慢性重型肝炎和肝细胞肝癌患者肝移植围手术期T淋巴细胞亚群及其功能的变化

Changes of T lymphocyte subset and its function in patients with chronic severe hepatitis or hepatocellular carcinoma during the perioperation of liver transplantation

目的探讨慢性重型肝炎(慢重肝)和肝细胞肝癌(肝癌)患者肝移植围手术期T淋巴细胞亚群及其功能的变化。方法本组前瞻性研究对象为2008年9月至2009年7月在中山大学附属第三医院肝移植中心行同种异体原位肝移植术的20例患者。根据术前原发病将患者分为两组。慢重肝肝移植组(重肝组)10例,其中男8例,女2例;年龄(42±7)岁。肝癌肝移植组(肝癌组)10例,其中男9例,女1例;年龄(47±7)岁。选取同期在本院体检的10例健康人作为对照组(健康组)。患者均签署知情同意书,符合医学伦理学规定。两组肝移植患者分别在手术开始前30 min(术前)、术后1、3、7、14 d共5个时间点留取新鲜血液标本,健康组于入组时留取1次新鲜血标本。用血液标本检测外周血T淋巴细胞百分率(T细胞百分率)、CD4+CD25+Foxp3+调节性T细胞(Treg)占CD4+淋巴细胞百分率(Treg百分率)、Foxp3信使核糖核酸(mRNA)含量、γ-干扰素(IFN-γ)、白介素(IL)-4表达水平。患者免疫学检测指标比较采用方差分析或t检验。结果与健康组比较,重肝组术前T细胞百分率和IFN-γ/IL-4比值明显降低(LSD-t=4.431,3.045;P<0.05),而Treg百分率、Foxp3 mRNA明显升高(LSD-t=5.667,3.663;P<0.05)。与健康组比较,肝癌组术前T细胞百分率明显降低(LSD-t=4.464,P<0.05),而Treg百分率、Foxp3 mRNA和IFN-γ/IL-4比值差异无统计学意义(LSD-t=0.778,1.010,1.698;P>0.05)。重肝组Treg百分率和Foxp3 mRNA表达均明显高于肝癌组(LSD-t=4.253,3.117;P<0.05)。两组患者术后1 d外周血T细胞百分率明显下降,术后7 d开始回升,并在术后14 d恢复至术前水平。重肝组术后Treg百分率和Foxp3 mRNA含量均逐渐下降,至术后14 d与术前比较差异有统计学意义(t=3.265,2.842;P<0.05);肝癌组术后Treg百分率和Foxp3 mRNA含量也有下降趋势,其中Foxp3 mRNA术后14 d与术前比较,差异有统计学意义(t=2.580,P<0.05);重肝组术后各时间点Treg百分率和Foxp3 mRNA含量均明显高于肝癌组(P<0.05)。重肝组术后3 d的IFN-γ/IL-4比值明显高于术前(t=2.261,P<0.05)。肝癌组术后各时间点(术后1、3、7、14 d)IFN-γ/IL-4比值与术前比较,差异无统计学意义(P>0.05)。结论慢重肝和肝癌患者围手术期机体免疫状态存在明显差异,检测T淋巴细胞亚群和功能的变化有望成为反映患者免疫功能的重要手段,为制定个体化免疫抑制方案提供依据。

Objective To investigate the changes of T lymphocyte subset and its function in patients with chronic severe hepatitis or hepatocellular carcinoma （HCC） during the perioperation of liver transplantation. Methods Twenty patients who underwent orthotopic liver transplantation in the Third Affiliated Hospital of Sun Yat-sen University from September 2008 to July 2009 were enrolled in this prospective study. According to their primary disease, the patients were divided into 2 groups： SH group [patients with chronic severe hepatitis, n=10; 8 males and 2 females, mean age of （42±7） years old] and HCC group [patients with HCC, n=10; 9 males and 1 female, mean age of （47±7） years old]. Ten healthy volunteers received physical examination at the same period in the hospital were selected as the control group. The informed consents of all patients were obtained and the ethical committee approval was received. Patients in the SH group and HCC group were drawn blood sample at the time points of 30 min pre-transplantation, and 1, 3, 7, 14 d post-transplantation, while patients in the control group were taken blood sample when enrolled in the study. Parameters including percentage of T lymphocyte （T cell percentage）, CD4＋CD25＋Foxp3＋ regulatory T cell vs CD4＋ lymphocyte （Treg percentage）, Foxp3 mRNA level, IFN-7 and IL-4 level in peripheral blood were detected. The immunological detection indexes between two groups were compared using ANOVA analysis or t test. Results Before the transplantation, T cell percentage and IFN-7/IL-4 ratio in SH group were significantly lower, compared with that in the control group （LSD-t=4.431, 3.045; P〈0.05）, while Treg percentage and Foxp3 mRNA level raised significantly （LSD-t=5.667, 3.663; P〈0.05）. T cell percentage before transplantation in HCC group was significantly lower, compared with that in the control group （LSD-t=4.464, P〈0.05）, but there were no significant differences in Treg percentage, Foxp3 mRNA level and IFN-7/IL-4 ratio between two groups （LSD-t=0.778, 1.010, 1.698; P〉0.05）. Treg percentage and Foxp3 mRNA level in SH group were both significantly higher than that in the HCC group （LSD-t=4.253, 3.117; P〈0.05）. T cell percentage in both groups significantly decreased on day 1 after operation, and began to increase on day 7 and recovered to the preoperative level on day 14 after operation. Treg percentage and Foxp3 mRNA level decreased gradually in SH group after operation, and the value on day 14 after operation was significantly lower than that of pre-transplantation （t=3.265, 2.842; P〈0.05）; Treg percentage and Foxp3 mRNA level also decreased in HCC group after transplantation, and Foxp3 mRNA level on day 14 after transplantation was significantly lower than that of pre-transplantation （t=2.580, P〈0.05）. Treg percentage and Foxp3 mRNA level in SH group were all significantly higher compared with those in HCC group at different time point after operation（P〈0.05）. IFN-γ/IL-4 ratio in SH group significantly increased on day 3 after operation, compared with that of pre-transplantation （t=2.261, P〈0.05）. There were no significant differences about IFN-γ/IL-4 ratio in HCC group between pre-transplantation and each time point after transplantation （P〉0.05）. Conclusions The immune status between patients with severe hepatitis and patients with hepatocelluar carcinoma are obviously different. Detecting the change of T lymphocyte subgroup and its function is helpful to determine the immune status of patients and to make individual immunosuppressant protocol.