ADMA對(duì)單核細(xì)胞來(lái)源的樹(shù)突狀細(xì)胞成熟和免疫的影響
發(fā)布時(shí)間：2018-09-14 12:50
【摘要】： 【目的】 非對(duì)稱性二甲基精氨酸(Asymmetrical dimethylarginine ADMA)是一種內(nèi)源性一氧化氮合酶(Nitric Oxide Synthase,NOS)抑制劑,血漿ADMA水平的升高可以預(yù)測(cè)冠心病的突發(fā)事件和病死率,是心血管疾病的獨(dú)立危險(xiǎn)因子,動(dòng)脈粥樣硬化(artherosclerosis,AS)目前被認(rèn)為是一種炎癥和免疫性疾病。本研究通過(guò)觀察非對(duì)稱性二甲基精氨酸(ADMA)對(duì)樹(shù)突狀細(xì)胞(dendritic cells,DCs)成熟及免疫的影響來(lái)探討AS形成的可能機(jī)制。 【方法】 1、各種細(xì)胞的分離和培養(yǎng)::(1)用淋巴細(xì)胞分離液從健康成人外周血白細(xì)胞懸液分離單個(gè)核細(xì)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培養(yǎng)基培養(yǎng),第5天收集懸浮細(xì)胞作為未成熟DC(immature DC,imDC) 2、細(xì)胞干預(yù):用1、8、16umol/L的ADMA干預(yù)未成熟DC 24 h。 3、各種指標(biāo)的檢測(cè):用流式細(xì)胞術(shù)檢測(cè)DC細(xì)胞表面分子的表達(dá)、吞噬能力及DC的凋亡,用混合淋巴細(xì)胞反應(yīng)檢測(cè)成熟DC刺激T淋巴細(xì)胞增殖的能力,用ELISA檢測(cè)DC細(xì)胞因子的分泌。 【結(jié)果】 1.DC的培養(yǎng)和觀察:在倒置顯微鏡下見(jiàn)DC懸浮生長(zhǎng),聚集成簇,有大量胞質(zhì)突起。 2.ADMA對(duì)DCs表型的影響: mDC表面CD86、CD83、HLA-DR的表達(dá)較imDC明顯升高,但CDla變化不大;用ADMA干預(yù)后,細(xì)胞表面分子的表達(dá)與未用ADMA干預(yù)的相比設(shè)有明顯差異,但在ADMA病理濃度下,CDla的平均熒光強(qiáng)度降低,因CDla是DC成熟標(biāo)志,說(shuō)明在生理濃度下ADMA并不誘導(dǎo)DC分化,在病理濃度下ADMA可以抑制DC成熟, 3.ADMA刺激DC對(duì)T淋巴細(xì)胞增殖作用: 將經(jīng)不同濃度ADMA刺激的mDC與T淋巴細(xì)胞以1:10比例混合培養(yǎng)5天,用MTT法來(lái)檢測(cè)ADMA對(duì)MLR的影響。結(jié)果如圖所示,ADMA干預(yù)組的MTT數(shù)值明顯小于LPS組(P＜0.05,P＜0.01);用16umol/L的ADMA干預(yù)產(chǎn)生與對(duì)照組有差異的MLR(P＜0.01);ADMA濃度為16umol/L時(shí),T淋巴細(xì)胞增殖反而不明顯;說(shuō)明ADMA刺激DC對(duì)T淋巴細(xì)胞增殖有抑制作用。 4.DCs吞噬功能的檢測(cè): 經(jīng)ADMA干預(yù)后的imDC及未經(jīng)干預(yù)的mDC,與FITC-Dextran在4℃(作陰性對(duì)照)或37℃共孵育30 min,用流式細(xì)胞術(shù)檢測(cè)細(xì)胞的吞噬能力,發(fā)現(xiàn)生理濃度ADMA對(duì)DC的吞噬能力無(wú)明顯影響;病理濃度ADMA對(duì)DC的吞噬能力有抑制作用,而用LPS干預(yù)DC的吞噬能力明顯低于對(duì)照組,說(shuō)明成熟DC的吞噬能力較未成熟DC降低 5.DCs凋亡的檢測(cè): 經(jīng)ADMA干預(yù)后的DC,用流式細(xì)胞術(shù)檢測(cè)凋亡的百分比,發(fā)現(xiàn)16μmol/LADMA.濃度組凋亡的百分比比其它組明顯增高(P＜0.05,P＜0.01) 6.DCs的細(xì)胞因子的分泌功能檢測(cè): 經(jīng)ADMA干預(yù)后的DC,用ELISA檢測(cè)IL-12細(xì)胞因子的分泌量,結(jié)果發(fā)現(xiàn)經(jīng)100ng/mL LPS誘導(dǎo)得到的mDC IL-12細(xì)胞因子的分泌量明顯增多(P＜0.01),ADMA干預(yù)組在濃度為8μmol/L和16μmol/L時(shí)IL-12細(xì)胞因子的分泌量明顯減少(P＜0.01) 經(jīng)ADMA干預(yù)后的DC,用ELISA檢測(cè)TNF-α分泌量,結(jié)果發(fā)現(xiàn)經(jīng)100 ng/mLLPS誘導(dǎo)得到的mDC及不加任何刺激因子TNF-α分泌量明顯增多(P＜0.01),ADMA干預(yù)組在濃度為16μmol/L時(shí)TNF-α分泌量明顯減少(P＜0.01), 【結(jié)論】 1.生理濃度ADMA并不刺激DC成熟及分化;但病理濃度ADMA抑制DC成熟; 2.病理濃度ADMA抑制DC誘導(dǎo)的T淋巴細(xì)胞增殖; 3.病理濃度ADMA抑制DC的吞噬功能; 4.病理濃度ADMA誘導(dǎo)DC凋亡; 5.病理生理濃度ADMA抑制DC分泌IL-12細(xì)胞因子、TNF-α及IL-10細(xì)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R392
本文編號(hào)：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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 郭濤,王志平,段建敏;膀胱癌患者一氧化氮對(duì)外周血樹(shù)突狀細(xì)胞的影響[J];第四軍醫(yī)大學(xué)學(xué)報(bào);2005年19期
，本文編號(hào)：2242760
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