脓毒症患者外周血微小RNA-155和调节性T细胞表达的关系

Expression of microRNA-155 and regulative T cell in sepsis patients and their relationship

目的 探讨微小RNA-155（miR-155）对脓毒症患者外周血CD4 ＋CD25＋调节性T细胞（Treg）的调节作用,从而了解miR-155在脓毒症发病中的作用机制.方法 采用回顾性研究方法,选取江苏大学第四附属医院急诊科和重症监护病房（ICU）脓毒症患者60例（轻度20例、中度20例、重度20例）及20例健康对照者.于确诊后2h内取静脉血,采用流式细胞仪检测外周血CD4＋CD25＋ Treg细胞表达；采用实时荧光定量聚合酶链反应（RT-PCR）检测miR-155、Foxp3mRNA表达；采用酶联免疫吸附试验（ELISA）检测白细胞介素-10（IL-10）水平.结果 脓毒症患者外周血Treg表达率和miR-155、Foxp3mRNA表达以及IL-10水平均明显高于健康对照组[Treg：（2.89±1.13）％比（2.32±0.91）％,t=10.540,P=0.002; miR-155：1.19±0.48比0.80±0.33,t=8.605,P=0.006; Foxp3 mRNA：0.18±0.08比0.13±0.03,t=6.862,P=0.008; IL-10（ng/L）：56.89±17.28比33.24±11.93,t=12.742,P=0.001]；并且随着急性生理学与慢性健康状况评分系统Ⅱ（APACHEⅡ）评分增加而升高,重度组[Treg：（3.05±1.21）％,miR-155：1.36±0.79,Foxp3 mRNA：0.21±0.10,IL-10（ng/L）：62.82±21.38]、中度组[Treg：（2.86±0.88）％,miR-155：1.25±0.56,Foxp3 mRNA：0.17±0.08,IL-10（ng/L）：56.38±19.65]、轻度组[Treg：（2.61±0.87）％,miR-155：0.94±0.52,Foxp3 mRNA：0.15±0.05,IL-10（ng/L）：45.43±14.40]之间两两比较差异均有统计学意义（均P＜0.01）.死亡组各指标明显高于存活组[Treg：（3.46±1.53）％比（2.85±1.03）％,t=14.250,P=0.005; miR-155：1.41±0.85比1.16±0.76,t=11.875,P=0.006;Foxp3mRNA：0.24±0.11比0.17±0.09,t=8.795,P=0.001; IL-10 （ng/L）：65.47±23.58比51.70±16.86,t=16.313,P=0.001].miR-155表达与Treg和Foxp3mRNA表达水平均呈正相关（r1=0.635、P=0.007,r2=0.671、P2=0.005）.结论 miR-155参与对Treg细胞增殖的调节,在脓毒症免疫失衡机制中发挥一定的作用.

Objective To investigate the adjustment effect of microRNA-155 on CD4＋CD25＋ regulative T cell （Treg） in peripheral blood of sepsis patients, and to elucidate the role of miR-155 in the pathogenesis of sepsis. Methods A retrospective study was corducted. 60 sepsis patients （mild u=20, moderate n=20, and severe n=20） from emergency room or intensive care unit （ICU） of the Fourth Hospital of Jiangsu University were enrolled. 20 healthy volunteers were enrolled as controls. Real-time tluorescent quantitation polymerase chain reaction （qRT-PCR） was used to detect the levels of miR-155 and Foxp3 mRNA expressions in peripheral blood. CD4＋CD25＋ Treg cells in peripheral blood were identified by flow cytometry. Peripheral interleukin-10 （IL-10） was measured by enzyme-linked immunosorbent assay （ELISA）. Results The expressions of miR-155, Treg, Foxp3 mRNA and the level of IL-10 were higher in the patients with sepsis than those in healthy control group [Treg： （2.89 ± 1.13 ）% vs. （2.32 ± 0.91 ）%, t=10.540, P=0.002; miR-155：1.19 ±0.48 vs. 0.80 ±0.33, t=8.605, P=0.006; Foxp3 mRNA： 0.18 ±0.08 vs. 0.13 ±0.03, t=6.862, P=0.008; IL-10 （ng/L）： 56.89 ± 17.28 vs. 33.24 ± 11.93, t=12.742, P=0.001]. These variables were elevated gradually with the elevation of acute physiology and chronic health evaluation II （APACHE II ） score. The expressions of Treg, miR-155, Foxp3 mRNA and the level of IL-10 were （3.05 ± 1.21）%, 1.36 ± 0.79, 0.21 ± 0.10, （62.82 ± 21.38） ng/L in severe sepsis group; they were （2.86 ± 0.88）%, 1.25 ± 0.56, 0.17 ± 0.08, （56.38 ± 19.65） ng/L in moderate group; they were （2.61 ± 0.87）%, 0.94 ± 0.52, 0.15 ± 0.05, （45.43 ± 14.40） ng/L in mild group. The values showed significant statistical difference among the mild, moderate and severe sepsis groups （all P〈 0.01 ）. Above values were significantly higher in non-survival group than those in survival group [ Treg： （3.46 ± 1.53）% vs. （2.85 ± 1.03）%, t=14.250, P=0.005; miR-155：1.41 ±0.85 vs. 1.16 ±0.76, t=11.875, P=0.006; Foxp3 mRNA： 0.24 ± 0.11 vs. 0.17 ± 0.09, t=8.795,P=0.001 ; IL-10 （ng/L） ： 65.47 ± 23.58 vs. 51.70 ± 16.86, t= 16.313, P=0.0013. The expression of miR-155 was positively correlated with the expression of CD4＋CD25＋ Treg and Foxp3 mRNA （r1=0.635, P1=0.007; r2=0.671, P2=0.005）. Conclusion The result of this study suggest that miR-155 is involved in the cell proliferation regulation of CD4 ＋CD25 ＋ Treg cells, and play some role in the immunological dissonance in sepsis.