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目的探讨采用异戊烯焦磷酸(IPP)刺激人外周血单个核细胞(PBMCs)特异性扩增γδT细胞,观察其在体外对HBV复制和HBeAg表达的影响,并评估应用γδT细胞过继回输治疗乙型肝炎的疗效。方法在体外应用IPP和IL-2刺激培养人外周血PBMCs8天,采用流式细胞术检测γδT细胞百分率及细胞表面穿孔素、粒酶B、NKG2D和CD44表达量;采用ELISA法检测培养上清IFN-γ、TNF-α和IL-12含量;将γδT细胞与HepG2215细胞按比例共孵育后,检测上清HBeAg和HBV DNA水平。将培养成功的γδT细胞回输治疗慢性乙型肝炎患者50例,12个月后观察疗效。结果人外周血PBMCs在培养前γδT细胞数占5.12%,CD44表达5.13%。在培养8天后则分别升至91.27%和94.00%;γδT细胞表面穿孔素、粒酶B、NKG2D受体表达量(分别为57.1%、71.6%和71.7%)明显高于培养前(分别为0.2%、1.1%和1.3%);γδT细胞分泌IFN-γ和IL-12量明显增加;扩增的γδT细胞能够抑制HepG2215细胞HBV DNA复制和HBeAg的表达;γδT细胞治疗慢性乙型肝炎患者,HBeAg转阴率为54.3%(25/46),HBV DNA转阴率为66.0%(33/50)。结论乙型肝炎患者PBMC经刺激培养获得的γδT细胞免疫功能增强,初步观察表明有一定的临床治疗作用。
Objective To investigate the specific amplification of γδT cells induced by isopentenyl pyrophosphate (IPP) in human peripheral blood mononuclear cells (PBMCs), and to observe their effects on HBV replication and HBeAg expression in vitro and to evaluate the effect of adoptive transfer of γδT cells Hepatitis B curative effect. Methods PBMCs were cultured with IPP and IL-2 for 8 days in vitro. The percentages of γδT cells and the expressions of perforin, granzyme B, NKG2D and CD44 were measured by flow cytometry. The expression of IFN-γ γ, TNF-α and IL-12. After the γδT cells and HepG2215 cells were incubated in proportion, the levels of HBeAg and HBV DNA in the supernatant were detected. Successfully cultured γδT cells were transfused in 50 patients with chronic hepatitis B and the effects were observed after 12 months. Results The percentage of γδT cells in peripheral blood PBMCs before culture was 5.12% and that of CD44 was 5.13%. And increased to 91.27% and 94.00% respectively after cultured for 8 days. The expression of perforin, granzyme B and NKG2D receptors on γδT cells (57.1%, 71.6% and 71.7%, respectively) was significantly higher than that before culture (0.2 %, 1.1% and 1.3%, respectively). The amount of IFN-γ and IL-12 secreted by γδT cells was significantly increased. The expanded γδT cells could inhibit the HBV DNA replication and HBeAg expression in HepG2215 cells. In patients with chronic hepatitis B treated by γδT cells, HBeAg The negative conversion rate was 54.3% (25/46), and the negative conversion rate of HBV DNA was 66.0% (33/50). Conclusion The immune function of γδT cells obtained from stimulated PBMC of patients with hepatitis B is enhanced. The preliminary observation indicates that there is a certain clinical therapeutic effect.