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背景:未成熟树突状细胞(dendritic cells,DC)可以诱导免疫耐受形成,在器官移植领域具有广阔应用前景。目前采用的诱导未成熟树突状细胞的方法仍存在一些不足,寻求新的诱导方法十分必要。目的:观察丁酸钠对人外周血来源的树突状细胞的成熟状态和免疫学功能的影响。设计:观察对比,体外细胞学实验。单位:中山大学附属第二医院肝胆外科。材料:人外周血样品取自中山大学身体健康的大学生志愿捐献者(年龄20~23岁),共5份,每份100mL,均在献血后2h内进行外周血单个核细胞和淋巴细胞分离。方法:实验于2006-09/2007-03在中山大学附属第二医院医学研究中心完成。①人外周血单个核细胞采用粒细胞-巨噬细胞集落刺激因子和白细胞介素4诱导成为未成熟树突状细胞。②诱导6d后,加入组蛋白去乙酰化酶抑制剂丁酸钠1mmol/L诱导,以促成熟因子脂多糖1mg/L为对照;设不加促成熟因子为空白对照组。③在诱导0d时加入丁酸钠,6d时加入脂多糖再次刺激。主要观察指标:动态观察细胞形态学变化,流式细胞仪检测DC表型,FITC标记的Dextran检测DC内吞能力,采用酶联免疫吸附法检测白细胞介素12分泌,混合淋巴细胞反应检测DC刺激淋巴细胞增殖能力。结果:①DC形态:在丁酸钠作用下,DC聚集现象明显减少。②细胞表型检测结果:丁酸钠促成熟组常规方法诱导的未成熟DC的CD80,CD83,HLA-DR表达均低于脂多糖组,差异有显著性意义(P<0.01)。采用丁酸钠法诱导的未成熟DC,在第6天时加入促成熟因子脂多糖后CD80,CD83,HLA-DR表达仍处于低水平。③DC内吞能力:常规方法诱导的未成熟DC,用LPS诱导成熟后,其内吞能力均低于空白对照组和丁酸钠促成熟组,差异有非常显著性意义(P<0.01);而采用丁酸钠法诱导的DC,其内吞能力最强,高于其他各组,差异有非常显著性意义(P<0.01)。④DC分泌白细胞介素12:常规诱导法用LPS诱导DC成熟后的白细胞介素12分泌水平高于对照组、丁酸钠促成熟组及丁酸钠诱导法,差异有非常显著性意义(P<0.01)。⑤DC诱导的MLR:常规诱导法用LPS诱导的成熟DC刺激淋巴细胞的增殖能力显著高于对照组、丁酸钠促成熟组及丁酸钠诱导法,差异有非常显著性意义(P<0.01)。结论:丁酸钠可以抑制DC成熟,稳定诱导未成熟DC生成,该DC不能被促成熟因子激活,在诱导移植免疫耐受方面具有潜在应用前景。
BACKGROUND: Immature dendritic cells (DCs) can induce the formation of immune tolerance and have broad application prospects in the field of organ transplantation. Currently there are still some shortcomings in the method of inducing immature dendritic cells. It is necessary to find a new induction method. Objective: To observe the effect of sodium butyrate on the maturation status and immunological function of dendritic cells derived from human peripheral blood. Design: Observed contrast, in vitro cytology experiments. Unit: Second Affiliated Hospital of Sun Yat-sen University of hepatobiliary surgery. MATERIALS: Human peripheral blood samples were obtained from 5 volunteers (aged from 20 to 23 years old) from Sun Yat-sen University. Five samples of 100mL each were collected for separation of peripheral blood mononuclear cells and lymphocytes within 2h after blood donation. METHODS: The experiment was performed at the Medical Research Center of the Second Affiliated Hospital of Sun Yat-sen University from September 2006 to March 2007. ① human peripheral blood mononuclear cells using granulocyte-macrophage colony stimulating factor and interleukin-4 induced immature dendritic cells. ② After 6 days of induction, histamine deacetylase inhibitor sodium butyrate (1 mmol / L) was added to induce the maturation factor lipopolysaccharide 1 mg / L as control; no maturation factor was added as blank control. Â '¢ 0d in the induction of sodium butyrate added 6d lipopolysaccharide stimulation again. MAIN OUTCOME MEASURES: The morphological changes of cells were observed dynamically. The DC phenotypes were detected by flow cytometry. The endocytosis of DCs was detected by FITC-labeled Dextran. The secretion of interleukin-12 (IL-12) was detected by enzyme-linked immunosorbent assay Lymphocyte proliferation ability. Results: ①DC morphology: Under the action of sodium butyrate, the aggregation of DCs decreased significantly. ② Cell phenotype test results: The expressions of CD80, CD83 and HLA-DR in immature DC induced by sodium butyrate in mature group were lower than those in lipopolysaccharide group, the difference was significant (P <0.01). Immature DCs induced by sodium butyrate showed low levels of CD80, CD83 and HLA-DR on the sixth day after adding the pro-mature factor lipopolysaccharide. (3) DC endocytic ability: The immature DCs induced by conventional methods showed a significant difference (P <0.01) when the LPS induced maturation was lower than that of the blank control group and sodium butyrate-induced maturation group DC induced by sodium butyrate had the highest endocytosis capacity, higher than the other groups, the difference was significant (P <0.01). ④ DC secrete interleukin 12: IL-12 secreted by LPS-induced DC maturation is higher than that of control group, sodium butyrate-induced maturation group and sodium butyrate induction method, the difference has a very significant difference (P < 0.01). ⑤ DC-induced MLR: The proliferative capacity of lymphocytes stimulated by LPS-induced mature DCs was significantly higher than that of the control group by conventional induction method. The difference was significant (P <0.01) . CONCLUSION: Sodium butyrate can inhibit the maturation of DC and induce the immature DC to form stably. DC can not be activated by mature factor and has potential application in inducing transplantation tolerance.