慢性乙型肝炎患者外周血T淋巴细胞表面共刺激分子表达量变化及其意义

The significance of the co-stimulatory molecules expression atternation in the peripharal T lymphocytes of patients with chronic hepatitis B infection

目的探讨慢性乙型肝炎患者外周血T淋巴细胞表面共刺激分子的表达量变化及其意义。方法临床病例对照研究。收集2012年10月至2013年11月在苏州大学附属第一医院感染科住院的慢性乙型肝炎患者82例，其中男50例，女32例，年龄（42．25±14．22）岁，健康对照组30名来自本院预防保健科，其中男15例，女15例，年龄（42．32±3．74）岁；采用流式细胞术和定量PCR等技术测定慢性乙型肝炎患者外周血T淋巴细胞表面CD28、CTLA4、PD．1、Tim一3的表达、T淋巴细胞亚群（CD4＋、CD8＋、CD4＋／CD8＋），其中未经抗病毒及相关免疫治疗的有40例，经过抗病毒治疗有效的有30例，无效的12例，并结合HBV病毒载量、乙型肝炎e抗原和丙氨酸转氨酶等指标作综合分析。组间比较采用单因素方差分析和Kruskal—WallisH检验，两样本均数的比较采用t检验和Mann—WhitneyU检验，相关分析用Spearman＋S检验。结果CD4＋CD28＋组间表达量健康对照组最高[404．65（331．65—536．09）]，治疗无效组最低[277．15（249．90—344．25）]，差异有统计学意义（H=29．81，P〈0．001）；CD4＋PD一1＋组间表达量未治疗组最高[32．89（29．69～39．69）]，治疗无效组最低[19．26（11．90—20．56）]，差异有统计学意义（H=43．13，P〈0．001）；CD8＋PD．1＋组间表达量未治疗组最高[15．47（12．50～17．78）]，健康对照组最低[3．05（1．41～3．97）]，差异有统计学意义（H=56．60，P〈0．001）；CD4＋Tim-3＋组间表达量治疗无效组最高[199．62（55．61—239．45）]、健康对照组最低[70．62（53．88—112．32）]，差异有统计学意义（H=41．03，P〈0．001）；CD8＋Tim-3＋组间表达量未治疗组最高[82．50（78．69～84．58）]，健康对照组最低[3．07（1．56—6．87）]，差异有统计学意义（H=74．84，P〈0．001）；与低病毒载量组相比，治疗前CD8＋PD-1＋在高病毒载量组中的表达量[17．87（13．38—20．94）]升高，差异有统计学意义（U=25．00，P〈0．001），治疗后CD8＋PD-1＋在高病毒载量组中的表达量[16．95（5．39～18．27）]升高，差异有统计学意义（U=63．50，P〈0．001）；治疗后PD-1表达量与ALT也有明显的正相关性，其中CD4＋PD-1＋（r=0．689，P〈0．001）、CD8＋PD-1＋（r=0．751，P〈0．001）。结论在慢性乙型肝炎患者抗病毒治疗过程中，可能伴随有CD28、PD．1、CTLA-4和Tim-3的异常表达。共刺激分子的检测可为临床合理治疗慢性乙肝提供新线索。（中华检验医学杂志，2014,37：105－109）

Objective To investigate the significance of changes in expression of co-stimulatory molecules on T lymphocytes in patients with chronic hepatitis B （CHB） infection. Methods In a casecontrol study, a total of 82 CHB cases including 50 male cases and 32 female cases （the mean age was 42. 32 ±3.74） were enrolled in the First Affiliated Hospital to Soochow University from October 2012 to November 2013, together with 30 cases health control age was 42. 32 ± 3.74）. Patients were divided into （15 male cases and 15 female cases, and the mean three groups： 40 cases of non-treated, 30 cases of effective-treated and 12 cases of ineffective-treated with anti-viral drugs and immune-related therapy. The expression levels of CD28, CTLA-4, PD-1, Tim-3 on T cells subset from peripheral blood were determined by Flow Cytometry. Serum load of HBV DNA was detected by Real-time PCR and the serology markers such as HBeAg and ALT were detected by conventional methods The Kruskal-Wallis test was used to analysis groups comparison. Independent samples t-test and Mann-Whitney U test were used to two sample comparison. Spearman＇s rank correlation test was used to analyze the correlation. Results The expression levels of CD28, CTLA-4 and PD-1 on CD4 in health control group was the highest compared to ineffective- treated group [404.65 （331.65 -536.09） vs 277. 15 （249.90-344.25） （H=29.81, P〈0.001）; 32.89 （29.69-39.69） vs 19.26 （11.90 -20.56） （H=43.13, P 〈0.001）, respeetivelyJ. The expression level of PD-1 on CD8 in pre-treated group was the highest ,while expression level in health control group was the lowest [ 15.47 （ 12.50 - 17. 78 ） vs 3.05 （ 1.41 - 3.97 ） （ H = 56. 60, P 〈 0. 001 ） ]. Similarly, the expression level of Tim-3 on CD4 in ineffective-treated group was the highest, while Tim expression on CD4 in health control group was the lowest [ 199.62 （55.61 - 239. 45 ） vs 70. 62 （ 53.88 - 112. 32） （H =-41.03, P 〈 0. 001 ）1. The expression level of Tim-3 on CD8 in pre-treated group was the highest, while it was the lowest in health control group [ 82. 50 （78. 69 - 84. 58） vs 3.07 （ 1.56 - 6. 87 ） （ H =74. 84, P 〈0. 001）]. Compared to the group with low viral load, the expression level of PD-1 on CD8 both in the pre-treated group with high viral load and the post-treated group was significantly increased [17.87 （13.38-20.94） （U=25.00, P〈0.001）; 16.95 （5.39-18.27） （U=63.50, P〈0.001）, respectively]. Importantly, in the post-treated group, the PD-1 expression on CIM T （r = 0. 689, P 〈 0. 001 ） and on CD8 T （ r = 0. 751, P 〈 0. 001 ） was also positively correlated with ALT. Conclusions The abnormal expression of these co-stimulatory molecules may be present in the antiviral treatment process of CHB. It may provide new clues for the reasonable treatment of CHB. （Chin J Lab Med,2014,37：105-109）