【作者】 冯旭阳；

【导师】 王海昌；

【作者基本信息】 第四军医大学 ， 内科学， 2009， 博士

【摘要】 研究背景动脉粥样硬化是慢性炎症性疾病。高脂血症时,血浆低密度脂蛋白(LDL)进入动脉壁氧化成为氧化低密度脂蛋白(oxLDL),被巨噬细胞吞噬后转化为泡沫细胞,发生动脉粥样硬化。既往研究证实,天然属性IgM亚类抗体通过封闭oxLDL与巨噬细胞结合的表位,抑制巨噬细胞对oxLDL的吞噬,从而降低泡沫细胞与动脉粥样硬化斑块的形成。临床研究发现血清高浓度脂多糖/LPS与动脉粥样硬化发病密切相关,但机理尚不十分明确。目前还没有关于高脂饮食饲养的正常小鼠是否能够诱导产生天然属性抗氧化低密度脂蛋白IgM亚类抗体,从而能影响动脉粥样硬化发生发展;体内或者体外应用这些天然抗体是否有保护作用;以及在LPS血清浓度升高提高动脉粥样硬化的发病过程中,这些天然抗体是否参与尚未见报道。基于以上,我们设计并完成了相关实验。目的:1.制备并鉴定天然属性的抗LDL及抗oxLDL IgM亚类抗体,为研究LDL和oxLDL及其天然抗体在动脉粥样硬化发生发展中的作用奠定基础。2.探讨天然属性的抗oxLDL IgM亚类抗体对巨噬细胞与oxLDL结合的影响,了解其在动脉粥样硬化发病机制中的作用。3.以apoE基因敲除小鼠为研究对象,复制动脉粥样硬化模型,研究天然属性抗oxLDL IgM亚类抗体对apoE基因敲除小鼠血脂、血oxLDL和主动脉粥样硬化斑块形成的影响。4.探讨脂多糖活化对巨噬细胞与oxLDL结合的影响,了解脂多糖活化对天然IgM亚类抗体在动脉粥样硬化中的保护作用的破坏机理,进一步了解天然抗体在动脉粥样硬化中的作用。方法:1.给予饲养在无特殊病原体条件下Babl/c小鼠高胆固醇饮食,4周后取脾细胞直接与SP2/0细胞融合,以纯化的LDL及oxLDL为抗原对阳性杂交瘤细胞生长孔进行间接ELISA筛选。鉴定杂交瘤上清的免疫球蛋白亚类、亚型,进而采用免疫印迹、免疫沉淀法和ELISA法对获得的抗体进行免疫学反应性鉴定。2.体外培养小鼠巨噬细胞系;纯化IgM抗体3A6并将其与Na125I标记的oxLDL作用形成免疫复合物。通过油红O染色及细胞结合同位素标记oxLDL,以观察3A6对巨噬细胞对oxLDL吞噬的封闭作用。3. 18只8周龄apoE基因敲除小鼠随机分为对照组(腹腔注射PBS/2ml/周);5G8组:(腹腔注射天然属性抗低密度脂蛋白IgM亚类抗体5G8/10μg/g体重,2ml/周)和3A6组(腹腔注射天然属性抗氧化低密度脂蛋白IgM抗体3A6/10μg/g体重,2ml/周)。高脂饲料饲养16周后行处死,分离血清,测定血清脂质含量及血浆oxLDL含量,小鼠原位灌注固定,石蜡包埋,连续切片,HE染色,对各个切面的动脉粥样斑块面积进行分析。4. LPS刺激巨噬细胞后,通过油红O染色及细胞结合同位素标记oxLDL,观察IgM抗体3A6在LPS活化巨噬细胞情况下对oxLDL吞噬地影响。分别利用TLR4的中和性单抗, p38MAPK阻抑剂SB203580和NF-κB阻抑剂PDTC作用及Fcα/μ受体进行RNA干扰后,利用实时定量RT-PCR和流式细胞术观察Fcα/μ受体mRNA转录及蛋白表达情况。结果:1.杂交瘤细胞分泌的抗LDL及抗oxLDL的天然抗体通过ELISA法被筛选出来,可以与LDL或oxLDL发生高亲和力结合,经过4次克隆化,最终获得2株稳定分泌天然属性抗LDL抗体,命名为5G8和2H7,及1株稳定分泌天然属性抗oxLDL抗体,命名为3A6,3株抗体均属于IgM亚类,无交叉反应,可以满足免疫印迹、免疫沉淀、ELISA等实验要求。2.天然属性抗oxLDL IgM亚类抗体3A6,在体外试验中能够抑制巨噬细胞与铜氧化的LDL的结合,从而降低泡沫细胞的形成。3. apoE基因敲除小鼠高脂饲料喂养16周后,三组之间的各项血脂及oxLDL含量无明显差异;HE染色结果显示:三组均出现动脉粥样硬化斑块,但3A6组可显著降低胸主动脉斑块面积和校正面积,与对照组和5G8组比较差异有显著性。4. 3A6不能抑制脂多糖活化的巨噬细胞对铜氧化的LDL的结合,并且促进铜氧化的LDL介导的泡沫细胞的生成。我们分别利用3A6 F(ab′)2、Fcα/μ受体RNA干扰或者利用不识别oxLDL的IgM抑制了oxLDL-IgM与脂多糖活化的巨噬细胞的结合,意味着Fcα/μ受体对这个过程是起作用的。同时,脂多糖促进Fcα/μ受体的表达有时间和剂量的依赖性,TLR4特异性中和抗体的封闭作用可减少LPS的作用。另外,脂多糖诱导的p38MAPK磷酸化和NF-κB 65的转位促进了Fcα/μ受体表达的上调。当使用p38MAPK阻抑剂SB203580和NF-κB阻抑剂PDTC,可降低了Fcα/μ受体表达的上调。结论:1.抗LDL及抗oxLDL IgM亚类单抗的制备为研究天然抗体在体内脂质代谢和相关心脑血管疾病如动脉粥样硬化等发生发展中的作用提供了重要的研究工具。2.天然属性抗oxLDL IgM亚类抗体3A6降低了泡沫细胞的形成,可能对动脉粥样硬化形成有预防作用。3.天然属性抗oxLDL IgM亚类抗体3A6对apoE基因敲除小鼠血脂及血浆oxLDL含量无明显影响;但对apoE基因敲除小鼠胸主动脉粥样硬化斑块形成有抑制作用。4. 3A6不能抑制脂多糖活化的巨噬细胞对oxLDL的摄取,并且通过其Fc段与巨噬细胞的结合而促进了oxLDL介导的泡沫细胞的生成。脂多糖通过TLR-4受体活化了p38MAPK和NF-κB通路,并且上调了Fcα/μ受体在巨噬细胞表面的表达。5.我们的研究结果证实了LPS浓度升高加重动脉粥样硬化形成的内在机制。更多还原

【Abstract】 BackgroundAtherosclerosis is now widely recognized as a chronic inflammatory disease. In condition of high blood lipids, plasma Low-density lipoprotein (LDL) infiltrates into the artery wall and becomes oxidized LDL (oxLDL); oxLDL is uptaken by macrophages and macrophages transform into foam cells, which can cause dramatic atherosclerotic effect. A number of in vitro studies have suggested that these IgM antibodies blocked the uptake of oxLDL by macrophages and thus could prevent foam cells formation in vivo. Clinic researches proved that chronic infection (serum high level lipopolysaccharide) had close relationship with atherosclerosis pathogenesis, but the mechanisms were not clear.No data were released of whether the normal mice fed with high fat diet produced natural antibodies against oxLDL to influence development of atherosclerosis and whether these natural antibodies played protective roles when applied in vitro or in vivo, and whether these auto-antibodies were involved during course of increased mortality of atherosclerosis induced by increased infection. Based on the above, we designed and fulfilled the following experiments.Objective:1. To Generate and identify the production of natural mouse IgM monoclonal antibodies(mAbs) against LDL and oxLDL with high specificity and activity and establish foundation for the research on LDL and oxidized oxLDL and natural antibody in atherosclerosis progression.2. To explore the role and the exact molecular mechanisms of natural anti-oxLDL IgM monoclonal antibody played and involved in pathogenesis of atherosclerosis underlying the action of IgM antibody in the binding of oxLDL to macrophages.3. By applying apoE gene knock-out mice as the animal model ,artherosclerosis was induced by high fat food.Experiments were executed on purpose to study the effects of natural anti-oxLDL antibody 3A6 on serum lipids, blood plasma oxLDL and lesion of apoE gene knock-out mice.4. To explore the role and the exact molecular mechanisms of natural anti-oxLDL IgM monoclonal antibody on pathogenesis of atherosclerosis underlying the action of bacterial lipopolysaccharide (LPS) activation in the binding of oxLDL to macrophages.Methods:1. BALB/c mice were breed in specific pathogen free conditions and fed with high cholesterol diet; the splenocytes from these mice were directly fused with Sp2/0 myeloma cells by using standard hybridoma production techniques. Hybridomas were selected on the basis of the supernatant’s ability to bind with native LDL and oxLDL in ELISA. Selected hybridomas were identified by Western blot, Immunoblotting and ELISA.2. Murine macrophage cell line RAW-264.7 was cultured in vitro. OxLDL specific monoclonal antibody 3A6 with IgM isotype was purified and utilized to form complex with Na125I-conjugated oxLDL. Influence of 3A6 on formation of foam cells was observed by Oil Red O staining and the affinity of Na125I-conjugated oxLDL.3. 18 apoE gene knock-out mice at the age of 8 weeks were randomly divided into 3 groups:the control group (PBS was intraperitoneal injected 2ml once per-week); 5G8 group(5G8 was intraperitoneal injected 10μg/g weight,2ml per-week) and 3A6 group(3A6 was intraperitoneal injected 10μg/g weight,2ml per-week),then all of the animal were euthanized after fed by high fat food for 16 weeks. Blood was sampled from the eye.The serum lipids and blood plasma oxLDL was detected.Tissue was fixed by in situ liquid flow.Thoracic aorta harvested and fixed in 10% formalin,followed by paraffin imbedding,serial sections,and HE staining. The area of aortic AS plaque in every sectioned was analyzed.4. After LPS stimulation on macrophages, anti-TLR4 neutralizing mAb, p38MAPK specific inhibitor SB203580,NF-κB specific inhibitor PDTC or RNAi targeting Fcα/μreceptor were applied, respectively. The mRNA transcription and protein expression of Fcα/μreceptor in macrophages were studied by real-time RT-PCR and flow cytometry. Similarly, influence of 3A6 on formation of foam cells was observed by Oil Red O staining and the affinity of Na125I-conjugated oxLDL.Results:1. Resulting hybridomas producing anti-LDL and anti-oxLDL antibodies were screened by enzyme-linked immunosorbent assay (ELISA) and isotyped. As a result, two hybridoma cell lines, named 5G8 and 2H7 were developed, which could screte anti-LDL MAbs stably, and one hybridoma cell lines, named 3A6 was developed, which could screte anti-oxLDL MAbs stably. All of them belonged to IgM subclass, no cross reactions were found between these MAbs. The specificity of MAb was determined based on its activity in Western blot, Immunoblotting and ELISA analysis.2. Natural anti-oxLDL IgM monoclonal antibody 3A6 specifically inhibited the binding of CuoxLDL to na?ve macrophages in vitro.3. After the apoE gene knock-out mice being fed with high fat diet for 16 weeks,there was no significant difference in serum lipids and blood plasma oxLDL among 3 groups, but obvious atherosclerotic lesion was found in their aortic root. The HE staining indicated that the thoracic aorta plaque area and correct area of the apoE-/-+3A6 group were significant reduced than the apoE-/-+PBS group and the apoE-/-+5G8 group.4. 3A6 failed to inhibit the binding of CuoxLDL to LPS-activated macrophages and promoted the formation of CuoxLDL-mediated foam macrophages. Furthermore, 3A6 F (ab′) 2 or pre-incubation with un-related IgM inhibited the binding of 3A6/CuoxLDL complex to LPS-activated macrophages, suggesting that the Fcα/μreceptor may be responsible for the binding of 3A6/CuoxLDL complex to LPS-activated macrophages. Indeed, LPS up-regulated the expression of Fcα/μreceptor in macrophages in a dose- and time-dependent manner, which was diminished by treatment with anti-TLR4 neutralizing mAb. In addition, LPS induced the phosphorylation of p38MAPK and translocation of NF-κB p65, contributing to the up-regulated expression of Fcα/μreceptor in macrophages as treatment with specific inhibitor for p38MAPK (SB203580) or NF-κB (PDTC) attenuated the up-regulation of Fcα/μreceptor expression induced by LPS in macrophages.Conclusions:1. The production of natural mouse IgM monoclonal antibodies (MAbs) anti-LDL and anti-oxLDL with high specificity and activity were generated by using standard hybridoma production techniques, which could provide a potential value for the research on lipid metabolisms and atherosclerosis progression.2. Natural anti-oxLDL IgM monoclonal antibody 3A6 specifically inhibited the binding of CuoxLDL to na?ve macrophages and foam cell formation in vitro.3. Natural anti-oxLDL antibody 3A6 has no effect on serum lipids and blood plasma oxLDL of apoE gene knock-out mice, but inhibits formation of the thoracic aorta atherosclerosisas plaque.4. LPS, through the TLR4 receptor, activated the p38MAPK and NF-κB pathways and up-regulated the expression of Fcα/μreceptor in macrophages, which promoted the binding of 3A6/CuoxLDL complex to macrophages through binding with Fc fragments and the formation of foam macrophages.5. Our findings provide a new explanation why increased LPS concentration deteriorates the pathogenesis of atherosclerosis.更多还原