the CD8^＋ T lymphocyte of the asthmatic patients was 24. 20%, not significant different from those of the asthmatic patients in clinical remission and healthy controls （26. 10% and 32. 20% respectively, Z =0. 09, P 〉0. 05）. The expression level of cyclin E of the CD8^＋ T lymphocyte of the asthmatic patients was 9. 30%, significant higher than those of the asthmatic patients in clinical remission and healthy controls （5.60% and 3.50% respectively, Z =4. 91, P 〉0. 05）. （3） The expression level of PI3K-110αof the CD4^＋ T lymphocyte of the asthmatic patients was 7. 60%, significant higher than those of the asthmatic patients in clinical remission and healthy controls （6. 40% and 3.30% respectively, Z = 9. 04, P 〈 0. 05 ）. The expression level of STST6 of the CD4^＋ T lymphocyte of the asthmatic patients was 8. 20%, significant higher than those of the asthmatic patients in clinical remission and healthy controls （2. 70% and 1.90% respectively, Z = 18.08, P 〉 0.01 ）. The expression levels of PI3K-110α and STST6 of the CD8^＋ T lymphocytes of the asthmatic patients were not significant different from those of the asthmatic patients in clinical remission and healthy controls （ Z = 4. 91 and 5.70, both P 〉 0. 05）. Conclusion There is excessive proliferation of CD4^＋T lymphocytes in the patients with clinical asthma remission, which may be related to the abnormal expression of CCRP （ cyclin D, cyclin E, and P27^kipl） , PI3K, and STAT6. A