辅助性T淋巴细胞9在慢性丙型肝炎初治患者中的变化及临床意义

Change and clinical significance of T helper 9 cells in previously untreated patients with chronic hepatitis C

目的观察辅助性T淋巴细胞9(Th9)、IL-9及相关转录因子在慢性丙型肝炎初治患者中的变化,评估与临床指标的相关性。方法选取2018年12月-2019年7月于海南省人民医院就诊的慢性丙型肝炎初治患者29例和健康对照者15例,慢性丙型肝炎患者接受索磷布韦/维帕他韦抗病毒治疗12周,分离血浆和外周血单个核细胞(PBMC)。流式细胞术检测PBMC中CD3+CD4+IL-9+的Th9比例,ELISA检测血浆中IL-9水平,实时定量PCR法检测PBMC中IL-9和转录因子PU.1、Foxo1的mRNA相对表达量。两组间比较采用t检验或配对t检验,采用Pearson检验进行相关性分析。结果慢性丙型肝炎初治患者外周血Th9比例明显低于健康对照者(0.92%±0.14%vs 1.14%±0.21%,t=4.31,P<0.001),血浆IL-9水平亦明显低于健康对照者[(248.27±66.97)pg/ml vs(309.02±88.48)pg/ml,t=2.63,P=0.012]。慢性丙型肝炎初治患者PBMC中IL-9和PU.1 mRNA相对表达量均明显低于健康对照者(t值分别为20.67、23.21,P值均<0.001),但Foxo1 mRNA相对表达量在慢性丙型肝炎初治患者和健康对照者之间的差异无统计学意义(t=0.704,P>0.05)。慢性丙型肝炎初治患者外周血Th9比例、IL-9水平、IL-9 mRNA和PU.1 mRNA与HCV RNA呈负相关(r值分别为-0.46、-0.38、-0.52、-0.41,P值均<0.05),但与ALT水平无明显相关性(P值均>0.05)。索磷布韦/维帕他韦抗病毒治疗使29例慢性丙型肝炎患者均达到病毒学应答,抗病毒治疗后外周血Th9比例和PU.1 mRNA均明显高于基线(t值分别为2.20、6.52,P值均<0.05),但血浆IL-9水平和IL-9 mRNA相对表达量在基线和治疗后差异均无统计学意义(P值均>0.05)。结论慢性HCV感染可能抑制Th9活化,Th9可能参与了HCV感染慢性化。

Objective To investigate the changes of T helper 9(Th9)cells,interleukin-9(IL-9),and related transcription factors in previously untreated patients with chronic hepatitis C,as well as their association with clinical indices.Methods A total of 29 previously untreated patients with chronic hepatitis C who attended Hainan Provincial People’s Hospital from December 2018 to July 2019 were enrolled,and 15 healthy individuals were enrolled as healthy controls.The patients with chronic hepatitis C received sofosbuvir/velpatasvir antiviral therapy for 12 weeks,and then plasma and peripheral mononuclear cells(PBMCs)were isolated.Flow cytometry was used to measure the percentage of CD3+CD4+IL-9+Th9 cells in PBMCs;ELISA was used to measure the plasma level of IL-9;quantitative real-time PCR was used to measure the relative mRNA expression of IL-9 and the transcription factors PU.1 and Foxo1 in PBMCs.The t-test or the paired t-test was used for comparison between two groups,and a Pearson correlation analysis was used to investigate correlation.Results Compared with the healthy controls,the previously untreated chronic hepatitis C patients had significantly lower percentage of peripheral Th9 cells(0.92%±0.14%vs 1.14%±0.21%,t=4.31,P<0.001)and plasma IL-9 level(248.2±66.97 pg/ml vs 309.02±88.48 pg/ml,t=2.63,P=0.012).The previously untreated chronic hepatitis C patients had significantly lower relative mRNA expression of IL-9 and PU.1 than the healthy controls(t=20.67 and 23.21,both P<0.001),while there was no significant difference in the relative mRNA expression of Foxo1 between the previously untreated chronic hepatitis C patients and the healthy controls(P>0.05).In the previously untreated chronic hepatitis C patients,the percentage of peripheral Th9 cells,IL-9 level,and mRNA expression of IL-9 and PU.1 were negatively correlated with HCV RNA(r=-0.46,-0.38,-0.52,and-0.41,all P<0.05),but they were not correlated with the level of alanine aminotransferase(all P>0.05).Sofosbuvir/velpatasvir antiviral therapy achieved virologic response in 29 chronic hepatitis C patients,and the percentage of peripheral Th9 cells and the mRNA expression of PU.1 after antiviral therapy were significantly higher than those at baseline(t=2.20 and 6.52,both P<0.05),while there were no significant changes in the plasma level of IL-9 and the relative mRNA expression of IL-9 from baseline to after treatment(both P>0.05).Conclusion Chronic hepatitis C virus infection may suppress the activation of Th9 cells,suggesting that Th9 cells might be involved in the chronicity of hepatitis C virus infection.