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原发性胆汁性肝硬化患者外周血单个核细胞在体外对脂多糖刺激的反应
Immune Response of Peripheral Blood Mononuclear Cells to Lipopolysaccharide in vitro in Patients with Primary Biliary Cirrhosis
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摘要:目的 通过观察原发性胆汁性肝硬化(primary biliary cirrhosis, PBC)患者外周血单个核细胞(peripheral blood mononuclear cells, PBMCs)在体外自然状况及在脂多糖(lipopolysaccharide, LPS)刺激下分泌细胞因子的特点, 探讨LPS和细胞因子在PBC发病机制中的作用。方法 采集PBC、原发性干燥综合征(primary Sj gren sjösyndrome, pSS)患者及健康对照者的PBMCs, 分别置于加或不加0.01mg/ml LPS的RPMI1640完全培养基中培养, ELISA法检测PB-MCs上清液中的细胞因子——白细胞介素(interleukin, IL)-1β、IL-2、IL-6、IL-10、肿瘤坏死因子α(tumor necrosisfactor-α, TNF-α)和干扰素γ(interferon-γ, IFN-γ)的水平。结果 (1) 新鲜提取的PBMCs与液氮冻存复苏后的PBMCs, 在体外对LPS刺激的反应具有明显差异。(2)在无LPS刺激培养的PBMCs上清液中, PBC患者的IL-2水平高于健康对照者, 差异有统计学意义(P < 0.05)。(3)在LPS刺激培养的PBMCs上清液中, PBC患者的IL-1β、IL-2和TNF-α水平高于健康对照者, 差异有统计学意义(P < 0.05)。(4) LPS刺激后, PBC患者PBMCs上清液的IL-1β升高幅度和IL-2降低幅度与健康对照者比较, 差异有统计学意义(P < 0.05)。(5) PBC与pSS比较, 无LPS刺激时各细胞因子水平差异无统计学意义; LPS刺激后PBC患者IL-6高于pSS患者, 差异有统计学意义(P < 0.05)。(6) IFN-γ在PBC患者PBMCs上清液中几乎检测不到, 将LPS浓度提高至0.1和1.0mg/ml仍未检测到IFN-γ。结论 PBC发病与细菌感染有关, LPS在PBC的发病中起一定作用; 多种细胞因子与PBC发病有关, 其中以Th1型细胞因子为主。Abstract:Objective To explore the roles of lipopolysaccharide (LPS) and cytokines in the pathogenesis of primary biliary cirrhosis (PBC).Methods The peripheral blood mononuclear cells (PBMCs) were collected from PBC patients, patients with primary Sjögren's syndrome (pSS), and healthy controls and then cultured in vitro in RPMI 1640 medium with or without LPS. The immune responses of PBMCs to 0.01mg/ml LPS in vitro were investigated by measuring the levels of cytokines(IL-1β, IL-2, IL-6, IL-10, TNF-α, IFN-γ) in supernates using enzyme-linked immunosorbent assay (ELISA).Results The response of freshly extracted PBMCs to LPS were remarkably different from that of the resuscitated PBMCs. In the supernates of PBMCs cultured without LPS for 24 hours in vitro, the level of IL-2 was significantly higher in PBC patients than in healthy controls (P < 0.05). In the supernates of PBMCs cultured with 0.01mg/ml LPS for 24 hours in vitro, the levels of IL-1β, IL-2, and TNF-α were significantly higher in PBC patients than in healthy controls (P < 0.05). After the stimulation of LPS, the elevation rate of IL-1β and decrease rate of IL-2 in PBC patients were significantly higher than those in healthy controls (P < 0.05). Compared with the pSS patients, the levels of cytokines in PBC patients showed no difference in culture media without LPS, while IL-6 level was significantly increased in the supernates cultured with LPS in PBC patients. IFN-γ were seldomly detected in the the supernates of PBMCs from PBC patients, even after the LPS concentrations were increased to 0.1 and 1.0 mg/ml.Conclusions The occurence of PBC is related to bacterial infections, in which LPS may play an important role. Many cytokines are involved in the pathogenesis of PBC, especially Th1 subtype.
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