IL-27对ox-LDL介导人树突状细胞免疫成熟作用的研究
发布时间：2018-09-18 11:59
【摘要】：冠心病(coronary heart disease CHD)是20世纪以来威胁人类生命健康的最主要疾病之一,而且其发病率越来越高,其发病原因及防治方法一直是社会关注的焦点。冠心病的诊治方法日渐完善,但其发病机制却未十分明了。动脉粥样硬化(atherosclerosis AS)是心脑血管疾病发病的主要病理基础。AS的形成机制学说主要有脂质沉淀、损伤修复、血栓形成等。众所周知,心脏病家族史、性别、高龄、吸烟、高血压病、糖尿病、高脂血症等是AS的高危因素,近年研究表明感染因素如肺炎衣原体、幽门螺旋杆菌、巨细胞病毒等也与CHD有重要关系。急性冠脉综合征是冠心病的一个重要进展阶段,而易损斑块是急性冠脉综合征的主要病理基础。国内外的研究发现,炎症反应在易损斑块的进展中发挥着重要的作用,是斑块不稳定及容易破裂的主要原因,因而CHD可能是一种冠状动脉慢性炎症及全身免疫性疾病。 树突状细胞(Dendritic cell, DCs)在免疫过程中主要起抗原呈递作用,是体内功能最强的专职抗原呈递细胞。极少量的DCs即可强烈激活T淋巴细胞启动特异性细胞免疫反应。国内外研究发现,正常动脉内膜存在一种血管相关淋巴组织(VALT),散在分布着一些由免疫活性细胞和抗原呈递细胞组成的细胞群,对血管组织中可能有害的内源性或外源性抗原进行监视和筛查。研究发现,DCs在AS病变中聚集明显增加,DCs与T淋巴细胞共同出现于AS的病变薄弱位置。当DC遇到内源性或外源性抗原时,可将抗原呈递给T细胞,使其活化增殖,分泌细胞因子,然后启动一系列免疫反应,导致AS的发生发展。最近的研究显示氧化修饰的低密度脂蛋白可通过激活DCs介导的获得性免疫反应,促进AS病变的进展。 研究发现,氧化型低密度脂蛋白(oxidized low density lipoprotein, ox-LDL)是一种内源性免疫反应激活剂,可引起血管内皮功能失调、参与泡沫细胞形成、促进血管平滑肌细胞增殖及诱导凋亡等。已有的研究表明,ox-LDL在动脉粥样硬化局部沉积后,促进DC与血管内皮粘附,并且作为自身抗原刺激局部产生抗氧化型低密度脂蛋白抗体,促进DC分化成熟,并激活T淋巴细胞,参与动脉粥样硬化局部免疫炎症病变。适度ox-LDL可促进单核细胞来源的树突状细胞成熟,增强其激活T细胞的功能,增加DC分泌细胞因子IL-2、IL-12、IL-18、INF-γ(?)(?)TNFα等,并且这些作用随ox-LDL浓度和氧化程度的增高而增强,但是过高的浓度却会导致DCs凋亡 越来越多研究发现,多种炎症标志物参与了CHD的发生、发展及预后,如IL-6、IL-10、IL-18、IL-12及超敏C反应蛋白(high sensitive C-reactive protein, hs-CRP)等。IL-27 (Interleukin-27, IL-27)是新近发现的属于IL-6/IL-12家族细胞因子,由p28和EBI3组成,具有促进及抑制免疫反应的双重作用。由于IL-27在免疫反应和免疫耐受的调节中扮演了不可或缺的角色,因此在炎症性、自身免疫性、感染性、肿瘤性疾病中均发现IL-27的表达及功能变化。但是在冠心病的研究中却十分稀少,本研究通过检测不同类型冠心病患者的血浆IL-27水平,了解CHD患者血浆IL-27的变化,并探讨它们之间的关系。 IL-27主要由活化的DC细胞产生,单核细胞、巨噬细胞、NK细胞等也可以分泌一定量的IL-27,当DCs表面的Toll样受体(TLRs)受到病原体相关分子模式(PAMP)刺激后会分泌IL-27。人外周血单核细胞来源的DC表达IL-27受体(EBI3和p28组成的异二聚体),提示IL-27能够对单核细胞来源的DC产生影响,它可以反馈作用于DCs,促进DCs表面共刺激分子的表达以及提高其激活辅助性T细胞(Th)的能力。国外研究学者发现WSX-1敲除的小鼠中DC在体外受到LPS刺激后表面分子CD80/CD86表达上调,诱导Thl细胞增殖和产生IFN-γ的能力也大大提高。 DCs是联系天然防御功能和获得性免疫的关键,与CHD发生发展密切相关,那么在CHD发生发展的机制中,IL-27是否通过作用于DCs发挥免疫调节功能?IL-27处理后DCs的功能状态如何,是否会影响其功能蛋白及免疫调节分子的分泌?本文旨在研究IL-27对体外扩增DC免疫活性的影响,为IL-27结合DC用于CHD免疫机制研究提供依据。 本课题分为三个部分,第一部分：通过ELISA法及免疫比浊法分别检测不同类型冠状动脉粥样硬化性心脏病(简称冠心病,CHD)患者血浆白介素27(IL-27)及超敏C反应蛋白(hs-CRP)的水平,并进行比较,以探讨IL-27是否与冠心病的进展以及粥样斑块的稳定密切相关。第二部分：将人PBMC来源的DC作为实验模型,以OX-LDL作为抗原诱导DC成熟,用RT—PCR的方法测定DC产生IL-27亚基的情况,探讨人体内IL-27的由来。第三部分：将人PBMC来源的DC作为实验模型,以OX-LDL作为抗原诱导DC成熟,用流式细胞技术及ELISA方法,观察DC的成熟程度和分泌细胞因子的能力,并进一步应用IL-27进行干预研究。本课题通过研究IL-27对体外扩增DC免疫活性的影响,为IL-27结合DC用于CHD免疫机制研究提供依据,为AS发生发展的免疫学介导学说提供实验依据,具有深远的意义。结果分述如下： 1不同CHD患者血浆中IL-27及hs-CRP水平具有差异性 1.1入选各组病例一般临床资料比较无显著性差异 临床入选病例共88例,其中对照组28例,SAP组10例,UAP组25例,AMI组 25例,以上各组一般临床资料的比较无显著性差异,具有可比性。 1.2不同CHD患者血浆中IL-27水平具有差异性 CHD组血浆IL-27水平明显高于对照组；SAP组及UAP组血浆IL-27水平无差异；UAP组及SAP组血浆IL-27水平高于AMI组。 1.3不同CHD患者血浆中hs-CRP具有差异性 CHD组患者血浆hs-CRP水平高于对照组；UAP组血浆hs-CRP水平高于SAP组；AMI组均高于UAP及SAP组。 1.4 hs-CRP、IL-27在CHD中表达的关系 将88例患者血清中hs-CRP与IL-27进行相关分析,结果显示IL-27与hs-CRP呈正相关,r=0.308,P0.01。 2 ox-LDL诱导人DC细胞成熟,用RT-PCR的方法测定DC的IL-27亚基mRNA表达 将加ox-LDL分为四组(n=3)：(1)对照组：DC+PBS；(2)DC+ox-LDL(100mg/L,作用8小时)组；(3)DC+0x-LDL(100mg/L,作用16小时)组；(4)DC+ox-LDL(100mg/L,作用24小时)组。 2.1 DC的镜下鉴定： 倒置相差显微镜及扫描电镜下,从健康人外周血中分离的PBMC经ox-LDL诱导后成为细胞形态不规则、表面毛刺状突起、细胞质丰富的成熟DC。 2.2用RT-PCR的方法检测DC的IL-27亚基(P28及EBI3)mRNA表达 ox-LDL上调DC的IL-27亚基(P28及EBI3)mRNA的表达,ox-LDL刺激组P28及EBI3mRNA表达均比对照组高,并有时间依赖性的特点。 3 IL-27对ox-LDL介导树突状细胞免疫成熟作用的研究 将加IL-27分为三组(n=5),均作用24小时：(1)对照组：DC+PBS；(2)DC+ox-LDL(100mg/L组；(3)DC+ox-LDL(100mg/L)+IL-27(100ng/mL)组。 3.1 DC的镜下鉴定： 倒置相差显微镜及扫描电镜下观察,从外周血中分离的PBMC经ox-LDL及IL-27诱导后成为细胞形态不规则、表面毛刺状突起、细胞质丰富的成熟DC。 3.2用流式细胞仪检测对DC细胞表型的影响 3.2.1 ox-LDL上调DC细胞表型表达 流式细胞仪检测人外周血PBMC来源的DC表型HLA-DR.CD83及CD86。ox-LDL组HLA-DR.CD83及CD86表型表达较对照组高,说明ox-LDL可刺激外周血PBMC来源的DC成熟,具有一定的抗原呈递功能。 3.2.2 IL-27上调ox-LDL诱导成熟的DC表型的表达 加入IL-27后DC表面的HLA-DR、CD83及CD86表达高于ox-LDL组,说明IL-27可进一步促进ox-LDL刺激外周血PBMC来源的DC成熟。 3.3对DC培养上清液细胞因子IL-6、IL-10及IL-12p70的影响 3.3.1 ox-LDL上调DC培养上清液细胞因子IL-6及IL-10的表达,下调IL-12p70的表达。 ox-LDL组DC培养上清液细胞因子IL-6及IL-10的表达均比对照组高,IL-12p70表达比对照组低。说明ox-LDL刺激DC免疫成熟后,促进Thl型细胞因子IL-6分泌,具有Th1型免疫反应的能力。 3.3.2 IL—27上调ox-LDL诱导成熟的DC表达IL-6,下调IL-10的表达。 用ox-LDL诱导DC成熟后,加入IL-27刺激,DC培养上清液细胞因子IL-6表达比ox-LDL高,IL-10的表达比ox-LDL低。说明IL-27具有一定促进Thl型免疫反应及抑制Th2型免疫反应的能力。 通过上述三个部分的实验,我们可以得出以下结论： 1、不同类型CHD患者血浆IL-27及hs-CRP均较对照组升高,说明与病情相关,可作为CHD病情评估的参考指标； 2、ox-LDL上调DC的IL-27亚基(P28及EBI3)mRNA的表达,说明ox-LDL能够使活化的人PBMC来源的DC分泌IL-27。 3、ox-LDL可促进DC免疫分化成熟。 4、ox-LDL可促进DC免疫成熟,并促进DC分泌IL-6及IL-10。 5、IL—27上调ox-LDL所诱导的DC的免疫成熟,并进一步促进DC分泌IL-6及抑制DC分泌IL-10。
[Abstract]:Coronary heart disease (CHD) is one of the most important diseases threatening human life and health since the 20th century, and its incidence is increasing. The causes and prevention methods of CHD have been the focus of social attention. As is known to all, family history of heart disease, sex, old age, smoking, hypertension, diabetes, hyperlipidemia and so on are high risk factors for AS. Recent studies have shown that infectious factors such as Chlamydia pneumoniae, pylorus and so on. Spirulina, cytomegalovirus and so on also have the important relations with CHD. Acute coronary syndrome is an important progress stage of coronary heart disease, and vulnerable plaque is the main pathological basis of acute coronary syndrome. Therefore, CHD may be a chronic inflammatory disease of the coronary artery and systemic immune diseases.
Dendritic cells (DCs) are the most powerful professional antigen presenting cells in vivo and play a major role in antigen presenting during the immune process. Very few DCs can activate T lymphocytes to initiate specific cellular immune responses. Domestic and foreign studies have found that there is a vascularly associated lymphoid tissue (VALT) in the intima of normal arteries. A number of immunocompetent cells and antigen presenting cells are distributed to monitor and screen for potentially harmful endogenous or exogenous antigens in vascular tissues. Studies have shown that DCs aggregate significantly in AS lesions, and DCs and T lymphocytes co-occur in the weaker sites of AS lesions. Sex antigens present antigens to T cells to activate and proliferate, secrete cytokines, and then initiate a series of immune responses leading to the development of AS. Recent studies have shown that oxidized low density lipoproteins can promote the progression of AS by activating DCs-mediated acquired immune responses.
It has been found that oxidized low density lipoprotein (ox-LDL) is an endogenous immune activator, which can cause endothelial dysfunction, participate in foam cell formation, promote the proliferation and induce apoptosis of vascular smooth muscle cells. Previous studies have shown that ox-LDL is localized after atherosclerosis. Promote DC adhesion to vascular endothelium, and as an autoantigen stimulate local production of anti-oxidative LDL antibodies, promote DC differentiation and maturation, and activate T lymphocytes, participate in atherosclerotic local immune inflammation. Increasing the secretion of cytokines IL-2, IL-12, IL-18, INF-gamma (?) TNFa and so on, and these effects increased with the increase of ox-LDL concentration and oxidation degree, but too high concentration would lead to apoptosis of DCs.
More and more studies have found that a variety of inflammatory markers are involved in the occurrence, development and prognosis of CHD, such as IL-6, IL-10, IL-18, IL-12 and high sensitive C-reactive protein (hs-CRP), etc. IL-27 (Interleukin-27, IL-27) is a newly discovered cytokine belonging to the IL-6/IL-12 family, which is composed of p28 and EBI3, and has promotive and inhibitory effects. Because IL-27 plays an indispensable role in the regulation of immune response and immune tolerance, the expression and function of IL-27 are found in inflammatory, autoimmune, infectious, and tumor diseases. However, it is rare in the study of coronary heart disease. This study examined different types of coronary artery disease. The level of plasma IL-27 in patients with heart disease was investigated, and the changes of plasma IL-27 in CHD patients were investigated, and the relationship between them was also discussed.
IL-27 is mainly produced by activated DC cells. Monocytes, macrophages, NK cells and so on can also secrete a certain amount of IL-27. Toll-like receptors (TLRs) on the surface of DCs can secrete IL-27 when stimulated by pathogen-associated molecular model (PAMP). Human peripheral blood monocyte-derived DC expresses IL-27 receptors (heterodimer composed of EBI3 and p28), suggesting that IL-27 is expressed in peripheral blood monocytes. -27 can affect monocyte-derived DC, which can feedback DCs, promote the expression of costimulatory molecules on DCs surface and enhance their ability to activate helper T cells (Th). Foreign researchers have found that in WSX-1 knockout mice, the expression of CD80/CD86 on DCs surface is up-regulated after LPS stimulation, and induces Thl cell proliferation. And the ability to produce IFN- gamma is also greatly improved.
DCs are the key link between natural defense and acquired immunity, and are closely related to the occurrence and development of CHD. Does IL-27 play an immunoregulatory role in the pathogenesis of CHD? What is the functional status of DCs after IL-27 treatment, and whether it affects the secretion of functional proteins and immunoregulatory molecules? The effect of L-27 on the DC activity in vitro was amplified, which provided a basis for the study of IL-27 combined with DC for the immune mechanism of CHD.
This study is divided into three parts. The first part is to detect the levels of interleukin-27 (IL-27) and high-sensitivity C-reactive protein (hs-CRP) in different types of coronary atherosclerotic heart disease (CHD) patients by ELISA and immunoturbidimetry, and compare them to explore whether IL-27 and coronary heart disease progress and atherosclerotic plaque. Part two: DC derived from human PBMC was used as an experimental model, OX-LDL was used as an antigen to induce DC maturation, and RT-PCR was used to determine the production of IL-27 subunit in DC. Part three: DC derived from human PBMC was used as an experimental model, OX-LDL was used as an antigen to induce DC maturation, and flow was used as an antigen to induce DC maturation. The maturity of DC and the ability of secreting cytokines were observed by cell-based immunoassay and ELISA, and the effect of IL-27 on the immune activity of DC was further studied. This study provides a basis for the study of the immune mechanism of IL-27 combined with DC in CHD and provides a basis for the immunological mediation theory of AS development. The results are of far-reaching significance. The results are as follows:
1 the levels of IL-27 and hs-CRP in plasma of different CHD patients are different.
1.1 there was no significant difference in clinical data between the selected groups.
A total of 88 clinical cases were selected, including 28 cases in the control group, 10 cases in group SAP, 25 cases in group UAP, and AMI group.
In 25 cases, there was no significant difference in general clinical data between the above groups.
1.2 the level of IL-27 in plasma of different CHD patients is different.
The level of IL-27 in plasma of CHD group was significantly higher than that of control group, and there was no difference between SAP group and UAP group. The level of IL-27 in plasma of UAP group and SAP group was higher than that of AMI group.
1.3 hs-CRP in plasma of different CHD patients is different.
The level of hs-CRP in plasma of CHD group was higher than that of control group, UAP group was higher than that of SAP group, AMI group was higher than that of UAP and SAP group.
Relationship between 1.4 hs-CRP and IL-27 expression in CHD
Correlation analysis of serum hs-CRP and IL-27 in 88 patients showed that IL-27 was positively correlated with hs-CRP, r=0.308, P 0.01.
2 ox-LDL induced the maturation of human DC cells, and mRNA expression of DC IL-27 subunit was determined by RT-PCR.
Ox-LDL was divided into four groups (n=3): (1) control group: DC + PBS; (2) DC + ox-LDL (100mg / L, 8 hours) group; (3) DC + 0x-LDL (100mg / L, 16 hours) group; (4) DC + ox-LDL (100mg / L, 24 hours) group.
2.1 DC for microscopic identification:
Under inverted phase contrast microscopy and scanning electron microscopy, PBMCs isolated from healthy human peripheral blood were induced by ox-LDL to become mature DC with irregular cell morphology, burr-like process on the surface and abundant cytoplasm.
2.2 the expression of DC IL-27 subunit (P28 and EBI3) mRNA was detected by RT-PCR.
Ox-LDL up-regulated the expression of IL-27 subunit (P28 and EBI3) mRNA in DC, and the expression of P28 and EBI3 mRNA in ox-LDL-stimulated group was higher than that in control group, and the expression was time-dependent.
Effect of 3 IL-27 on ox-LDL mediated immune maturation of dendritic cells
IL-27 was divided into three groups (n=5) for 24 hours: (1) control group: DC + PBS; (2) DC + ox-LDL group (100mg / L); and (3) DC + ox-LDL (100mg / L) + IL-27 (100ng / mL).
3.1 DC for microscopic identification:
Under inverted phase contrast microscope and scanning electron microscope, PBMCs isolated from peripheral blood were induced by ox-LDL and IL-27 to form mature DC with irregular morphology, burr-like process on the surface and abundant cytoplasm.
3.2 the effect of flow cytometry on the phenotype of DC cells.
3.2.1 ox-LDL upregulated DC cell phenotypic expression
The expression of HLA-DR.CD83 and CD86.ox-LDL phenotypes in PBMC-derived DC from human peripheral blood was higher than that in control group by flow cytometry.
3.2.2 IL-27 upregulated ox-LDL induced expression of mature DC phenotype.
After adding IL-27, the expression of HLA-DR, CD83 and CD86 on DC surface was higher than that in ox-LDL group, indicating that IL-27 could further promote the maturation of PBMC-derived DC stimulated by ox-LDL.
Effect of 3.3 on cytokines IL-6, IL-10 and IL-12p70 in supernatant of DC culture
3.3.1 ox-LDL increased the expression of cytokines IL-6 and IL-10 in DC supernatant, and down regulated the expression of IL-12p70.
The expression of cytokines IL-6 and IL-10 in DC supernatant of ox-LDL group was higher than that of control group, and IL-12p70 was lower than that of control group.
3.3.2 IL - 27 upregulated ox-LDL induced mature DC expression IL-6 and down regulated IL-10 expression.
After DC maturation was induced by ox-LDL and stimulated by IL-27, the expression of cytokine IL-6 in DC supernatant was higher than that of ox-LDL and IL-10 was lower than that of ox-LDL.
Through the above three parts of experiments, we can draw the following conclusions:
1. The levels of plasma IL-27 and hs-CRP in patients with different types of CHD were higher than those in the control group, indicating that the levels of IL-27 and hs-CRP were correlated with the severity of CHD.
2. ox-LDL up-regulates the expression of IL-27 subunit (P28 and EBI3) mRNA in DC, suggesting that ox-LDL can secrete IL-27 from activated PBMC-derived DC.
3, ox-LDL can promote immune differentiation and maturation of DC.
4, ox-LDL can promote DC immune maturation and promote DC to secrete IL-6 and IL-10..
5. IL-27 up-regulates the immune maturation of DC induced by ox-LDL and further promotes the secretion of IL-6 and inhibits the secretion of IL-10.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1
本文编号：2247835
[Abstract]:Coronary heart disease (CHD) is one of the most important diseases threatening human life and health since the 20th century, and its incidence is increasing. The causes and prevention methods of CHD have been the focus of social attention. As is known to all, family history of heart disease, sex, old age, smoking, hypertension, diabetes, hyperlipidemia and so on are high risk factors for AS. Recent studies have shown that infectious factors such as Chlamydia pneumoniae, pylorus and so on. Spirulina, cytomegalovirus and so on also have the important relations with CHD. Acute coronary syndrome is an important progress stage of coronary heart disease, and vulnerable plaque is the main pathological basis of acute coronary syndrome. Therefore, CHD may be a chronic inflammatory disease of the coronary artery and systemic immune diseases.
Dendritic cells (DCs) are the most powerful professional antigen presenting cells in vivo and play a major role in antigen presenting during the immune process. Very few DCs can activate T lymphocytes to initiate specific cellular immune responses. Domestic and foreign studies have found that there is a vascularly associated lymphoid tissue (VALT) in the intima of normal arteries. A number of immunocompetent cells and antigen presenting cells are distributed to monitor and screen for potentially harmful endogenous or exogenous antigens in vascular tissues. Studies have shown that DCs aggregate significantly in AS lesions, and DCs and T lymphocytes co-occur in the weaker sites of AS lesions. Sex antigens present antigens to T cells to activate and proliferate, secrete cytokines, and then initiate a series of immune responses leading to the development of AS. Recent studies have shown that oxidized low density lipoproteins can promote the progression of AS by activating DCs-mediated acquired immune responses.
It has been found that oxidized low density lipoprotein (ox-LDL) is an endogenous immune activator, which can cause endothelial dysfunction, participate in foam cell formation, promote the proliferation and induce apoptosis of vascular smooth muscle cells. Previous studies have shown that ox-LDL is localized after atherosclerosis. Promote DC adhesion to vascular endothelium, and as an autoantigen stimulate local production of anti-oxidative LDL antibodies, promote DC differentiation and maturation, and activate T lymphocytes, participate in atherosclerotic local immune inflammation. Increasing the secretion of cytokines IL-2, IL-12, IL-18, INF-gamma (?) TNFa and so on, and these effects increased with the increase of ox-LDL concentration and oxidation degree, but too high concentration would lead to apoptosis of DCs.
More and more studies have found that a variety of inflammatory markers are involved in the occurrence, development and prognosis of CHD, such as IL-6, IL-10, IL-18, IL-12 and high sensitive C-reactive protein (hs-CRP), etc. IL-27 (Interleukin-27, IL-27) is a newly discovered cytokine belonging to the IL-6/IL-12 family, which is composed of p28 and EBI3, and has promotive and inhibitory effects. Because IL-27 plays an indispensable role in the regulation of immune response and immune tolerance, the expression and function of IL-27 are found in inflammatory, autoimmune, infectious, and tumor diseases. However, it is rare in the study of coronary heart disease. This study examined different types of coronary artery disease. The level of plasma IL-27 in patients with heart disease was investigated, and the changes of plasma IL-27 in CHD patients were investigated, and the relationship between them was also discussed.
IL-27 is mainly produced by activated DC cells. Monocytes, macrophages, NK cells and so on can also secrete a certain amount of IL-27. Toll-like receptors (TLRs) on the surface of DCs can secrete IL-27 when stimulated by pathogen-associated molecular model (PAMP). Human peripheral blood monocyte-derived DC expresses IL-27 receptors (heterodimer composed of EBI3 and p28), suggesting that IL-27 is expressed in peripheral blood monocytes. -27 can affect monocyte-derived DC, which can feedback DCs, promote the expression of costimulatory molecules on DCs surface and enhance their ability to activate helper T cells (Th). Foreign researchers have found that in WSX-1 knockout mice, the expression of CD80/CD86 on DCs surface is up-regulated after LPS stimulation, and induces Thl cell proliferation. And the ability to produce IFN- gamma is also greatly improved.
DCs are the key link between natural defense and acquired immunity, and are closely related to the occurrence and development of CHD. Does IL-27 play an immunoregulatory role in the pathogenesis of CHD? What is the functional status of DCs after IL-27 treatment, and whether it affects the secretion of functional proteins and immunoregulatory molecules? The effect of L-27 on the DC activity in vitro was amplified, which provided a basis for the study of IL-27 combined with DC for the immune mechanism of CHD.
This study is divided into three parts. The first part is to detect the levels of interleukin-27 (IL-27) and high-sensitivity C-reactive protein (hs-CRP) in different types of coronary atherosclerotic heart disease (CHD) patients by ELISA and immunoturbidimetry, and compare them to explore whether IL-27 and coronary heart disease progress and atherosclerotic plaque. Part two: DC derived from human PBMC was used as an experimental model, OX-LDL was used as an antigen to induce DC maturation, and RT-PCR was used to determine the production of IL-27 subunit in DC. Part three: DC derived from human PBMC was used as an experimental model, OX-LDL was used as an antigen to induce DC maturation, and flow was used as an antigen to induce DC maturation. The maturity of DC and the ability of secreting cytokines were observed by cell-based immunoassay and ELISA, and the effect of IL-27 on the immune activity of DC was further studied. This study provides a basis for the study of the immune mechanism of IL-27 combined with DC in CHD and provides a basis for the immunological mediation theory of AS development. The results are of far-reaching significance. The results are as follows:
1 the levels of IL-27 and hs-CRP in plasma of different CHD patients are different.
1.1 there was no significant difference in clinical data between the selected groups.
A total of 88 clinical cases were selected, including 28 cases in the control group, 10 cases in group SAP, 25 cases in group UAP, and AMI group.
In 25 cases, there was no significant difference in general clinical data between the above groups.
1.2 the level of IL-27 in plasma of different CHD patients is different.
The level of IL-27 in plasma of CHD group was significantly higher than that of control group, and there was no difference between SAP group and UAP group. The level of IL-27 in plasma of UAP group and SAP group was higher than that of AMI group.
1.3 hs-CRP in plasma of different CHD patients is different.
The level of hs-CRP in plasma of CHD group was higher than that of control group, UAP group was higher than that of SAP group, AMI group was higher than that of UAP and SAP group.
Relationship between 1.4 hs-CRP and IL-27 expression in CHD
Correlation analysis of serum hs-CRP and IL-27 in 88 patients showed that IL-27 was positively correlated with hs-CRP, r=0.308, P 0.01.
2 ox-LDL induced the maturation of human DC cells, and mRNA expression of DC IL-27 subunit was determined by RT-PCR.
Ox-LDL was divided into four groups (n=3): (1) control group: DC + PBS; (2) DC + ox-LDL (100mg / L, 8 hours) group; (3) DC + 0x-LDL (100mg / L, 16 hours) group; (4) DC + ox-LDL (100mg / L, 24 hours) group.
2.1 DC for microscopic identification:
Under inverted phase contrast microscopy and scanning electron microscopy, PBMCs isolated from healthy human peripheral blood were induced by ox-LDL to become mature DC with irregular cell morphology, burr-like process on the surface and abundant cytoplasm.
2.2 the expression of DC IL-27 subunit (P28 and EBI3) mRNA was detected by RT-PCR.
Ox-LDL up-regulated the expression of IL-27 subunit (P28 and EBI3) mRNA in DC, and the expression of P28 and EBI3 mRNA in ox-LDL-stimulated group was higher than that in control group, and the expression was time-dependent.
Effect of 3 IL-27 on ox-LDL mediated immune maturation of dendritic cells
IL-27 was divided into three groups (n=5) for 24 hours: (1) control group: DC + PBS; (2) DC + ox-LDL group (100mg / L); and (3) DC + ox-LDL (100mg / L) + IL-27 (100ng / mL).
3.1 DC for microscopic identification:
Under inverted phase contrast microscope and scanning electron microscope, PBMCs isolated from peripheral blood were induced by ox-LDL and IL-27 to form mature DC with irregular morphology, burr-like process on the surface and abundant cytoplasm.
3.2 the effect of flow cytometry on the phenotype of DC cells.
3.2.1 ox-LDL upregulated DC cell phenotypic expression
The expression of HLA-DR.CD83 and CD86.ox-LDL phenotypes in PBMC-derived DC from human peripheral blood was higher than that in control group by flow cytometry.
3.2.2 IL-27 upregulated ox-LDL induced expression of mature DC phenotype.
After adding IL-27, the expression of HLA-DR, CD83 and CD86 on DC surface was higher than that in ox-LDL group, indicating that IL-27 could further promote the maturation of PBMC-derived DC stimulated by ox-LDL.
Effect of 3.3 on cytokines IL-6, IL-10 and IL-12p70 in supernatant of DC culture
3.3.1 ox-LDL increased the expression of cytokines IL-6 and IL-10 in DC supernatant, and down regulated the expression of IL-12p70.
The expression of cytokines IL-6 and IL-10 in DC supernatant of ox-LDL group was higher than that of control group, and IL-12p70 was lower than that of control group.
3.3.2 IL - 27 upregulated ox-LDL induced mature DC expression IL-6 and down regulated IL-10 expression.
After DC maturation was induced by ox-LDL and stimulated by IL-27, the expression of cytokine IL-6 in DC supernatant was higher than that of ox-LDL and IL-10 was lower than that of ox-LDL.
Through the above three parts of experiments, we can draw the following conclusions:
1. The levels of plasma IL-27 and hs-CRP in patients with different types of CHD were higher than those in the control group, indicating that the levels of IL-27 and hs-CRP were correlated with the severity of CHD.
2. ox-LDL up-regulates the expression of IL-27 subunit (P28 and EBI3) mRNA in DC, suggesting that ox-LDL can secrete IL-27 from activated PBMC-derived DC.
3, ox-LDL can promote immune differentiation and maturation of DC.
4, ox-LDL can promote DC immune maturation and promote DC to secrete IL-6 and IL-10..
5. IL-27 up-regulates the immune maturation of DC induced by ox-LDL and further promotes the secretion of IL-6 and inhibits the secretion of IL-10.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1
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