探讨树突状细胞在冠心病免疫反应中的作用机制
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摘要
研究背景
冠状动脉粥样硬化性心脏病(Coronary Atherosclerosis heart disease，CHD)是目前人类发病率最高、危害最大的疾病之一。其发病机理非常复杂，对CHD发病机制的研究及其防治策略是当今医学领域的重要课题之一。曾有多种学说从不同角度来阐明CHD的病因，新近有学者发现自身免疫在CHD中可能发挥着重要作用，CHD符合自身免疫性疾病(autoimmune disease，AID)有以下条件：①在CHD患者的血液及受累的组织中存在炎性递质或免疫物质；②在CHD患者体内检出自身抗体；③在受累组织中可见参与自身免疫的特异细胞，如T淋巴细胞。
抗原提呈细胞(antigen-presenting cell，APC)是指能捕捉、加工、处理抗原，并将抗原提呈给抗原特异性淋巴细胞的一类免疫细胞，根据细胞表面是否组成性表达MHC-Ⅱ类抗原和其他参与T细胞激活的协同刺激分子，可将APC分为专职和兼职两类，淋巴细胞与APC直接接触则是启动免疫应答的起始环节。DC是目前发现的机体内功能最强的专职抗原提呈细胞，极少量的DC即可强烈激活T淋巴细胞启动特异性细胞免疫反应，其激活T淋巴细胞的能力是巨噬细胞或B细胞的100～10000倍，T淋巴细胞的激活在CHD起始和斑块的稳定性中起重要的作用。
氧化低密度脂蛋白(Oxidized low density lipoprotein，Ox-LDL)最初因为其较低密度脂蛋白(Low density lipoprotein，LDL)有更强的致泡沫细胞作用及细胞毒性，成为CHD研究的热点，后继的研究发现，LDL在氧化过程中伴随着一系列的脂质及蛋白的分解和重新组合过程，产生新的表位，具有类似异体蛋白的免疫原性。已有的临床研究发现Ox-LDL除了损伤内皮细胞和平滑肌外，还对其他的单核细胞具有趋化性能。Ox-LDL与DC清道夫受体结合后，可以形成泡沫细胞，启动包括产生炎性细胞因子在内的一连串细胞内事件，触发和放大炎症反应。Ox-LDL免疫原性的作用多集中在引起血管内皮细胞CD40／CD40L的高表达上，关于Ox-LDL对于DC的作用机制目前尚无定论。
他汀类药物用于治疗CHD已有肯定的疗效，其药理机制是竞争性抑制3-羟基3-甲基戊二酰辅酶A(3-hydroxy-3-methylglutaryl coenzyme A，HMG-CoA)还原酶抑制剂，抑制甲羟戊酸的产生，从而抑制胆固醇合成。阿托伐他汀是最新一代HMG-CoA还原酶抑制剂，一系列大型一、二级冠心病预防研究结果均显示阿托伐他汀类药物对血脂紊乱及冠状动脉事件危险性的理想防治效果，降脂益处已无可争辩。近年来，阿托伐他汀的非调脂作用越来越引起关注，临床研究发现，心脏移植患者经阿托伐他汀治疗后，1年成活率明显增加，移植血管病事件和急性重症移植排斥事件明显减少，提示阿托伐他汀类药物具有免疫调节作用。Hwang等给予高胆固醇血症患者阿托法他汀治疗后，CD40L和P-选择素水平显著降低，明显抑制CD40／CD40L系统的高表达作用，这恰好与Ox-LDL免疫原性的作用相反，从而肯定了阿托法他汀的免疫调节作用。
本实验拟在细胞生物学、分子免疫水平，观察在冠心病发病重要因素之一的Ox-LDL刺激下，DC成熟及免疫功能的变化；探讨在最强的专职抗原提呈细胞—DC途径上免疫调节作用，从而为临床防治CHD提供免疫学理论。
目的
1、探讨Ox-LDL对DC成熟及免疫功能的影响。
2、探讨阿托伐他汀对Ox-LDL作用下的DC成熟和免疫应答反应的调节作用。
对象与方法
1、对象
门诊体检健康人20例，男性12例，女性8例，平均年龄(40.5±8.7岁)。取其外周血20ml。
2、方法
2．1 Ox-LDL的制备与DiⅠ标记
采用超高速密度梯度离心法从健康人新鲜血液分离出LDL，测定蛋白含量，并将蛋白终浓度调整为500ug／mL，加入终浓度为10mmol／mL的CuSO_4溶液中氧化，在终溶度为100μmol／L的EDTA溶液中终止氧化，获得Ox-LDL，以DiⅠ标记Ox-LDL备用。
2．2 DC的培养及干预
淋巴细胞分离液密度梯度离心法分离出外周血单个核细胞(PBMC)，用完全RPMI1640(含10％FCS，rhGM-CSF 100万单位／L，rhIL-4 50万单位／L)培养液，置CO2孵箱中培养5天，给予分组干预后，继续培养48h后，收集DC细胞，培养过程中以倒置显微镜观察细胞形态学变化。
2．3实验分组：
(1)对照组：无干预因素培养
(2) Ox-LDL组：Ox-LDL与DC共同培养48h
(3)阿托伐他汀组：阿托伐他汀+Ox-LDL与DC共同培养48h
2．4混合T淋巴细胞增值反应：以DC为刺激细胞，测定DC刺激同种异体T淋巴细胞增殖情况。
2．5流式细胞学(FACS)分析：收集DC细胞，用流式细胞仪检测DC表面相关分化抗原的表达情况。
2．6 IL-12浓度的检测：ELISA双抗夹心法检测。
3、统计学方法：应用SPSS13.0软件系统分析，所有计量资料以均数±标准差((?)±s)表示，比较采用配对t检验。P＜0.05为差异有统计学意义。
结果
1、DC形态学上的观察：倒置相差显微镜下，(1) PBS组：培养第5天，树突样结构清晰，胞质丰富：(2)Ox-LDL组：DC与DiⅠ标记的Ox-LDL共同孵育48h后，DC逐渐发育变大，胞浆逐渐丰富，变的红染，呈不规则多型状态，并有较多伪足伸出，表现出树突形状；(3)阿托伐他汀组：阿托伐他汀与DC及DiⅠ标记的Ox-LDL共同孵育48h后，吞噬Ox-LDL的DC数目减少，且胞浆红染程度减轻。
2、混合T淋巴细胞反应：Ox-LDL干预后DC刺激T淋巴细胞增值反应增强，Ox-LDL组的A值是(3.9±0.8)与对照组(1.1±0.3)比较，P=0.000，差异有统计学意义：而阿托伐他汀和Ox-LDL共同干预DC后T淋巴细胞增值反应减弱，阿托伐他汀和Ox-LDL组的A值为(1.2±0.4)与Ox-LDL组比较，P=0.000，差异有统计学意义。
3、表型的影响收集干预48h的悬浮细胞，经FACS分析显示，与对照组相比，经Ox-LDL处理的DC其表面分子CD1a和HLA-DR的表达增加，均可刺激T淋巴细胞增值，与对照组比较[(64.5±18.2)vs(40.1±17.2)P=0.000]和[(89.8±5.7)vs(69.9±17.7)P=0.000]差异均有统计学意义；CD80和CD86的表达虽有轻度的增加，但与对照组比较分别为[(72.5±1.9)vs(60.7±23.8)P=0.063]和[(66.5±18.4)vs(74.9±18.6)P=0.159]差异尚无统计学意义。而阿托伐他汀处理的DC与Ox-LDL组比较其表面分子的表达CD1a和HLA-DR的表达明显减少，与Ox-LDL组比较分别为[(55.2±12.7)vs(64.5±18.2)P=0.038]和[(89.8±57)vs(71.0±11.6)P=0.000]，差异有统计学意义。
结论
1、外周血PBMC中的贴壁细胞培养7天后，在含粒细胞巨噬细胞集落刺激因子(GM-CSF)、白介素4(IL-4)条件下培养，均可成功诱导为DC。
2、Ox-LDL与DC共同孵育后，可使DC表面MHC-Ⅱ类分子高表达，从而具有较强的摄取抗原的能力，激活未致敏的T细胞，启动初次免疫应答。
3、阿托伐他汀依赖性地抑制Ox-LDL引起的生物学效应及可能的细胞内信号转导，可明显抑制DC的免疫活性，这推测是他汀类药物免疫调节治疗冠心病的新机制。
Background
Coronary Atherosclerosis heart disease(CHD) is one of the diseases with thehighest incidence rate and most disservice at preasent. It becames one of theconsequence question for discussion nowdays how the pathogenetic to CHD goes onand how to prevent and cure it. Therefor there were several theories to interpret theetiopathogenisis of CHD from difference points. Howevre some reseachers found thatautoimmune was a key factor to the etiopathogenisis of CHD. That is to say CHD isan autoimmune disease for some reasons. Firstly some phlegmasia transmitters andimmune substance were found in the blood and affected tissues with CHD patients.Secondly some autologous antibodies were detected with CHD patients. Thirdly therewere some special immune cells such as T leukomomocyte cell which participateautoimmune in affected tissues.
The initiation step to priming immune response is the immediate contactbetween leukomomocyte cell and antigen presenting cell(APC). APC is a kind ofspecial immune cell who can capture and process antigens, then present thoseantigens to antigenic special leukomomocyte cell. Acroding to whether MHC-Ⅱmolecules and its co-stimulus molecules were expressed in the surface ofcells or not, APC included two kinds of cells which was special and unspecial APC.Dendritic cell is the most functional APC in our bodys. T leukomomocyte cells couldbe intensively activited to priming special immune response with slight dendritic cells.And its capability to active T leukomomocyte cell was as much 100～10000 times asmacrophaga and B leukomomocyte cells. As we know the activationg of Tleukomomocyte cell was key to form plaques in CHD.
More and more researchs focused on Oxidized low density lipoprotein(Ox-LDL)with more cytotoxicitys which could result in more foam cells developed. Howeverfollowing researches showed us that LDL had some immunogenicitys similar toforeign proteins caused, which LDL was descompounded and reassociated inoxidative process and some new epiposition formed. Some finished clinicalresearches had drawed conclusions that Ox-LDL could not only damage endothelialcells and smooth muscle cells but also show some chemotaxis to histomonocytessome else. The binding between Ox-LDL and its receptor insurface of macrophagecould induce a series of incidents inner cells, such as generating inflamm cytokinesand magnifing inflamm responses. Although Ox-LDL had some immunogenicityssimilar to foreign proteins caused, its functions focued on that it could cause thehighly express of CD40/CD40L insurface of blood vessel endothelium cell. Howeverthere was no clear machanism of Ox-LDL to dendritic cells.It's well known that wecould use statins to cure CHD. And its pharmaco-mechanism is that statins couldcompetitively inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reducingenzyme. So that the synthesis of cholesterol decreased because of mevalonate reduced.Atorvastatin is the newest HMG-CoA reducing enzyme inhibitor. Several serious ofclinical researches discovered that atorvastatin has ideal effect on balancing blood-fatdisorders.
Our studies will maily be focus on observing the immune function changes ofdendritic cells excited with Ox-LDL in the level of cytobiology and molecule immue.Then to discuss how the Ox-LDL influences the immunological regulation ofdendritic cells which were the special antigen presenting cell with most capability. Sothat some immune theorys in our studies could offer a suppourt to the clinialprevention and cure CHD.
Objective
1. To determine whether Ox-LDL could influence DC maturing and immunefunction.
2. To investigate whether Atorvastatin could regulate adaptive immunity whichwas elicited by Ox-LDL
Subjects and Method
1、Subjects
Twenty healthy persons is verification by medical examination that include twelvemails and 8 femails.Their mean age is 40.5±8.7 years.
2、Method
2.1 Preparation of Ox-LDL and DiI-labelled
We separated and quantitatified the LDL from blood of healthy individuals by Superhigh density gradient centrifugation, and adjusted it to a final concentration of500ug/mL,then incubated with 10mmol/mL CuSO4.The oxidation was stopped with100μmol/L EDTA. Finally, we labeled the Ox-LDL with DiI.
2.2 Culture and interference of DC
Periperil blood mononuclear cell (PBMC) were isolated by density gradientcentrifugation, and cultured with RPMI-1640(containing 1% FCS, rhGM-CSF1,000,000 U/L, rhIL-4,500,000U/L)at an atmosphere of 5%CO2 for 5 days.Afterbeing taken into several groups, with different interferences and culturing for another 2h, DC were collected. Culture changes in cell morphology were observed under aninverted microscope.
2.3 Groups
(1) control group: DC were cultured without any treatment
(2) Ox-LDL group : DC were cultured with Ox-LDL for 48h
(3) Atorvastatin group: DC were cultured both with Atorvastatin and Ox-LDL
2.4 Proliferation of lymphocytes assay
Proliferation of allogeneic T lymphocyte were evaluated after incubation withDC which served as a stimulant.
2.5 Flow cytometry analysis
DC were collected and the expression of relative differentiation antigens on thesurface of DC were analyzed on a flow meter.
2.6 Detection of IL-12
Double mAb sandwich ELISA was applied in quantitation of IL-12.
3、Statistical analysis: SPSS 10.0 statistical soft was used.The data was shown as((?)±s), The compared of two groups was tested by paired sample t-test,
Results
1、Observation on morphology of DC
Several phenomenons were observed under an inverted microscope: (1)control group: There were distinct dendritic structures and abundant cytoplasm in DCafter cultured for 5 days. (2)Ox-LDL group :The volume of DC grew bigger andbigger with more abundant cytoplasm, dyed red, and became irregular morphous withmany pseudopodia. (3) Atorvastatin group: The number of DC whichphagocytosed Ox-LDL diminished, and the dyeing of DC became lighter afterincubating with both Atorvastatin and Ox-LDL libeled with DiI.
2、Proliferation of lymphocytes assay
Differences in proliferation of T lymphocytes between treated with Ox-LDLgroup (3.9±0.8) and the control group (1.0±0.4) were found to be statisticallysignificant (P＜0.05) .And the same result (P＜0.05) also occurred between the grouptreated both with Atorvastatin and Ox-LDL(1.3±0.2)and treated with Ox-LDL.
3、Influence of phenotype
Collected the suspension cells treated for 48h, and detected themolecules expressed on the cell surface by FACS analysis. The results showedthat, compared with the negative control and PBS group, there was higherexpression of CD1a and HLA-DR on the cell surface of Ox-LDLtreated group,both of which could stimulate the proliferation of T lymphocytes. Differencebetween the control and treated group (35.3±10.1 vs 69.8±9.1) and (11.3 vs. 70.5±92.3±5.0) was statistically significant (P＜0.05); although compared with the controlgroup,there was mild expression of CD80 and CD86 (63.5±7.7 vs 88.0±7.9) and(10.2 vs. 61.3±80.84±1.12 P＜0.01),difference was not statistically significant (P＞0.05). Compared with Ox-LDL treated group, there was a significant decrese on theexpression of CDla (69.8±9.1 vs 55.5±12.4) and HLA-DR (92.3±5.0 vs66.8±5.8) on DC treated with atorvastatin.The difference was statistically significant(P＜0.05).
Conclusion
1. The adherent cells of PBMC in peripheral blood could be induced to be DCafter culturing with GM-CSF or IL-4 for 7 days.
2. After incubated with Ox-LDL, there was high expression of MHC classⅡmolecules on the cell surface of DC, which could enhance its capacity to uptake ofantigens, and activate T cells and initial immune response.
3. Atorvastatin can inhibit immunocompetence of DC obviously, by inhibitingbiological effects of Ox-LDL and probably by participating the intracellular signal transduction. This presumption is a new mechanism of immune modulationtherapy of coronary heart diseases.
冠状动脉粥样硬化性心脏病(Coronary Atherosclerosis heart disease，CHD)是目前人类发病率最高、危害最大的疾病之一。其发病机理非常复杂，对CHD发病机制的研究及其防治策略是当今医学领域的重要课题之一。曾有多种学说从不同角度来阐明CHD的病因，新近有学者发现自身免疫在CHD中可能发挥着重要作用，CHD符合自身免疫性疾病(autoimmune disease，AID)有以下条件：①在CHD患者的血液及受累的组织中存在炎性递质或免疫物质；②在CHD患者体内检出自身抗体；③在受累组织中可见参与自身免疫的特异细胞，如T淋巴细胞。
抗原提呈细胞(antigen-presenting cell，APC)是指能捕捉、加工、处理抗原，并将抗原提呈给抗原特异性淋巴细胞的一类免疫细胞，根据细胞表面是否组成性表达MHC-Ⅱ类抗原和其他参与T细胞激活的协同刺激分子，可将APC分为专职和兼职两类，淋巴细胞与APC直接接触则是启动免疫应答的起始环节。DC是目前发现的机体内功能最强的专职抗原提呈细胞，极少量的DC即可强烈激活T淋巴细胞启动特异性细胞免疫反应，其激活T淋巴细胞的能力是巨噬细胞或B细胞的100～10000倍，T淋巴细胞的激活在CHD起始和斑块的稳定性中起重要的作用。
氧化低密度脂蛋白(Oxidized low density lipoprotein，Ox-LDL)最初因为其较低密度脂蛋白(Low density lipoprotein，LDL)有更强的致泡沫细胞作用及细胞毒性，成为CHD研究的热点，后继的研究发现，LDL在氧化过程中伴随着一系列的脂质及蛋白的分解和重新组合过程，产生新的表位，具有类似异体蛋白的免疫原性。已有的临床研究发现Ox-LDL除了损伤内皮细胞和平滑肌外，还对其他的单核细胞具有趋化性能。Ox-LDL与DC清道夫受体结合后，可以形成泡沫细胞，启动包括产生炎性细胞因子在内的一连串细胞内事件，触发和放大炎症反应。Ox-LDL免疫原性的作用多集中在引起血管内皮细胞CD40／CD40L的高表达上，关于Ox-LDL对于DC的作用机制目前尚无定论。
他汀类药物用于治疗CHD已有肯定的疗效，其药理机制是竞争性抑制3-羟基3-甲基戊二酰辅酶A(3-hydroxy-3-methylglutaryl coenzyme A，HMG-CoA)还原酶抑制剂，抑制甲羟戊酸的产生，从而抑制胆固醇合成。阿托伐他汀是最新一代HMG-CoA还原酶抑制剂，一系列大型一、二级冠心病预防研究结果均显示阿托伐他汀类药物对血脂紊乱及冠状动脉事件危险性的理想防治效果，降脂益处已无可争辩。近年来，阿托伐他汀的非调脂作用越来越引起关注，临床研究发现，心脏移植患者经阿托伐他汀治疗后，1年成活率明显增加，移植血管病事件和急性重症移植排斥事件明显减少，提示阿托伐他汀类药物具有免疫调节作用。Hwang等给予高胆固醇血症患者阿托法他汀治疗后，CD40L和P-选择素水平显著降低，明显抑制CD40／CD40L系统的高表达作用，这恰好与Ox-LDL免疫原性的作用相反，从而肯定了阿托法他汀的免疫调节作用。
本实验拟在细胞生物学、分子免疫水平，观察在冠心病发病重要因素之一的Ox-LDL刺激下，DC成熟及免疫功能的变化；探讨在最强的专职抗原提呈细胞—DC途径上免疫调节作用，从而为临床防治CHD提供免疫学理论。
目的
1、探讨Ox-LDL对DC成熟及免疫功能的影响。
2、探讨阿托伐他汀对Ox-LDL作用下的DC成熟和免疫应答反应的调节作用。
对象与方法
1、对象
门诊体检健康人20例，男性12例，女性8例，平均年龄(40.5±8.7岁)。取其外周血20ml。
2、方法
2．1 Ox-LDL的制备与DiⅠ标记
采用超高速密度梯度离心法从健康人新鲜血液分离出LDL，测定蛋白含量，并将蛋白终浓度调整为500ug／mL，加入终浓度为10mmol／mL的CuSO_4溶液中氧化，在终溶度为100μmol／L的EDTA溶液中终止氧化，获得Ox-LDL，以DiⅠ标记Ox-LDL备用。
2．2 DC的培养及干预
淋巴细胞分离液密度梯度离心法分离出外周血单个核细胞(PBMC)，用完全RPMI1640(含10％FCS，rhGM-CSF 100万单位／L，rhIL-4 50万单位／L)培养液，置CO2孵箱中培养5天，给予分组干预后，继续培养48h后，收集DC细胞，培养过程中以倒置显微镜观察细胞形态学变化。
2．3实验分组：
(1)对照组：无干预因素培养
(2) Ox-LDL组：Ox-LDL与DC共同培养48h
(3)阿托伐他汀组：阿托伐他汀+Ox-LDL与DC共同培养48h
2．4混合T淋巴细胞增值反应：以DC为刺激细胞，测定DC刺激同种异体T淋巴细胞增殖情况。
2．5流式细胞学(FACS)分析：收集DC细胞，用流式细胞仪检测DC表面相关分化抗原的表达情况。
2．6 IL-12浓度的检测：ELISA双抗夹心法检测。
3、统计学方法：应用SPSS13.0软件系统分析，所有计量资料以均数±标准差((?)±s)表示，比较采用配对t检验。P＜0.05为差异有统计学意义。
结果
1、DC形态学上的观察：倒置相差显微镜下，(1) PBS组：培养第5天，树突样结构清晰，胞质丰富：(2)Ox-LDL组：DC与DiⅠ标记的Ox-LDL共同孵育48h后，DC逐渐发育变大，胞浆逐渐丰富，变的红染，呈不规则多型状态，并有较多伪足伸出，表现出树突形状；(3)阿托伐他汀组：阿托伐他汀与DC及DiⅠ标记的Ox-LDL共同孵育48h后，吞噬Ox-LDL的DC数目减少，且胞浆红染程度减轻。
2、混合T淋巴细胞反应：Ox-LDL干预后DC刺激T淋巴细胞增值反应增强，Ox-LDL组的A值是(3.9±0.8)与对照组(1.1±0.3)比较，P=0.000，差异有统计学意义：而阿托伐他汀和Ox-LDL共同干预DC后T淋巴细胞增值反应减弱，阿托伐他汀和Ox-LDL组的A值为(1.2±0.4)与Ox-LDL组比较，P=0.000，差异有统计学意义。
3、表型的影响收集干预48h的悬浮细胞，经FACS分析显示，与对照组相比，经Ox-LDL处理的DC其表面分子CD1a和HLA-DR的表达增加，均可刺激T淋巴细胞增值，与对照组比较[(64.5±18.2)vs(40.1±17.2)P=0.000]和[(89.8±5.7)vs(69.9±17.7)P=0.000]差异均有统计学意义；CD80和CD86的表达虽有轻度的增加，但与对照组比较分别为[(72.5±1.9)vs(60.7±23.8)P=0.063]和[(66.5±18.4)vs(74.9±18.6)P=0.159]差异尚无统计学意义。而阿托伐他汀处理的DC与Ox-LDL组比较其表面分子的表达CD1a和HLA-DR的表达明显减少，与Ox-LDL组比较分别为[(55.2±12.7)vs(64.5±18.2)P=0.038]和[(89.8±57)vs(71.0±11.6)P=0.000]，差异有统计学意义。
结论
1、外周血PBMC中的贴壁细胞培养7天后，在含粒细胞巨噬细胞集落刺激因子(GM-CSF)、白介素4(IL-4)条件下培养，均可成功诱导为DC。
2、Ox-LDL与DC共同孵育后，可使DC表面MHC-Ⅱ类分子高表达，从而具有较强的摄取抗原的能力，激活未致敏的T细胞，启动初次免疫应答。
3、阿托伐他汀依赖性地抑制Ox-LDL引起的生物学效应及可能的细胞内信号转导，可明显抑制DC的免疫活性，这推测是他汀类药物免疫调节治疗冠心病的新机制。
Background
Coronary Atherosclerosis heart disease(CHD) is one of the diseases with thehighest incidence rate and most disservice at preasent. It becames one of theconsequence question for discussion nowdays how the pathogenetic to CHD goes onand how to prevent and cure it. Therefor there were several theories to interpret theetiopathogenisis of CHD from difference points. Howevre some reseachers found thatautoimmune was a key factor to the etiopathogenisis of CHD. That is to say CHD isan autoimmune disease for some reasons. Firstly some phlegmasia transmitters andimmune substance were found in the blood and affected tissues with CHD patients.Secondly some autologous antibodies were detected with CHD patients. Thirdly therewere some special immune cells such as T leukomomocyte cell which participateautoimmune in affected tissues.
The initiation step to priming immune response is the immediate contactbetween leukomomocyte cell and antigen presenting cell(APC). APC is a kind ofspecial immune cell who can capture and process antigens, then present thoseantigens to antigenic special leukomomocyte cell. Acroding to whether MHC-Ⅱmolecules and its co-stimulus molecules were expressed in the surface ofcells or not, APC included two kinds of cells which was special and unspecial APC.Dendritic cell is the most functional APC in our bodys. T leukomomocyte cells couldbe intensively activited to priming special immune response with slight dendritic cells.And its capability to active T leukomomocyte cell was as much 100～10000 times asmacrophaga and B leukomomocyte cells. As we know the activationg of Tleukomomocyte cell was key to form plaques in CHD.
More and more researchs focused on Oxidized low density lipoprotein(Ox-LDL)with more cytotoxicitys which could result in more foam cells developed. Howeverfollowing researches showed us that LDL had some immunogenicitys similar toforeign proteins caused, which LDL was descompounded and reassociated inoxidative process and some new epiposition formed. Some finished clinicalresearches had drawed conclusions that Ox-LDL could not only damage endothelialcells and smooth muscle cells but also show some chemotaxis to histomonocytessome else. The binding between Ox-LDL and its receptor insurface of macrophagecould induce a series of incidents inner cells, such as generating inflamm cytokinesand magnifing inflamm responses. Although Ox-LDL had some immunogenicityssimilar to foreign proteins caused, its functions focued on that it could cause thehighly express of CD40/CD40L insurface of blood vessel endothelium cell. Howeverthere was no clear machanism of Ox-LDL to dendritic cells.It's well known that wecould use statins to cure CHD. And its pharmaco-mechanism is that statins couldcompetitively inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reducingenzyme. So that the synthesis of cholesterol decreased because of mevalonate reduced.Atorvastatin is the newest HMG-CoA reducing enzyme inhibitor. Several serious ofclinical researches discovered that atorvastatin has ideal effect on balancing blood-fatdisorders.
Our studies will maily be focus on observing the immune function changes ofdendritic cells excited with Ox-LDL in the level of cytobiology and molecule immue.Then to discuss how the Ox-LDL influences the immunological regulation ofdendritic cells which were the special antigen presenting cell with most capability. Sothat some immune theorys in our studies could offer a suppourt to the clinialprevention and cure CHD.
Objective
1. To determine whether Ox-LDL could influence DC maturing and immunefunction.
2. To investigate whether Atorvastatin could regulate adaptive immunity whichwas elicited by Ox-LDL
Subjects and Method
1、Subjects
Twenty healthy persons is verification by medical examination that include twelvemails and 8 femails.Their mean age is 40.5±8.7 years.
2、Method
2.1 Preparation of Ox-LDL and DiI-labelled
We separated and quantitatified the LDL from blood of healthy individuals by Superhigh density gradient centrifugation, and adjusted it to a final concentration of500ug/mL,then incubated with 10mmol/mL CuSO4.The oxidation was stopped with100μmol/L EDTA. Finally, we labeled the Ox-LDL with DiI.
2.2 Culture and interference of DC
Periperil blood mononuclear cell (PBMC) were isolated by density gradientcentrifugation, and cultured with RPMI-1640(containing 1% FCS, rhGM-CSF1,000,000 U/L, rhIL-4,500,000U/L)at an atmosphere of 5%CO2 for 5 days.Afterbeing taken into several groups, with different interferences and culturing for another 2h, DC were collected. Culture changes in cell morphology were observed under aninverted microscope.
2.3 Groups
(1) control group: DC were cultured without any treatment
(2) Ox-LDL group : DC were cultured with Ox-LDL for 48h
(3) Atorvastatin group: DC were cultured both with Atorvastatin and Ox-LDL
2.4 Proliferation of lymphocytes assay
Proliferation of allogeneic T lymphocyte were evaluated after incubation withDC which served as a stimulant.
2.5 Flow cytometry analysis
DC were collected and the expression of relative differentiation antigens on thesurface of DC were analyzed on a flow meter.
2.6 Detection of IL-12
Double mAb sandwich ELISA was applied in quantitation of IL-12.
3、Statistical analysis: SPSS 10.0 statistical soft was used.The data was shown as((?)±s), The compared of two groups was tested by paired sample t-test,
Results
1、Observation on morphology of DC
Several phenomenons were observed under an inverted microscope: (1)control group: There were distinct dendritic structures and abundant cytoplasm in DCafter cultured for 5 days. (2)Ox-LDL group :The volume of DC grew bigger andbigger with more abundant cytoplasm, dyed red, and became irregular morphous withmany pseudopodia. (3) Atorvastatin group: The number of DC whichphagocytosed Ox-LDL diminished, and the dyeing of DC became lighter afterincubating with both Atorvastatin and Ox-LDL libeled with DiI.
2、Proliferation of lymphocytes assay
Differences in proliferation of T lymphocytes between treated with Ox-LDLgroup (3.9±0.8) and the control group (1.0±0.4) were found to be statisticallysignificant (P＜0.05) .And the same result (P＜0.05) also occurred between the grouptreated both with Atorvastatin and Ox-LDL(1.3±0.2)and treated with Ox-LDL.
3、Influence of phenotype
Collected the suspension cells treated for 48h, and detected themolecules expressed on the cell surface by FACS analysis. The results showedthat, compared with the negative control and PBS group, there was higherexpression of CD1a and HLA-DR on the cell surface of Ox-LDLtreated group,both of which could stimulate the proliferation of T lymphocytes. Differencebetween the control and treated group (35.3±10.1 vs 69.8±9.1) and (11.3 vs. 70.5±92.3±5.0) was statistically significant (P＜0.05); although compared with the controlgroup,there was mild expression of CD80 and CD86 (63.5±7.7 vs 88.0±7.9) and(10.2 vs. 61.3±80.84±1.12 P＜0.01),difference was not statistically significant (P＞0.05). Compared with Ox-LDL treated group, there was a significant decrese on theexpression of CDla (69.8±9.1 vs 55.5±12.4) and HLA-DR (92.3±5.0 vs66.8±5.8) on DC treated with atorvastatin.The difference was statistically significant(P＜0.05).
Conclusion
1. The adherent cells of PBMC in peripheral blood could be induced to be DCafter culturing with GM-CSF or IL-4 for 7 days.
2. After incubated with Ox-LDL, there was high expression of MHC classⅡmolecules on the cell surface of DC, which could enhance its capacity to uptake ofantigens, and activate T cells and initial immune response.
3. Atorvastatin can inhibit immunocompetence of DC obviously, by inhibitingbiological effects of Ox-LDL and probably by participating the intracellular signal transduction. This presumption is a new mechanism of immune modulationtherapy of coronary heart diseases.
引文
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