论文标题:慢性HBV感染者和乙肝疫苗无应答者CD28表达及相关细胞因子的研究
Study of CD28 molecule expression and levels of cytokines in patients wiht chronic hepatitis B virus infection and hepatitis B vaccinenon-responders
论文作者 黄茵
论文导师 陈智,论文学位 硕士,论文专业 内科学（传染病）
论文单位 浙江大学,点击次数 100,论文页数 57页File Size1658k
2001-05-01论文网 http://www.lw23.com/lunwen_100543082/ 乙型肝炎病毒; CD28; B7; 细胞因子
Hepatitis B virus; CD28; B7; cytokines
乙型肝炎病毒（hepatitis B virus，HBV）持续感染机理尚未完全清楚，目前认为与宿主免疫应答低下或形成免疫耐受，以及Th1/Th2细胞失衡等密切相关。现已知免疫应答中，T细胞表面的CD28分子与其配体B7结合，在防止形成免疫耐受方面起重要作用；白细胞介素-12（IL-12）、干扰素-γ（IFN-γ）和白细胞介素-10（IL-10）等对Th1/Th2平衡有调节作用。另外，乙肝疫苗接种者中约有5％～10％的人不能产生中和抗体（抗-HBs），其机理还不十分明确。通过了解慢性HBV感染者CD28表达及血清IL-12、IFN-γ和IL-10水平，以及探讨接种乙肝疫苗免疫失败的可能原因，有助于进一步阐明HBV感染慢性化的机制，并为慢性乙肝的预防、诊断和治疗提供一定的理论和实验依据。 第一部分：慢性HBV感染者外周血T细胞CD28表达及血清IL-12，IFN-γ和IL-10水平的研究 为了解慢性HBV感染者外周血T细胞表面CD28分子表达及血清IL-12、IFN-γ和IL-10水平与血清HBeAg、丙氨酸转移酶（ALT）水平之间的关系，探讨HBV持续感染过程中的免疫病理学特点，我们采用流式细胞测定技术（fluoresceinactivated cell sorter，FACS）和酶标双抗体夹心法（EIA），分别检测112例血清HBeAg和ALT水平不同的慢性HBV感染者外周血淋巴细胞中CD4~+、CD8~+细胞CD28分子的表达情况，及血清IL-12、IFN-γ和IL-10水平。 结果显示，HBeAg阳性/ALT正常组CD4~+CD28~+、CD28~+、CD4~+和CD8~+细胞I 浙江大学硕士学位论文D 亚群百分数（分别为 27．03％、44．42％、29．06O和 27．86o）均比正常对照组（分l 别为 34．35％，53．68％，37．37％和 33．00％）减少，差异有显著意义（均 P＜0刀1）。DHBeAg阳性／ALT异常组、HBeAg阴性／ALT异常组CD28”细胞亚群百分数（62．15％D 和 62．20o），及 CDS”CD28”细胞亚群百分数（26．69o和 26．17O）分别比对照组】（53．68％和19．33％）增高（均尸刃刀1）：CD矿T细胞上CD沈表达率比对照组增高，差异有显著意义（分另P＜0刀1，P＜0刀引；血清几一12、f：Nf：N一Y和1卜10水平D 也都比对照组增高，差异均有显著意义（均尸＜0刀1）。各组＊＊8丫＊28”细胞比对－照减少，差异均有显著意义（P＜0．of或P＜0．05）。各组 CD4”、CDS”T细胞上 CD28D 表达率均比对照组增高。l 因此我们认为，血清 HBeAg阳性及肝功能正常的 HBV慢性感染者处于细胞l 免疫应答低下或耐受状态。Thl 与仆 细胞均参与肝细胞的免疫病理损伤，ICDS”CD28”T细胞的 CTL应答起主要作用。血清 IL10水平增高是 HBV持续感l 染的重要因素之一，而 T细胞 CD28分于表达减少或 CDS＋CD28细胞增多可能均 l 不是主要原＠。临床上检测 HBV感染者外w＆ CD4“CD28”、CDS”CD28”T mm 及血清 IL刁，IFN十和 IL10水平，有助于了解其机体免疫应答水平、特点及免疫 性肝损伤程度，以指导疾病的治疗和预后。D 第二部分：乙肝疫苗抗体无应答与 B7．CD28共刺激途径及 IL12、互L10的相 l 为了从共刺激倍号B7（CD80，CD86）－CD28以及相关细胞因于方面探讨导致乙l 肝疫苗兔疫失败的机制，我们对15例乙肝疫苗兔疫成功者（应答组）和 15例免 D 疫夫败者（无应答组）分离外周血单个核细胞（peripheral blood mononuclear cells， PBMC），给予非特异性植物血凝素（PHA）刺激及特异性重组乙肝表而抗原D （rHBsAg）刺激，进行体外细胞培养。用流式细胞术（FACS）检测 PBMC表面 D CD80、CD86表达和 CD4”、CDS”细胞CD28表达，酶标双抗夹心法（ELISA）检 0 测培养细胞上清儿－12和 IL－10水平，四甲基偶氮哇盐（MTT）法检测培养细胞 D ZI］浙江大学硕士学位论文l 结果显示，在 PHA刺激下，PBMC表达 CD80、CD86分子的百分数，CD4＋、ICDS”细胞表达CD28分于的白分数，培养上清IL12和IL．10水平，以及PBMCl 的增殖反应等，无应答组和应答组之间差异均无显著意义（P＞005）。但经 MSAgl 刺激后，无应答组与应答组比较，PBMC表达 CD80和 CD86的百分数均减少，D 差异百显者怠义（分别P＜0刀5，尸＜0．01）；培养上清1卜12和IL．10水平均降低，l 差异有显著意义（均 P＜0刀5）；PBMC
The mechanism of chronic hepatitis B virus (HBV) infection remains unclear. It is considered to be related to immune tolerance of HBV and imbalance of the T helper (Th)1/Th2 subset. It is well known that B7-CD28 T-cell costimulation plays a very important role in preventing from immunologic tolerance, and that interleukin-12 (IL-12), IFN-gamma (IFN-y), interleukin-10 (IL-10) are significant factors to regulate the balance of Thl/Th2 subset. In addition, the mechanism that about 5%~10% of hepatitis B (HB) vaccinees could not produce anti-HBs is not clear utterly. In order to farther clarify the mechanism of HBV persistent infection and offer some grounds for diagnosing, treating and preventing from chronic hepatitis B, we have studied the CD28 molecule expression on T lymphocytes and the serum levels of IL-12, IFN-y and IL-10 in patients with chronic HBV infection and HB vaccine non-responders. Part one:Study of CD28 molecule expression on T lymphocytes and levels of IL-12, IFN-γand IL-10 in patients with chronic HBV infectionWe have studied the CD28 molecule expression on CD4+/CD8+ lymphocytes in peripheral blood lymphocytes (PBL) and the serum levels of IL-12, IFN-y and IL-10 inpatients with chronic HBV infection, and combined with the levels of HBeAg and alanine transferase (ALT) in serums to explore the immunopathological characteristic of chronic HBV infection. CD28 molecule expression on CD4+/CD8+ lymphocytes in PBL was detected by using flow cytometry, and levels of cytokines above-mentioned in serums were determined by enzyme linked immunoassay (ElA) for 112 cases with different levels of HBeAg and ALT in serums and 30 normal controls.It showed that the percentage of CD4+CD28+, CD28+, CD4+ and CD8+ lymphocyte subsets of patients with chronic HBV infection with HBeAg positive/ALT normal was significantly lower than that of normal controls, respectively (27.03%, 44.42%, 29.06% and 27.86% vs 34.35%, 53.68%, 37.37% and 33.00%. all .PO.01). Then the percentage of CD28+ and CD8+CD28+ lymphocyte subsets of cases with HBeAg positive/ALT abnormal (62.15% and 26.69%) and cases with HBeAg negative/ALT abnormal (62.20% and 26.17%) was significantly higher than that of normal controls (53.68% and 19.33%, all P<0.01). The serum levels of IL-12, IFN-y, IL-10 in ALT abnormal groups were also higher compared to the normal levels, respectively (all P<0.01). Otherwise, the percentage of CD8+CD28" subset of all groups of patients (HBeAg positive/ALT abnormal, HBeAg positive/ALT normal, HBeAg negative/ALT abnormal and HBeAg negative/ALT normal) was lower than that of normal controls, and the expression rate of CD28 molecule on CD4+/CD8+ T cells of groups of patients was higher than those of controls.It was concluded that the patients with chronic HBV infection with HBeAg positive and ALT normal were probably at the stage of immune tolerance of HBV, and Thl and Th2 cellular immunity were all concerned with immunopathologic injury of hepatocytes with persistent HBV infection, and the CTL response mediated by CD8+CD28+ lymphocyte subset played an important role in the damage of hepatocytes.In addition, the higher serum level of IL-10 may be associated with the chronicity ofHBV infection, and the lack of CD28 molecule expression on T cells or increasedamount of CD8+CD28" subset was not the main factor possibly.Part two:Study on the relationship between B7-CD28 costimulation/ IL-12,IL-10and non-response to hepatitis B vaccineIn order to quest for the mechanism of immunization failure of HB vaccine by analyzing B7 (CD80, CD86)-CD28 molecule expression and the levels of IL-12 and IL-10, we have isolated peripheral blood mononuclear cells (PBMCs) from 15 HB vaccine responders and 15 non-responders who were adults without HBV infection, and incubated them in the presence of PHA as well as purified recombinant hepatitis B surface antigen (rHBsAg). CD80, CD86 and CD28 molecule expression on PBMCs was detected by using flow cytometry, and levels of cytokines above-mentioned in the super
|
