ADMA对单核细胞来源的树突状细胞成熟和免疫的影响
发布时间：2018-09-14 12:50
【摘要】： 【目的】 非对称性二甲基精氨酸(Asymmetrical dimethylarginine ADMA)是一种内源性一氧化氮合酶(Nitric Oxide Synthase,NOS)抑制剂,血浆ADMA水平的升高可以预测冠心病的突发事件和病死率,是心血管疾病的独立危险因子,动脉粥样硬化(artherosclerosis,AS)目前被认为是一种炎症和免疫性疾病。本研究通过观察非对称性二甲基精氨酸(ADMA)对树突状细胞(dendritic cells,DCs)成熟及免疫的影响来探讨AS形成的可能机制。 【方法】 1、各种细胞的分离和培养::(1)用淋巴细胞分离液从健康成人外周血白细胞悬液分离单个核细胞,用含20 ng/ml rhGM-CSF(recombinant human granulocytemacrophage-colony stimulating factor)、10 ng/ml rhIL-4(recombinant humaninterleukin-4)、10%胎牛血清(fetal bovine serum,FBS)的RPMl1640培养基培养,第5天收集悬浮细胞作为未成熟DC(immature DC,imDC) 2、细胞干预:用1、8、16umol/L的ADMA干预未成熟DC 24 h。 3、各种指标的检测:用流式细胞术检测DC细胞表面分子的表达、吞噬能力及DC的凋亡,用混合淋巴细胞反应检测成熟DC刺激T淋巴细胞增殖的能力,用ELISA检测DC细胞因子的分泌。 【结果】 1.DC的培养和观察:在倒置显微镜下见DC悬浮生长,聚集成簇,有大量胞质突起。 2.ADMA对DCs表型的影响: mDC表面CD86、CD83、HLA-DR的表达较imDC明显升高,但CDla变化不大;用ADMA干预后,细胞表面分子的表达与未用ADMA干预的相比设有明显差异,但在ADMA病理浓度下,CDla的平均荧光强度降低,因CDla是DC成熟标志,说明在生理浓度下ADMA并不诱导DC分化,在病理浓度下ADMA可以抑制DC成熟, 3.ADMA刺激DC对T淋巴细胞增殖作用: 将经不同浓度ADMA刺激的mDC与T淋巴细胞以1:10比例混合培养5天,用MTT法来检测ADMA对MLR的影响。结果如图所示,ADMA干预组的MTT数值明显小于LPS组(P＜0.05,P＜0.01);用16umol/L的ADMA干预产生与对照组有差异的MLR(P＜0.01);ADMA浓度为16umol/L时,T淋巴细胞增殖反而不明显;说明ADMA刺激DC对T淋巴细胞增殖有抑制作用。 4.DCs吞噬功能的检测: 经ADMA干预后的imDC及未经干预的mDC,与FITC-Dextran在4℃(作阴性对照)或37℃共孵育30 min,用流式细胞术检测细胞的吞噬能力,发现生理浓度ADMA对DC的吞噬能力无明显影响;病理浓度ADMA对DC的吞噬能力有抑制作用,而用LPS干预DC的吞噬能力明显低于对照组,说明成熟DC的吞噬能力较未成熟DC降低 5.DCs凋亡的检测: 经ADMA干预后的DC,用流式细胞术检测凋亡的百分比,发现16μmol/LADMA.浓度组凋亡的百分比比其它组明显增高(P＜0.05,P＜0.01) 6.DCs的细胞因子的分泌功能检测: 经ADMA干预后的DC,用ELISA检测IL-12细胞因子的分泌量,结果发现经100ng/mL LPS诱导得到的mDC IL-12细胞因子的分泌量明显增多(P＜0.01),ADMA干预组在浓度为8μmol/L和16μmol/L时IL-12细胞因子的分泌量明显减少(P＜0.01) 经ADMA干预后的DC,用ELISA检测TNF-α分泌量,结果发现经100 ng/mLLPS诱导得到的mDC及不加任何刺激因子TNF-α分泌量明显增多(P＜0.01),ADMA干预组在浓度为16μmol/L时TNF-α分泌量明显减少(P＜0.01), 【结论】 1.生理浓度ADMA并不刺激DC成熟及分化;但病理浓度ADMA抑制DC成熟; 2.病理浓度ADMA抑制DC诱导的T淋巴细胞增殖; 3.病理浓度ADMA抑制DC的吞噬功能; 4.病理浓度ADMA诱导DC凋亡; 5.病理生理浓度ADMA抑制DC分泌IL-12细胞因子、TNF-α及IL-10细胞因子。
[Abstract]:[Objective]
Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor. Elevated plasma ADMA levels can predict sudden events and mortality in coronary heart disease. It is an independent risk factor for cardiovascular disease. Atherosclerosis (AS) is currently the leading cause of cardiovascular disease. This study investigated the possible mechanism of AS formation by observing the effects of asymmetric dimethylarginine (ADMA) on the maturation and immunity of dendritic cells (DCs).
[method]
1. Isolation and culture of various kinds of cells: (1) Mononuclear cells were isolated from peripheral blood leukocyte suspension of healthy adults with lymphocyte isolation solution. Mononuclear cells were isolated with 20 ng/ml rhGM-CSF (recombinant human granulocyte acrophage-colony stimulating factor), 10 ng/ml rhIL-4 (recombinant human interleukin eukin-4) and 10% fetal bovine serum (FBS). The RPMl1640 medium was cultured for fifth days to collect suspension cells as immature DC (immature DC, imDC).
2, cell intervention: intervention of immature DC 24 h. with 1,8,16umol/L ADMA.
3. Detection of various indicators: Flow cytometry was used to detect the expression of DC cell surface molecules, phagocytosis and DC apoptosis, mixed lymphocyte reaction was used to detect the ability of mature DC to stimulate T lymphocyte proliferation, and ELISA was used to detect the secretion of DC cytokines.
[results]
1.DC culture and observation: under inverted microscope, DC was suspended and clustered into clusters with large cytoplasmic processes.
Effects of 2.ADMA on DCs phenotype:
The expression of CD86, CD83 and HLA-DR on the surface of mDC was significantly higher than that of imDC, but the change of CDla was not significant. After ADMA treatment, the expression of cell surface molecules was significantly different from that without ADMA treatment, but the average fluorescence intensity of CDla decreased under the pathological concentration of ADMA, because CDla was a marker of DC maturation, indicating that ADMA did not induce DC differentiation at physiological concentration. ADMA can inhibit DC maturation at pathological level.
3.ADMA stimulates DC on proliferation of T lymphocytes:
MTT assay was used to detect the effect of ADMA on MLR. Results As shown in the figure, the MTT value of ADMA group was significantly lower than that of LPS group (P The proliferation of Ba cells was not obvious, indicating that ADMA stimulates DC to inhibit the proliferation of T lymphocytes.
Detection of 4.DCs phagocytosis function:
ImDC and mDC after ADMA treatment were incubated with FITC-Dextran at 4 C (negative control) or 37 It was significantly lower than the control group, indicating that the phagocytosis of mature DC was lower than that of immature DC.
Detection of apoptosis in 5.DCs:
The percentage of apoptosis in DC treated with ADMA was detected by flow cytometry. It was found that the percentage of apoptosis in 16 micromol/LADMA concentration group was significantly higher than that in other groups (P < 0.05, P < 0.01).
The cytokine secretion function of 6.DCs was detected:
After ADMA intervention, the secretion of IL-12 cytokines in DC was detected by ELISA. The results showed that the secretion of IL-12 cytokines in mDC induced by 100ng/mL LPS increased significantly (P < 0.01). In ADMA intervention group, the secretion of IL-12 cytokines decreased significantly (P < 0.01).
After ADMA intervention, the secretion of TNF-alpha was detected by ELISA. The results showed that the secretion of TNF-alpha and mDC induced by 100 ng/mLLPS increased significantly (P
[Conclusion]
1. physiological concentration of ADMA did not stimulate DC maturation and differentiation, but pathological concentration ADMA inhibited DC maturation.
2. the pathological concentration of ADMA inhibited the proliferation of DC induced T lymphocytes.
3. the pathological concentration of ADMA inhibited the phagocytosis of DC.
4. pathological concentration of ADMA induced DC apoptosis.
5. the pathophysiological concentration of ADMA inhibits DC secretion of IL-12 cytokines, TNF- alpha and IL-10 cytokines.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
本文编号：2242760
[Abstract]:[Objective]
Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor. Elevated plasma ADMA levels can predict sudden events and mortality in coronary heart disease. It is an independent risk factor for cardiovascular disease. Atherosclerosis (AS) is currently the leading cause of cardiovascular disease. This study investigated the possible mechanism of AS formation by observing the effects of asymmetric dimethylarginine (ADMA) on the maturation and immunity of dendritic cells (DCs).
[method]
1. Isolation and culture of various kinds of cells: (1) Mononuclear cells were isolated from peripheral blood leukocyte suspension of healthy adults with lymphocyte isolation solution. Mononuclear cells were isolated with 20 ng/ml rhGM-CSF (recombinant human granulocyte acrophage-colony stimulating factor), 10 ng/ml rhIL-4 (recombinant human interleukin eukin-4) and 10% fetal bovine serum (FBS). The RPMl1640 medium was cultured for fifth days to collect suspension cells as immature DC (immature DC, imDC).
2, cell intervention: intervention of immature DC 24 h. with 1,8,16umol/L ADMA.
3. Detection of various indicators: Flow cytometry was used to detect the expression of DC cell surface molecules, phagocytosis and DC apoptosis, mixed lymphocyte reaction was used to detect the ability of mature DC to stimulate T lymphocyte proliferation, and ELISA was used to detect the secretion of DC cytokines.
[results]
1.DC culture and observation: under inverted microscope, DC was suspended and clustered into clusters with large cytoplasmic processes.
Effects of 2.ADMA on DCs phenotype:
The expression of CD86, CD83 and HLA-DR on the surface of mDC was significantly higher than that of imDC, but the change of CDla was not significant. After ADMA treatment, the expression of cell surface molecules was significantly different from that without ADMA treatment, but the average fluorescence intensity of CDla decreased under the pathological concentration of ADMA, because CDla was a marker of DC maturation, indicating that ADMA did not induce DC differentiation at physiological concentration. ADMA can inhibit DC maturation at pathological level.
3.ADMA stimulates DC on proliferation of T lymphocytes:
MTT assay was used to detect the effect of ADMA on MLR. Results As shown in the figure, the MTT value of ADMA group was significantly lower than that of LPS group (P The proliferation of Ba cells was not obvious, indicating that ADMA stimulates DC to inhibit the proliferation of T lymphocytes.
Detection of 4.DCs phagocytosis function:
ImDC and mDC after ADMA treatment were incubated with FITC-Dextran at 4 C (negative control) or 37 It was significantly lower than the control group, indicating that the phagocytosis of mature DC was lower than that of immature DC.
Detection of apoptosis in 5.DCs:
The percentage of apoptosis in DC treated with ADMA was detected by flow cytometry. It was found that the percentage of apoptosis in 16 micromol/LADMA concentration group was significantly higher than that in other groups (P < 0.05, P < 0.01).
The cytokine secretion function of 6.DCs was detected:
After ADMA intervention, the secretion of IL-12 cytokines in DC was detected by ELISA. The results showed that the secretion of IL-12 cytokines in mDC induced by 100ng/mL LPS increased significantly (P < 0.01). In ADMA intervention group, the secretion of IL-12 cytokines decreased significantly (P < 0.01).
After ADMA intervention, the secretion of TNF-alpha was detected by ELISA. The results showed that the secretion of TNF-alpha and mDC induced by 100 ng/mLLPS increased significantly (P
[Conclusion]
1. physiological concentration of ADMA did not stimulate DC maturation and differentiation, but pathological concentration ADMA inhibited DC maturation.
2. the pathological concentration of ADMA inhibited the proliferation of DC induced T lymphocytes.
3. the pathological concentration of ADMA inhibited the phagocytosis of DC.
4. pathological concentration of ADMA induced DC apoptosis.
5. the pathophysiological concentration of ADMA inhibits DC secretion of IL-12 cytokines, TNF- alpha and IL-10 cytokines.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
【参考文献】
相关期刊论文 前1条
1 郭涛,王志平,段建敏;膀胱癌患者一氧化氮对外周血树突状细胞的影响[J];第四军医大学学报;2005年19期
，本文编号：2242760
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