慢性乙型重型肝炎患者外周血单个核细胞衍生的树突状细胞表型与功能

目的 通过检测慢性乙型重型肝炎(CSHB)患者外周血单个核细胞(PBMC)衍生的树突状细胞(MoDC)经聚肌胞苷酸刺激后表型变化及细胞因子分泌情况,了解MoDC的免疫调节功能及其在慢性乙型肝炎(CHB)重症化中的作用.方法 采集CSHB组患者37例、CHB组患者20例和健康对照者20例的外周血,分离PBMC,免疫磁珠细胞分选法获得纯化的PBMC,体外培养诱导为未成熟树突状细胞(iDC),聚肌胞苷酸刺激为成熟树突状细胞(mDC).流式细胞术检测iDC及mDC细胞表面人类白细胞DR抗原(HLA-DR)、CD83、CD86、CD80的平均荧光强度(MFI);ELISA测定聚肌胞苷酸刺激后12、24、48 h的MoDC培养上清液中IL-12、IL-6、TNF-α分泌水平.多组间比较采用单因素方差分析,并进行方差齐性检验.结果 3组iDC表面HLA-DR、CD83、CD86、CD80的MFI比较,差异无统计学意义.CSHB组患者mDC表面HLA-DR、CD83、CD86、CD80的MFI与CHB组、健康对照组比较,显著低下(F值分别为59.73、13.95、34.80和73.02,均P＜0.05).聚肌胞苷酸刺激12、24、48 h时IL-12分泌量为:健康对照组＞CHB组＞CSHB组(F值分别为151.34、126.65、72.76,均P＜0.05),其中24 h时分泌量最高,分别为(48.2±7.6)、(56.7±11.8)、(97.8±16.2)ng/L.IL-6在上述3个时间点的分泌量为:CSHB组＞CHB组＞健康对照组(F值分别为92.50、86.89、64.57,均P＜0.05),其中12 h分泌量最高,分别为(1698.3±340.4)、(965.8±231.7)、(697.8±213.6)ng/L.TNF-α在上述3个时间点的分泌量为:CSHB组＞CHB组＞健康对照组(F值分别为58.66、122.36、44.73,均P＜0.05),24 h表达量分别为(19 672.7±4214.7)、(9946.1±2586.5)、(6659.2±955.8)ng/L.结论 CSHB患者MoDC成熟障碍,表现为IL-12分泌低下,而IL-6及TNF-α分泌亢进,可能是加剧肝脏炎性反应致重型肝炎的重要因素.

Study of phenotypes and functions of dendritic cells derived from the peripheral blood mononuclear cells of patients with chronic severe hepatitis B

Objective To understand the immune regulatory function of monocyte-derived dendritic cells (MoDC) in patients with chronic severe hepatitis B (CSHB) and its roles in the severe illness progression of chronic hepatitis B (CHB) by detecting surface phenotype of MoDC and expression level of cytokines in MoDC after polyl : C treatment. Methods The peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density gradient separation from 37 patients with CSHB, 20 patients with CHB, and 20 healthy controls (NC). Purified PBMC were acquired using immunomagnetic anti-CD14-beads. Then PBMC were induced to immature dendritic cell (iDC) in vitro. PolyI : C was added to induce DC maturation. The mean fluorescence intensity (MFI) of the phenotype marker molecules including HLA-DR, CD83, CD86 and CD80 on surface of iDC and mature DC (mDC) were detected by flow cytometry. The supernatants of MoDC culture were collected at 12,24 and 48 h after polyI : C treatment, respectively and the release levels of interleukin (IL)-12, IL-6and tumor necrosis factor (TNF)-α were determined by enzyme linked immunosorbent assay (ELISA). Comparisons among groups were done by single factor analysis of variance and homogeneity of variance was tested. Results There were no significant differences of phenotype marker molecules on cell surface of iDC, including HLA-DR, CD83, CD86 and CD80 in CSHB, CHB and NC groups.However, the expressions of HLA-DR, CD83, CD86 and CD80 on cell surface of mDC in CSHB group were lower than those in CHB and NC groups (F=59.73, 13.95, 34.80 and 73.02, respectively; all P＜0. 05). The secretions of IL-12 at three time points of 12 h, 24 h and 48 h after polyI : C treatment in group NC were higher than those in CHB and CSHB groups (F= 151.34, 126.65 and 72.76, respectively; P＜0.05), and peaked at 24 h which were (48.2±7.6), (56.7±11.8) and (97.8±16.2) ng/L, respectively. The secretions of IL-6 at the above three time points were CSHB＞CHB＞NC (F=92.50, 86.89 and 64.57, respectively; all P＜0. 05) and peaked at 12 h which were (1698.3±340.4), (965.8±231.7), (697.8±213.6) ng/L, respectively. The secretions of TNF-αat the above three time points were CSHB＞CHB＞NC (F=58.66, 122.36 and 44.73, respectively;all P＜0. 05) and were (19 672. 7±4214. 7), (9946. 1 ± 2586 5), (6659. 2±955. 8) ng/L,respectively at 24 h after treatment. Conclusions MoDCs of CSHB patients show mature defection and abnormal cytokine secretion. The expression level of IL-12 which mediates cellular immune is low.Meanwhile, the productions of IL-6 and TNF-α which mediate inflammatory response are up-regulated. This may be one of the major factors which lead to exacerbation of liver inflammation and ultimately development of severe hepatitis.