幽门螺杆菌细胞毒素相关蛋白A阳性菌株感染与颈动脉粥样硬化形成的相关性及可能机制研究
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摘要
高血压病、高脂血症、糖尿病和吸烟等是颈动脉粥样硬化形成公认的传统高危因素,但有的颈动脉粥样硬化患者并无明显传统高危因素,有的患者血管病变重而临床症状轻,这些现象无法用传统理论进行合理解释,近些年研究发现,肺炎衣原体、巨细胞病毒、单纯疱疹病毒、幽门螺杆菌(helicobacter pylori, Hp)等病原体感染在颈动脉粥样硬化的形成过程中有着重要作用。其中Hp是目前国内外研究热点,有研究认为,Hp感染与颈动脉粥样硬化形成有关,且只有细胞毒素相关蛋白A(cytotoxin-associated protein A, CagA)阳性菌株可能和动脉粥样硬化形成有关,可导致颈动脉粥样硬化斑块的不稳定。但又有研究进行了颈动脉粥样硬化斑块内Hp的相关检测,并没有发现局部Hp感染因子的存在,故认为Hp感染导致颈动脉粥样硬化的依据不充分。我国是Hp高感染率的国家,Hp CagA+菌株是主要感染菌株,加之我国脑血管疾病发病率呈日益升高的趋势,因此进行Hp CagA+菌株感染和颈动脉粥样硬化形成的相关性及可能机制研究具有重要的临床意义和社会价值。
目前理论认为,动脉粥样硬化是一种慢性炎症性疾病,炎症反应及免疫调节机制在其发生发展过程中起着至关重要的作用。超敏C-反应蛋白(high-sensitivity c-reactive protein, hs-CRP)作为全身性炎症反应的标志物,与内皮细胞功能衰竭相关,通过多种机制促进动脉粥样硬化的炎症反应,在动脉粥样硬化的形成和发展中发挥重要的作用。但有研究认为,Hp DNA阳性的动脉粥样硬化患者,血清CRP、IL-6水平均增高,同Hp DNA阴性组相比,并没有显著性差异。因此需要进一步探讨Hp CagA+菌株感染与hs-CRP、颈动脉粥样硬化之间的关系。被认为同CRP一样具有价值的炎症相关生物标记物YKL-40,又称人类软骨糖蛋白39(human cartilage gp-39, HCgp-39),属于18-糖基水解酶家族的成员,作为结缔组织细胞的刺激因子、强大的内皮细胞和平滑肌细胞的迁移因子,可促进结缔组织生长及内皮细胞移行,认为其在可能影响细胞迁移和组织重建促进动脉粥样硬化形成。Hp CagA+菌株感染后,除了产生炎症反应外,其菌体成分还具有高度的免疫原性,刺激机体产生强烈的免疫应答反应,且其菌体细胞壁成分脂多糖(1ipopolysaccharide, LPS)的受体,即细胞分化决定簇14(cell differentiation antigen 14, CD14),与LPS-LBP复合物结合而被活化。有研究发现,CD14浓度增高和Hp感染相关联,Hp感染者的游离CD14(sCD14)水平高于未合并感染者。CD14活化后诱导多种免疫调节蛋白或因子的编码基因活化及表达,调节免疫应答。此外还刺激巨噬细胞产生TNF-α、IL-1、IL-6、IFN、迁移抑制因子、血小板衍生的生长因子、二十烷类物质以及活性氧等,加重炎症反应及促进平滑肌细胞的迁移和增殖,导致动脉粥样硬化的发生和发展。目前国内外有关Hp CagA+菌株感染与YKL-40、CD14以及颈动脉粥样硬化形成的相关研究及报道较为少见。
本研究拟首先进行临床病例研究,收集临床颈动脉粥样硬化病例及颈动脉内膜切除术(carotid endarterectomy, CEA)病例,检测hs-CRP、YKL-40、CD14水平及Hp-IgG抗体,进行斑块内Hp DNA的检测,探讨Hp CagA+菌株感染与颈动脉粥样硬化的相关性及可能机制,以及Hp CagA+菌株感染导致颈动脉粥样硬化患者发生临床缺血性脑血管事件的可能机制。然后进行基础动物实验,应用Hp CagA+菌株干预高脂血症大兔,建立Hp CagA+菌株感染致兔颈动脉粥样硬化模型,进行血清Hp抗体、hs-CRP、血浆YKL-40水平检测、斑块内Hp DNA,进一步求证Hp CagA+菌株感染和颈动脉粥样硬化形成的相关性,Hp CagA+菌株感染是否可促进颈动脉粥样硬化的形成,深入探讨其可能机制,以期为颈动脉粥样硬化形成的病因学研究提供实验基础,为临床治疗颈动脉粥样硬化提供新的策略,从而完善其防治模式。
第一部分
颈动脉粥样硬化患者Hp CagA+菌株感染状况调查及其致病危险性分析
目的观察颈动脉粥样硬化病变以及Hp感染情况,检测血脂、血清hs-CRP、血浆YKL-40、血清sCD14水平,分析Hp及其Hp CagA+菌株感染与颈动脉粥样硬化之间的关系,探讨其感染导致颈动脉粥样硬化形成的可能机制
方法选择2008年1月～2008年10月期间在我院超声科行颈动脉彩超检查的知情同意者共计310例。详细记录所有研究对象的病历资料。按有无颈动脉粥样硬化病变分为对照组和颈动脉粥样硬化组。ELISA方法检测血清Hp-IgG抗体及Hp-CagA-IgG抗体。酶标法检测血脂、免疫散射比浊法检测血清hs-CRP、ELISA方法检测血浆YKL-40及血清sCD14水平。
结果1.颈动脉粥样硬化组Hp-IgG阳性率为62.50%,对照组Hp-IgG阳性率为38.73%,颈动脉粥样硬化组明显高于对照组(P<0.05),Hp感染者患颈动脉粥样硬化的相对危险性明显增高,在调整年龄、性别、体重、血脂、血压、血糖等危险因素后,其危险性仍明显增高。2.颈动脉粥样硬化组Hp-CagA-IgG+检出率为45.24%,对照组Hp-CagA-IgG+检出率为21.38%,颈动脉粥样硬化组明显高于对照组(P<0.05),Hp CagA+菌株感染者患颈动脉粥样硬化的危险性显著增高,Hp CagA-菌株感染并未明显增加患颈动脉粥样硬化的危险性。3.颈动脉粥样硬化组和对照组中,同Hp-CagA-菌株感染和不伴有Hp感染者相比,Hp-CagA+菌株感染者TC、TG、LDL-C明显增高、HDL-C明显降低(P<0.05)。同不伴有Hp感染者相比,Hp CagA-菌株感染者TC、TG、LDL-C增高、HDL-C降低,但无统计学意义(P>0.05)。4.颈动脉粥样硬化组和对照组中,同Hp CagA-菌株感染和不伴有Hp感染者相比,Hp CagA+菌株感染者血清hs-CRP、血浆YKL-40、血清sCD14均明显增高(P<0.05)。同不伴有Hp感染者相比,Hp CagA-菌株感染者血清hs-CRP、血浆YKL-40、血清sCD14均增高,但无统计学意义(P>0.05)。
结论1.本研究通过临床证实,Hp感染是颈动脉粥样硬化病变的重要危险因素。2.同Hp CagA-菌株感染相比较,Hp CagA+菌株感染患颈动脉粥样硬化的危险性更大。3.Hp CagA+菌株感染者可能通过影响血脂代谢和增强炎症反应来促进和加重颈动脉粥样硬化形成。
第二部分
颈动脉粥样硬化患者斑块内Hp DNA检测以及YKL-40、CD14蛋白表达的分析
目的通过检测颈动脉粥样硬化斑块内炎症物质YKL-40和CD14的蛋白表达高低以及斑块内Hp DNA的存在,观察Hp及其Hp CagA+菌株感染、斑块内Hp DNA存在与斑块内YKL-40、CD14蛋白表达之间的关系,探讨Hp感染与颈动脉粥样硬化形成的可能机制及Hp CagA+菌株感染与颈动脉粥样硬化发生的危险性。
方法自第一部分的310例研究对象中,选择经颈动脉彩超检查发现颈动脉粥样硬化狭窄、在我院血管外科行颈动脉内膜切除术(carotid endarterectomy,CEA)的54例患者。按Hp感染情况分为Hp感染组、不合并Hp感染组,其中按感染Hp菌株的类型分为Hp CagA+菌株感染组、Hp CagA-菌株感染组。采用PCR技术测定斑块内Hp的ureC DNA、斑块组织进行YKL-40、CD14的免疫组化染色及图像分析测定。
结果1.16例不伴有Hp感染者的斑块组织中均未发现Hp ureC DNA,32例Hp CagA+菌株感染者的斑块组织中发现17例患者ureC DNA阳性(检出率53.13%),6例Hp CagA-菌株感染者的斑块组织中发现2例患者ureC DNA阳性(检出率33.33%),提示Hp CagA+菌株感染患颈动脉粥样硬化的危险性更大。2.Hp CagA+菌株感染者的YKL-40、CD14的阳性染色面积比值明显高于Hp CagA-菌株感染和不合并Hp感染者(P<0.05),Hp CagA-菌株感染者的YKL-40、CD14的阳性染色面积比值高于不合并Hp感染者,但无统计学意义(P>0.05)。Hp CagA+菌株感染者及Hp CagA-菌株感染者中,Hp DNA检出者的YKL-40、CD14的阳性染色面积比值均明显高于未检出者(P<0.05)。
结论1.进一步证实,同Hp CagA-菌株感染者相比,Hp CagA+菌株感染者患颈动脉粥样硬化的危险性更大。2.Hp CagA+菌株感染者,可能通过增强粥样硬化斑块局部的炎症反应及免疫反应促进和加重颈动脉粥样硬化形成。3.颈动脉粥样硬化斑块中发现有Hp DNA的存在,局部Hp感染因子可导致局部炎症物质释放增多,从而影响颈动脉粥样硬化的形成。
第三部分Hp CagA+菌株感染与颈动脉粥样硬化所致急性缺血性脑血管疾病之间的关系探讨
目的通过检测外周血、颈动脉粥样斑块组织内的Hp-CagA-IgG抗体以及血清hs-CRP的水平,探讨Hp CagA+菌株感染与颈动脉粥样硬化所致急性缺血性脑血管疾病之间的关系。
方法自第一部分的310例研究对象中,选择在我院血管外科行CEA手术、合并Hp CagA+菌株感染的患者32例。按有无合并颈内动脉系统急性缺血性脑血管病,将入组病人分为有症状组和无症状组。应用ELISA方法检测外周血、颈动脉粥样斑块组织内的Hp-CagA-IgG抗体以及血清hs-CRP水平。
结果1.同无症状组相比,有症状组血清Hp-CagA-IgG抗体浓度明显增高,差别有统计学意义(P<0.05)。2.同无症状组相比,有症状组血清hs-CRP水平明显增高,差别有统计学意义(P<0.05)。3.同无症状组相比,有症状组颈动脉粥样硬化斑块中Hp-CagA-IgG抗体表达明显增高,差别有统计学意义(P<0.05)。
结论颈动脉粥样硬化患者Hp CagA+菌株感染严重时,可能通过增强全身及局部的炎症反应和免疫反应,导致血清、斑块内Hp-CagA-IgG抗体含量以及血清hs-CRP水平增高,加重颈动脉粥样硬化病变,影响颈动脉粥样硬化斑块的稳定性,引起临床缺血性脑血管事件的发生。
第四部分Hp CagA+菌株感染致兔颈动脉粥样硬化模型的建立
目的通过建立Hp CagA+菌株感染的兔颈动脉粥样硬化模型,探讨Hp CagA+菌株感染导致颈动脉粥样硬化形成的可能致病机制,为进一步的相关研究奠定实验基础。
方法健康雄性新西兰大白兔48只,随机分为对照组和实验组,其中对照组6只,普通饲料喂养,实验组42只,给予高脂饲料喂养。实验第6周时,酶标法检测血脂、颈动脉内中膜厚度(intima-media thickness, IMT)、肉眼及组织学观察,确定建立高脂血症兔模型。将成功建立的36只高脂血症大兔随机平均分为高脂饮食组、Hp感染组、Hp治疗组,每组各12只。所有实验动物继续高脂饲料喂养。Hp感染组及Hp治疗组经耳缘静脉接种,初次给予Hp感染,Hp治疗组同时给予抗Hp治疗,高脂饮食组以等量生理盐水替代SS1菌液作同样处理。于实验第8周(初次感染2周后),再次给予Hp感染及抗Hp治疗,感染途径、剂量、时间及抗Hp治疗方案同前。实验第8周各组随机抽取6只大兔,酶标法检测血脂、免疫散射比浊法检测血清hs-CRP、ELISA方法检测血浆YKL-40以及血清Hp-CagA-IgG抗体,同期行颈动脉彩超检测颈动脉IMT、观察有无斑块形成以及斑块情况,肉眼及组织学观察兔颈动脉病理学改变,并应用PCR方法检测斑块内Hp CagA DNA。实验第12周时,对各组剩余实验动物进行相同指标检测。
结果1.高脂喂养第6周时成功建立高脂血症兔模型;实验第8、12周,同实验第6周相比,血脂无明显升高或降低(P>0.05)。2.实验第8周,同高脂饮食组、Hp治疗组相比,Hp感染组大兔血TC、TG、LDL-C明显升高、HDL-C明显降低(P<0.05),hs-CRP、YKL-40明显升高(P<0.05),颈动脉IMT明显增厚(P<0.05),肉眼可见条片状脂纹形成,镜下可见内膜明显不规则增厚、多层含脂质的泡沫细胞(P<0.05);同高脂饮食组相比,Hp治疗组大兔血脂、hs-CRP、YKL-40、颈动脉IMT均无明显改变(P>0.05),病理组织学检测显示粥样硬化程度一致。3.实验第12周,同高脂饮食组和Hp治疗组以及实验第8周时检测指标相比,Hp感染组大兔血TC、TG、LDL-C明显升高、HDL-C明显降低(P<0.05),hs-CRP、YKL-40明显升高(P<0.05),颈动脉彩超、病理组织学检测显示粥样硬化明显;同实验第8周时检测指标相比,高脂饮食组和Hp治疗组大兔血脂、hs-CRP、YKL-40均无明显升高(P>0.05),颈动脉彩超及病理组织学显示粥样硬化病变较前明显;同高脂饮食组相比,Hp治疗组大兔血脂、hs-CRP、YKL-40、颈动脉IMT均无明显升高(P>0.05),病理组织学检测显示粥样硬化程度一致。
结论1.在高脂血症大兔基础上,经兔耳缘静脉注射Hp CagA+菌株,可成功建立Hp CagA+菌株感染致兔颈动脉粥样硬化模型。2.Hp CagA+菌株导致血脂代谢异常。3.Hp CagA+菌株感染后血清hs-CRP、血浆YKL-40水平升高。4.Hp CagA+菌株感染可能通过影响血脂代谢、加重炎症反应来促进颈动脉粥样硬化形成。
Hypertension disease, hyperlipemia, diabetes and smoking and so on are approved traditional high-risk factors on carotid atherosclerosis. But there were some carotid atherosclerotics without obvious traditional high-risk factors, and some patients with serious pathological change of carotid artery had slight clinical symptoms. These phenomenas were unable to be explained reasonably with the traditional theory. The researches indicated that Chlamydia pneumoniae, the cytomegalovirus, herpes simplex virus, Helicobacter pylori (Hp) and so on played important roles in carotid atherosclerosis. Recently, Hp is investigative focus on association between Hp and carotid atherosclerosis. Some researcher believed that there was association between Hp infection and carotid atherosclerosis, only cytotoxin-associated gene-A-positive Helicobacter pylori(Hp CagA+) strains was possible related with atherosclerosis formation, which would cause carotid atherosclerotic plaque unstable. But there was contrary opinion because there was not existence of Hp in carotid atherosclerotic plaque, therefore carotid atherosclerosis caused by Hp infection was not to be confidence. Cytotoxin-associated gene-A-positive Helicobacter pylori strains is the main strain in patients infected with Hp in China. Cerebrovascular disease incidence trends to be higher in our country. There is important clinical significance and social value in researching the association and possible mechanism between Hp CagA+ strains infection and carotid atherosclerosis.
It is believed that atherosclerosis is one kind of chronic inflammatory diseases, in which inflammatory reaction and mechanism of immune-mediating played important roles. Hs-CRP, which is regarded as the marker of systemic inflammatory response, is related with dysfunction in endothelial cells and accelerates inflammatory response and plays the vital role in process of atherogenesis through many kinds of mechanism. Some researches indicated that there was no significant difference of serum CRP and IL-6 in carotid atherosclerosis patients infected with Hp CagA+ strains in comparision with cytotoxin-associated gene-A-negative Helicobacter pylori(Hp CagA - )strains. So association between Hp CagA+ strains and carotid atherosclerosis or hs-CRP needs to be studyed furtherly. YKL-40, called humanity cartilage glycoprotein 39 (humancartilage-gp 39, HCgp-39), is belongs to a member of 18-sugar base hydroltyic enzyme family, which is equally value with CRP as inflammatory marker. As a modulator of connective tissue cells and a migration factor of formidable endothelial cells and smooth muscle cells, YKL-40 may promote the connective tissue growth and the endothelial cell migration to cause atherogenesis. Hp CagA+ strains infection induces inflammatory reaction and strong immune response. Cell differentiation antigen 14(CD14) is activated as acceptor of Lipopolysaccharide(LPS) after combined with complex of LPS-LBP. Some research demonstrated that the CD14 density augmentation connected with Hp infection. Studies have shown that the level of soluble cell differentiation antigen 14 (sCD14) in patients infected Hp is higher than that of person without Hp infection. After CD14 was activated, it adjusted immune response by mean of many kinds to induce immunity regulatory protein or code gene in factor to be activated and express. In addition, CD14 stimulated the macrophage to product TNF-α, IL-1, IL-6, IFN, migration inhibiting factor, blood platelet growth factor, petrosilane class material as well as the active oxygen and so on. Aforesaid factors aggravated inflammation response and promoted smooth muscle cell's migration and the multiplication and caused atherogenesis. The related research and report at home and abroad are rarer between YKL-40、CD14 with Hp CagA+ strain infection and carotid artery atherogenesis at present.
This research firstly carry on the clinical research, including collection of clinical carotid atherosclerotics and the carotid endarterectomy(CEA)cases, and detect hs-CRP, YKL-40, CD14 level and Hp-IgG antibody, Hp DNA in carotid atherosclerotic plaque, to investigate association and possible mechanism between Hp CagA+ strain infection and carotid atherosclerosis as well as clinical acute cerebrovascular accident occured in carotid atherosclerotics with Hp CagA+ strain infection.
PART ONE
Investigation on Hp CagA+ strain infection condition and pathogenesis risk analyze in patients of carotid atherosclerosis
Objective To observate pathological changes of carotid atherosclerosis as well as Hp infection situation, examinate blood fats, blood serum hs-CRP, blood plasma YKL-40, blood serum sCD14, analyze relations between Hp or Hp CagA+ strain and carotid atherosclerosis, and explore possible mechanism of carotid atherosclerosis caused by infection.
Method From January to October in 2008 in our hospital, a total of 310 participants undergoing color ultra supersonic were chosen for our study after informed and consented. All objects were detailedly recorded cases datum. Control group and carotid atherosclerosis group were divided according to with or without carotid atherosclerosis. Blood serum Hp-IgG antibody, Hp-CagA-IgG antibody, blood fats, hs-CRP, blood plasma YKL-40and blood serum sCD14 levels were examinated by enzyme sign law, enzyme linked immunosorbent assay (ELISA) and immunonephelometry respectively.
Result 1.Hp-IgG antibody positive rate was 62.50% in carotid atherosclerosis group, 38.73% in control group, positive rate in carotid atherosclerosis is obviously higher than that of control group(P<0.05). Relative risk was obviously high in person infected Hp. Its risk was still obviously high although adjustment hazard factor on age, sex, body weight, blood fats, blood pressure, blood sugar. 2.Hp-CagA-IgG antibody positive rate was 45.24% in carotid atherosclerosis group, 21.38% in control group, positive rate in carotid atherosclerosis group was obviously higher than that of control group(P<0.05). Relative risk was obviously high in persons infected Hp CagA+ strain, but persons infected Hp CagA- strains were no liability to catch carotid atherosclerosis. 3.In comparison with persons infected Hp CagA-strain and persons without Hp in all groups, Levels of TC, TG, LDL-C were obviously high in persons infected Hp CagA+ strain(P<0.05), HDL-C was obvious reducted(P<0.05). In comparison with person without Hp infection, Levels of TC, TG, LDL-C were high in persons infected Hp CagA- strains, HDL-C was low, but the difference was not significant from a statistical standpoint(P>0.05). 4.In comparison with persons infected Hp CagA-strain and persons without Hp infection in all groups, Levels of hs-CRP, YKL-40, sCD14 were obviously high in persons infected Hp CagA+ strain(P<0.05). In comparison with person without Hp infection, Levels of hs-CRP, YKL-40, sCD14 were high in persons infected Hp CagA - strains, but the difference was not significant from a statistical standpoint(P>0.05).
Conclusion 1.Hp infection is hazard factor of carotid atherosclerosis through clinical research. 2.Comparison with the Hp CagA-strain infection, the relative risk was obviously high in persons infected Hp CagA+ strain. 3.The Hp CagA+ strain infection may promote and aggravate carotid atherosclerosis through affecting blood fats metabolism and the enhancement inflammatory reaction.
PART TWO
Hp DNA detection and expression of YKL-40, CD14 in carotid atherosclerotic plaque
Objective Existence of Hp DNA and expression of YKL-40 and CD14 in carotid atherosclerotic plaque were detected.. Relation between YKL-40、CD14 and Hp CagA+ strain infection was observated to explore possible mechanism between Hp infection and carotid artery atherogenesis and risk between Hp CagA- strain infection and carotid atherosclerosis.
Method A total of 54 patients with carotid atherosclerotic stenosis undergoing carotid endarterectomy (CEA) in vascular surgery department of our hospital from the 310 objects in the first part of research were choosed. All the patients were divided into Hp infection group and no Hp infection group, and further devided into Hp CagA+ strain infection group and Hp CagA- strain infection group according to Hp infection situation. Hp ureC DNA, YKL-40、CD14 in carotid atherosclerotic plaque was detected by PCR technology, immunohistochemical stains and image analysis respectively.
Result 1.Hp ureC DNA were not detected in carotid atherosclerotic plaque of the 16 patients without Hp infection and were detected in 17 patients infected Hp CagA+ strain, the detectable rate is 53.13%. Among 6 patients infected Hp CagA- strain, 2 patients were detected for ureC DNA in mottling organization, the detectable rate is 33.33%. In terms of persons infected Hp CagA+ strain, relative risk was obviously high to catch carotid atherosclerosis. 2.Compared with patients infected Hp CagA- strain and patients without Hp infection, Levels of YKL-40、CD14 were obviously high in patients infected Hp CagA+ strain(P<0.05). Compared with no accompanied Hp infection in all groups, Levels of YKL-40、CD14 were high in persons infected Hp CagA- strain, but non-statistics significance (P>0.05). Positive dyeing area ratio of YKL-40, CD14 in patients with Hp DNA position were obviously higher than that of patients with Hp DNA negative(P<0.05).
Conclusion 1.Research furtherly certified that Hp CagA+ strain infection has more risk to carotid atherosclerosis compared with Hp CagA- strain infection. 2.The Hp CagA+ strain infection may promote and aggravate carotid artery atherogenesis through enhancement inflammation response and immunity in plaque. 3.Hp DNA were detected in atherosclerotic plaque, the partial Hp infection factor may cause the partial inflammation material release to increasing, thus influence on atherogenesis.
PART THREE
Research on relation between Hp CagA+ strain infection and acute ischemic cerebrovascular disease caused by carotid atherosclerosis
Objective To research the relation between Hp CagA+ strain infection and acute ischemic cerebrovascular disease caused by carotid atherosclerosis through detecting Hp-CagA-IgG antibody in blood and carotid atherosclerotic plaque and blood serum hs-CRP.
Method A total of 32 patients with carotid atherosclerotic sclerostenosis undergoing carotid endarterectomy complicating Hp CagA+ strain infection were selected. According to whether or not having acute ischemic cerebrovascular disease, all the patients were divided into symptomatic group and asymptomatic group. Hp-CagA-IgG antibody in blood and carotid atherosclerotic plaque were performed by Enzyme Linked Immunosorbent Assay (ELISA) and blood serum hs-CRP level was detected by immunonephelometry.
Result 1.Compared with asymptomatic group, the density of blood serum Hp-CagA-IgG antibody was obviously high in symptoms group and had statistics significance(P<0.05). 2.Comparison with no symptoms group, level of hs-CRP was obviously high in symptomatic group and had statistics significance(P<0.05). 3.Comparison with no symptoms group, the density of Hp-CagA-IgG antibody in carotid atherosclerotic plaque was obviously high in symptoms group and had statistics significance(P<0.05).
Conclusion Through strengthening the whole body and the partial inflammatory reaction and the immune response, Severe Hp infection possibly raises the contents of Hp-CagA-IgG antibody in blood and carotid atherosclerotic plaque as well as the blood serum hs-CRP to aggravates the carotid artery pathological change and affect carotid atherosclerotic plaque stability, which cause acute ischemic cerebrovascular disease.
PART FOUR
Establishment of a carotid arteriosclerosis model by transvenous injection of Hp CagA+ strain
Objective To investigate possible mechanism between Hp CagA+ strain infection and carotid arteriosclerosis with a carotid arteriosclerosis model, which is established on New Zealand white rabbit by intravenous infusion of Hp CagA+ strain.
Method A total of 48 healthy male New Zealand white rabbits were equally randomized into a control group of 6 and an experimental group of 42. Ordinary feed was feed in control group, and high fat feed was feed in experimental group. Blood fats, carotid intima-media thickness (IMT), macrography, histopathologic study were examinated respectively after 6th week to determine a hyperlipemia rabbit model establishment. The 36 hyperlipemia rabbit were equally randomized into high fat diet group, Hp infection group and Hp treatment group, which were feed with high fat feed. The rabbits in Hp infection group and Hp treatment group were firstly injected Hp CagA+ strain, the Hp treatment group simultaneously was feed the anti-Hp drugs, the high fat diet group was substitutes the SS1 vaccine by the isometric physiological saline to make similar process. When 8th week ( first infected 2 weeks later), Hp CagA+ strain was given again by the same way. And 6 rabbits were chooesed randomly from each group. Blood fats, blood serum hs-CRP, blood plasma YKL-40, Hp-CagA-IgG antibody as well as carotid IMT were detected by the way mentioned previously. Hp CagA DNA was detected by PCR method in carotid atherosclerotic plaque. The same indexes of all the surplus animals were detected on at 12th week.
Result 1.Hyperlipemia rabbit model were successful established when at 6th week. The blood fats did not obviously change (P>0.05) at 8th week, 12th week. 2.When at 8th week, TC, TG, LDL-C, hs-CRP, YKL-40 were obviously higher in Hp infection group compared with high fat diet group and Hp treatment group(P<0.05), at the same time HDL-C was low and carotid IMT was obvious accumulation(P<0.05). Fatty streak was seen. Thickening of arterial wall and multi-layer lipid-laden macrophages were seen under the mirror. They were more different from that of high fat diet group and Hp treatment group (P<0.05). Compared with high fat diet group, blood fats, hs-CRP, YKL-40, carotid artery IMT in Hp treatment group werenot obvious change (P>0.05) and extent of atherosclerosis was consistent. 3.TC, TG, LDL-C, hs-CRP, YKL-40 were obviously higher in Hp infection group at 12th week than that of high fat diet group and Hp treatment group at 8th week(P<0.05), at the same time HDL-C was low (P<0.05), and extent of atherosclerosis was severe. Compared with the same indexes of 8th week, blood fats, hs-CRP, YKL-40 werenot obvious change in high fat diet group and in Hp treatment group (P>0.05), extent of atherosclerosis was severe. Compared with high fat diet group, blood fats, hs-CRP, YKL-40, extent of atherosclerosis in Hp treatment group werenot obvious change (P>0.05).
Conclusion 1.Carotid arteriosclerosis model can be successfully established by intravenous injection of Helicobacter pylori on hyperlipidemia rabbit. 2.Hp CagA+ strain caused abnormal metabolism of blood fat. 3.Blood serum hs-CRP and blood plasma YKL-40 were elevated after Hp CagA+ strain infection. 4.The Hp CagA+ strain infection may promote and aggravate carotid arteriosclerosis through enhancement of inflammaory and immune response.
目前理论认为,动脉粥样硬化是一种慢性炎症性疾病,炎症反应及免疫调节机制在其发生发展过程中起着至关重要的作用。超敏C-反应蛋白(high-sensitivity c-reactive protein, hs-CRP)作为全身性炎症反应的标志物,与内皮细胞功能衰竭相关,通过多种机制促进动脉粥样硬化的炎症反应,在动脉粥样硬化的形成和发展中发挥重要的作用。但有研究认为,Hp DNA阳性的动脉粥样硬化患者,血清CRP、IL-6水平均增高,同Hp DNA阴性组相比,并没有显著性差异。因此需要进一步探讨Hp CagA+菌株感染与hs-CRP、颈动脉粥样硬化之间的关系。被认为同CRP一样具有价值的炎症相关生物标记物YKL-40,又称人类软骨糖蛋白39(human cartilage gp-39, HCgp-39),属于18-糖基水解酶家族的成员,作为结缔组织细胞的刺激因子、强大的内皮细胞和平滑肌细胞的迁移因子,可促进结缔组织生长及内皮细胞移行,认为其在可能影响细胞迁移和组织重建促进动脉粥样硬化形成。Hp CagA+菌株感染后,除了产生炎症反应外,其菌体成分还具有高度的免疫原性,刺激机体产生强烈的免疫应答反应,且其菌体细胞壁成分脂多糖(1ipopolysaccharide, LPS)的受体,即细胞分化决定簇14(cell differentiation antigen 14, CD14),与LPS-LBP复合物结合而被活化。有研究发现,CD14浓度增高和Hp感染相关联,Hp感染者的游离CD14(sCD14)水平高于未合并感染者。CD14活化后诱导多种免疫调节蛋白或因子的编码基因活化及表达,调节免疫应答。此外还刺激巨噬细胞产生TNF-α、IL-1、IL-6、IFN、迁移抑制因子、血小板衍生的生长因子、二十烷类物质以及活性氧等,加重炎症反应及促进平滑肌细胞的迁移和增殖,导致动脉粥样硬化的发生和发展。目前国内外有关Hp CagA+菌株感染与YKL-40、CD14以及颈动脉粥样硬化形成的相关研究及报道较为少见。
本研究拟首先进行临床病例研究,收集临床颈动脉粥样硬化病例及颈动脉内膜切除术(carotid endarterectomy, CEA)病例,检测hs-CRP、YKL-40、CD14水平及Hp-IgG抗体,进行斑块内Hp DNA的检测,探讨Hp CagA+菌株感染与颈动脉粥样硬化的相关性及可能机制,以及Hp CagA+菌株感染导致颈动脉粥样硬化患者发生临床缺血性脑血管事件的可能机制。然后进行基础动物实验,应用Hp CagA+菌株干预高脂血症大兔,建立Hp CagA+菌株感染致兔颈动脉粥样硬化模型,进行血清Hp抗体、hs-CRP、血浆YKL-40水平检测、斑块内Hp DNA,进一步求证Hp CagA+菌株感染和颈动脉粥样硬化形成的相关性,Hp CagA+菌株感染是否可促进颈动脉粥样硬化的形成,深入探讨其可能机制,以期为颈动脉粥样硬化形成的病因学研究提供实验基础,为临床治疗颈动脉粥样硬化提供新的策略,从而完善其防治模式。
第一部分
颈动脉粥样硬化患者Hp CagA+菌株感染状况调查及其致病危险性分析
目的观察颈动脉粥样硬化病变以及Hp感染情况,检测血脂、血清hs-CRP、血浆YKL-40、血清sCD14水平,分析Hp及其Hp CagA+菌株感染与颈动脉粥样硬化之间的关系,探讨其感染导致颈动脉粥样硬化形成的可能机制
方法选择2008年1月～2008年10月期间在我院超声科行颈动脉彩超检查的知情同意者共计310例。详细记录所有研究对象的病历资料。按有无颈动脉粥样硬化病变分为对照组和颈动脉粥样硬化组。ELISA方法检测血清Hp-IgG抗体及Hp-CagA-IgG抗体。酶标法检测血脂、免疫散射比浊法检测血清hs-CRP、ELISA方法检测血浆YKL-40及血清sCD14水平。
结果1.颈动脉粥样硬化组Hp-IgG阳性率为62.50%,对照组Hp-IgG阳性率为38.73%,颈动脉粥样硬化组明显高于对照组(P<0.05),Hp感染者患颈动脉粥样硬化的相对危险性明显增高,在调整年龄、性别、体重、血脂、血压、血糖等危险因素后,其危险性仍明显增高。2.颈动脉粥样硬化组Hp-CagA-IgG+检出率为45.24%,对照组Hp-CagA-IgG+检出率为21.38%,颈动脉粥样硬化组明显高于对照组(P<0.05),Hp CagA+菌株感染者患颈动脉粥样硬化的危险性显著增高,Hp CagA-菌株感染并未明显增加患颈动脉粥样硬化的危险性。3.颈动脉粥样硬化组和对照组中,同Hp-CagA-菌株感染和不伴有Hp感染者相比,Hp-CagA+菌株感染者TC、TG、LDL-C明显增高、HDL-C明显降低(P<0.05)。同不伴有Hp感染者相比,Hp CagA-菌株感染者TC、TG、LDL-C增高、HDL-C降低,但无统计学意义(P>0.05)。4.颈动脉粥样硬化组和对照组中,同Hp CagA-菌株感染和不伴有Hp感染者相比,Hp CagA+菌株感染者血清hs-CRP、血浆YKL-40、血清sCD14均明显增高(P<0.05)。同不伴有Hp感染者相比,Hp CagA-菌株感染者血清hs-CRP、血浆YKL-40、血清sCD14均增高,但无统计学意义(P>0.05)。
结论1.本研究通过临床证实,Hp感染是颈动脉粥样硬化病变的重要危险因素。2.同Hp CagA-菌株感染相比较,Hp CagA+菌株感染患颈动脉粥样硬化的危险性更大。3.Hp CagA+菌株感染者可能通过影响血脂代谢和增强炎症反应来促进和加重颈动脉粥样硬化形成。
第二部分
颈动脉粥样硬化患者斑块内Hp DNA检测以及YKL-40、CD14蛋白表达的分析
目的通过检测颈动脉粥样硬化斑块内炎症物质YKL-40和CD14的蛋白表达高低以及斑块内Hp DNA的存在,观察Hp及其Hp CagA+菌株感染、斑块内Hp DNA存在与斑块内YKL-40、CD14蛋白表达之间的关系,探讨Hp感染与颈动脉粥样硬化形成的可能机制及Hp CagA+菌株感染与颈动脉粥样硬化发生的危险性。
方法自第一部分的310例研究对象中,选择经颈动脉彩超检查发现颈动脉粥样硬化狭窄、在我院血管外科行颈动脉内膜切除术(carotid endarterectomy,CEA)的54例患者。按Hp感染情况分为Hp感染组、不合并Hp感染组,其中按感染Hp菌株的类型分为Hp CagA+菌株感染组、Hp CagA-菌株感染组。采用PCR技术测定斑块内Hp的ureC DNA、斑块组织进行YKL-40、CD14的免疫组化染色及图像分析测定。
结果1.16例不伴有Hp感染者的斑块组织中均未发现Hp ureC DNA,32例Hp CagA+菌株感染者的斑块组织中发现17例患者ureC DNA阳性(检出率53.13%),6例Hp CagA-菌株感染者的斑块组织中发现2例患者ureC DNA阳性(检出率33.33%),提示Hp CagA+菌株感染患颈动脉粥样硬化的危险性更大。2.Hp CagA+菌株感染者的YKL-40、CD14的阳性染色面积比值明显高于Hp CagA-菌株感染和不合并Hp感染者(P<0.05),Hp CagA-菌株感染者的YKL-40、CD14的阳性染色面积比值高于不合并Hp感染者,但无统计学意义(P>0.05)。Hp CagA+菌株感染者及Hp CagA-菌株感染者中,Hp DNA检出者的YKL-40、CD14的阳性染色面积比值均明显高于未检出者(P<0.05)。
结论1.进一步证实,同Hp CagA-菌株感染者相比,Hp CagA+菌株感染者患颈动脉粥样硬化的危险性更大。2.Hp CagA+菌株感染者,可能通过增强粥样硬化斑块局部的炎症反应及免疫反应促进和加重颈动脉粥样硬化形成。3.颈动脉粥样硬化斑块中发现有Hp DNA的存在,局部Hp感染因子可导致局部炎症物质释放增多,从而影响颈动脉粥样硬化的形成。
第三部分Hp CagA+菌株感染与颈动脉粥样硬化所致急性缺血性脑血管疾病之间的关系探讨
目的通过检测外周血、颈动脉粥样斑块组织内的Hp-CagA-IgG抗体以及血清hs-CRP的水平,探讨Hp CagA+菌株感染与颈动脉粥样硬化所致急性缺血性脑血管疾病之间的关系。
方法自第一部分的310例研究对象中,选择在我院血管外科行CEA手术、合并Hp CagA+菌株感染的患者32例。按有无合并颈内动脉系统急性缺血性脑血管病,将入组病人分为有症状组和无症状组。应用ELISA方法检测外周血、颈动脉粥样斑块组织内的Hp-CagA-IgG抗体以及血清hs-CRP水平。
结果1.同无症状组相比,有症状组血清Hp-CagA-IgG抗体浓度明显增高,差别有统计学意义(P<0.05)。2.同无症状组相比,有症状组血清hs-CRP水平明显增高,差别有统计学意义(P<0.05)。3.同无症状组相比,有症状组颈动脉粥样硬化斑块中Hp-CagA-IgG抗体表达明显增高,差别有统计学意义(P<0.05)。
结论颈动脉粥样硬化患者Hp CagA+菌株感染严重时,可能通过增强全身及局部的炎症反应和免疫反应,导致血清、斑块内Hp-CagA-IgG抗体含量以及血清hs-CRP水平增高,加重颈动脉粥样硬化病变,影响颈动脉粥样硬化斑块的稳定性,引起临床缺血性脑血管事件的发生。
第四部分Hp CagA+菌株感染致兔颈动脉粥样硬化模型的建立
目的通过建立Hp CagA+菌株感染的兔颈动脉粥样硬化模型,探讨Hp CagA+菌株感染导致颈动脉粥样硬化形成的可能致病机制,为进一步的相关研究奠定实验基础。
方法健康雄性新西兰大白兔48只,随机分为对照组和实验组,其中对照组6只,普通饲料喂养,实验组42只,给予高脂饲料喂养。实验第6周时,酶标法检测血脂、颈动脉内中膜厚度(intima-media thickness, IMT)、肉眼及组织学观察,确定建立高脂血症兔模型。将成功建立的36只高脂血症大兔随机平均分为高脂饮食组、Hp感染组、Hp治疗组,每组各12只。所有实验动物继续高脂饲料喂养。Hp感染组及Hp治疗组经耳缘静脉接种,初次给予Hp感染,Hp治疗组同时给予抗Hp治疗,高脂饮食组以等量生理盐水替代SS1菌液作同样处理。于实验第8周(初次感染2周后),再次给予Hp感染及抗Hp治疗,感染途径、剂量、时间及抗Hp治疗方案同前。实验第8周各组随机抽取6只大兔,酶标法检测血脂、免疫散射比浊法检测血清hs-CRP、ELISA方法检测血浆YKL-40以及血清Hp-CagA-IgG抗体,同期行颈动脉彩超检测颈动脉IMT、观察有无斑块形成以及斑块情况,肉眼及组织学观察兔颈动脉病理学改变,并应用PCR方法检测斑块内Hp CagA DNA。实验第12周时,对各组剩余实验动物进行相同指标检测。
结果1.高脂喂养第6周时成功建立高脂血症兔模型;实验第8、12周,同实验第6周相比,血脂无明显升高或降低(P>0.05)。2.实验第8周,同高脂饮食组、Hp治疗组相比,Hp感染组大兔血TC、TG、LDL-C明显升高、HDL-C明显降低(P<0.05),hs-CRP、YKL-40明显升高(P<0.05),颈动脉IMT明显增厚(P<0.05),肉眼可见条片状脂纹形成,镜下可见内膜明显不规则增厚、多层含脂质的泡沫细胞(P<0.05);同高脂饮食组相比,Hp治疗组大兔血脂、hs-CRP、YKL-40、颈动脉IMT均无明显改变(P>0.05),病理组织学检测显示粥样硬化程度一致。3.实验第12周,同高脂饮食组和Hp治疗组以及实验第8周时检测指标相比,Hp感染组大兔血TC、TG、LDL-C明显升高、HDL-C明显降低(P<0.05),hs-CRP、YKL-40明显升高(P<0.05),颈动脉彩超、病理组织学检测显示粥样硬化明显;同实验第8周时检测指标相比,高脂饮食组和Hp治疗组大兔血脂、hs-CRP、YKL-40均无明显升高(P>0.05),颈动脉彩超及病理组织学显示粥样硬化病变较前明显;同高脂饮食组相比,Hp治疗组大兔血脂、hs-CRP、YKL-40、颈动脉IMT均无明显升高(P>0.05),病理组织学检测显示粥样硬化程度一致。
结论1.在高脂血症大兔基础上,经兔耳缘静脉注射Hp CagA+菌株,可成功建立Hp CagA+菌株感染致兔颈动脉粥样硬化模型。2.Hp CagA+菌株导致血脂代谢异常。3.Hp CagA+菌株感染后血清hs-CRP、血浆YKL-40水平升高。4.Hp CagA+菌株感染可能通过影响血脂代谢、加重炎症反应来促进颈动脉粥样硬化形成。
Hypertension disease, hyperlipemia, diabetes and smoking and so on are approved traditional high-risk factors on carotid atherosclerosis. But there were some carotid atherosclerotics without obvious traditional high-risk factors, and some patients with serious pathological change of carotid artery had slight clinical symptoms. These phenomenas were unable to be explained reasonably with the traditional theory. The researches indicated that Chlamydia pneumoniae, the cytomegalovirus, herpes simplex virus, Helicobacter pylori (Hp) and so on played important roles in carotid atherosclerosis. Recently, Hp is investigative focus on association between Hp and carotid atherosclerosis. Some researcher believed that there was association between Hp infection and carotid atherosclerosis, only cytotoxin-associated gene-A-positive Helicobacter pylori(Hp CagA+) strains was possible related with atherosclerosis formation, which would cause carotid atherosclerotic plaque unstable. But there was contrary opinion because there was not existence of Hp in carotid atherosclerotic plaque, therefore carotid atherosclerosis caused by Hp infection was not to be confidence. Cytotoxin-associated gene-A-positive Helicobacter pylori strains is the main strain in patients infected with Hp in China. Cerebrovascular disease incidence trends to be higher in our country. There is important clinical significance and social value in researching the association and possible mechanism between Hp CagA+ strains infection and carotid atherosclerosis.
It is believed that atherosclerosis is one kind of chronic inflammatory diseases, in which inflammatory reaction and mechanism of immune-mediating played important roles. Hs-CRP, which is regarded as the marker of systemic inflammatory response, is related with dysfunction in endothelial cells and accelerates inflammatory response and plays the vital role in process of atherogenesis through many kinds of mechanism. Some researches indicated that there was no significant difference of serum CRP and IL-6 in carotid atherosclerosis patients infected with Hp CagA+ strains in comparision with cytotoxin-associated gene-A-negative Helicobacter pylori(Hp CagA - )strains. So association between Hp CagA+ strains and carotid atherosclerosis or hs-CRP needs to be studyed furtherly. YKL-40, called humanity cartilage glycoprotein 39 (humancartilage-gp 39, HCgp-39), is belongs to a member of 18-sugar base hydroltyic enzyme family, which is equally value with CRP as inflammatory marker. As a modulator of connective tissue cells and a migration factor of formidable endothelial cells and smooth muscle cells, YKL-40 may promote the connective tissue growth and the endothelial cell migration to cause atherogenesis. Hp CagA+ strains infection induces inflammatory reaction and strong immune response. Cell differentiation antigen 14(CD14) is activated as acceptor of Lipopolysaccharide(LPS) after combined with complex of LPS-LBP. Some research demonstrated that the CD14 density augmentation connected with Hp infection. Studies have shown that the level of soluble cell differentiation antigen 14 (sCD14) in patients infected Hp is higher than that of person without Hp infection. After CD14 was activated, it adjusted immune response by mean of many kinds to induce immunity regulatory protein or code gene in factor to be activated and express. In addition, CD14 stimulated the macrophage to product TNF-α, IL-1, IL-6, IFN, migration inhibiting factor, blood platelet growth factor, petrosilane class material as well as the active oxygen and so on. Aforesaid factors aggravated inflammation response and promoted smooth muscle cell's migration and the multiplication and caused atherogenesis. The related research and report at home and abroad are rarer between YKL-40、CD14 with Hp CagA+ strain infection and carotid artery atherogenesis at present.
This research firstly carry on the clinical research, including collection of clinical carotid atherosclerotics and the carotid endarterectomy(CEA)cases, and detect hs-CRP, YKL-40, CD14 level and Hp-IgG antibody, Hp DNA in carotid atherosclerotic plaque, to investigate association and possible mechanism between Hp CagA+ strain infection and carotid atherosclerosis as well as clinical acute cerebrovascular accident occured in carotid atherosclerotics with Hp CagA+ strain infection.
PART ONE
Investigation on Hp CagA+ strain infection condition and pathogenesis risk analyze in patients of carotid atherosclerosis
Objective To observate pathological changes of carotid atherosclerosis as well as Hp infection situation, examinate blood fats, blood serum hs-CRP, blood plasma YKL-40, blood serum sCD14, analyze relations between Hp or Hp CagA+ strain and carotid atherosclerosis, and explore possible mechanism of carotid atherosclerosis caused by infection.
Method From January to October in 2008 in our hospital, a total of 310 participants undergoing color ultra supersonic were chosen for our study after informed and consented. All objects were detailedly recorded cases datum. Control group and carotid atherosclerosis group were divided according to with or without carotid atherosclerosis. Blood serum Hp-IgG antibody, Hp-CagA-IgG antibody, blood fats, hs-CRP, blood plasma YKL-40and blood serum sCD14 levels were examinated by enzyme sign law, enzyme linked immunosorbent assay (ELISA) and immunonephelometry respectively.
Result 1.Hp-IgG antibody positive rate was 62.50% in carotid atherosclerosis group, 38.73% in control group, positive rate in carotid atherosclerosis is obviously higher than that of control group(P<0.05). Relative risk was obviously high in person infected Hp. Its risk was still obviously high although adjustment hazard factor on age, sex, body weight, blood fats, blood pressure, blood sugar. 2.Hp-CagA-IgG antibody positive rate was 45.24% in carotid atherosclerosis group, 21.38% in control group, positive rate in carotid atherosclerosis group was obviously higher than that of control group(P<0.05). Relative risk was obviously high in persons infected Hp CagA+ strain, but persons infected Hp CagA- strains were no liability to catch carotid atherosclerosis. 3.In comparison with persons infected Hp CagA-strain and persons without Hp in all groups, Levels of TC, TG, LDL-C were obviously high in persons infected Hp CagA+ strain(P<0.05), HDL-C was obvious reducted(P<0.05). In comparison with person without Hp infection, Levels of TC, TG, LDL-C were high in persons infected Hp CagA- strains, HDL-C was low, but the difference was not significant from a statistical standpoint(P>0.05). 4.In comparison with persons infected Hp CagA-strain and persons without Hp infection in all groups, Levels of hs-CRP, YKL-40, sCD14 were obviously high in persons infected Hp CagA+ strain(P<0.05). In comparison with person without Hp infection, Levels of hs-CRP, YKL-40, sCD14 were high in persons infected Hp CagA - strains, but the difference was not significant from a statistical standpoint(P>0.05).
Conclusion 1.Hp infection is hazard factor of carotid atherosclerosis through clinical research. 2.Comparison with the Hp CagA-strain infection, the relative risk was obviously high in persons infected Hp CagA+ strain. 3.The Hp CagA+ strain infection may promote and aggravate carotid atherosclerosis through affecting blood fats metabolism and the enhancement inflammatory reaction.
PART TWO
Hp DNA detection and expression of YKL-40, CD14 in carotid atherosclerotic plaque
Objective Existence of Hp DNA and expression of YKL-40 and CD14 in carotid atherosclerotic plaque were detected.. Relation between YKL-40、CD14 and Hp CagA+ strain infection was observated to explore possible mechanism between Hp infection and carotid artery atherogenesis and risk between Hp CagA- strain infection and carotid atherosclerosis.
Method A total of 54 patients with carotid atherosclerotic stenosis undergoing carotid endarterectomy (CEA) in vascular surgery department of our hospital from the 310 objects in the first part of research were choosed. All the patients were divided into Hp infection group and no Hp infection group, and further devided into Hp CagA+ strain infection group and Hp CagA- strain infection group according to Hp infection situation. Hp ureC DNA, YKL-40、CD14 in carotid atherosclerotic plaque was detected by PCR technology, immunohistochemical stains and image analysis respectively.
Result 1.Hp ureC DNA were not detected in carotid atherosclerotic plaque of the 16 patients without Hp infection and were detected in 17 patients infected Hp CagA+ strain, the detectable rate is 53.13%. Among 6 patients infected Hp CagA- strain, 2 patients were detected for ureC DNA in mottling organization, the detectable rate is 33.33%. In terms of persons infected Hp CagA+ strain, relative risk was obviously high to catch carotid atherosclerosis. 2.Compared with patients infected Hp CagA- strain and patients without Hp infection, Levels of YKL-40、CD14 were obviously high in patients infected Hp CagA+ strain(P<0.05). Compared with no accompanied Hp infection in all groups, Levels of YKL-40、CD14 were high in persons infected Hp CagA- strain, but non-statistics significance (P>0.05). Positive dyeing area ratio of YKL-40, CD14 in patients with Hp DNA position were obviously higher than that of patients with Hp DNA negative(P<0.05).
Conclusion 1.Research furtherly certified that Hp CagA+ strain infection has more risk to carotid atherosclerosis compared with Hp CagA- strain infection. 2.The Hp CagA+ strain infection may promote and aggravate carotid artery atherogenesis through enhancement inflammation response and immunity in plaque. 3.Hp DNA were detected in atherosclerotic plaque, the partial Hp infection factor may cause the partial inflammation material release to increasing, thus influence on atherogenesis.
PART THREE
Research on relation between Hp CagA+ strain infection and acute ischemic cerebrovascular disease caused by carotid atherosclerosis
Objective To research the relation between Hp CagA+ strain infection and acute ischemic cerebrovascular disease caused by carotid atherosclerosis through detecting Hp-CagA-IgG antibody in blood and carotid atherosclerotic plaque and blood serum hs-CRP.
Method A total of 32 patients with carotid atherosclerotic sclerostenosis undergoing carotid endarterectomy complicating Hp CagA+ strain infection were selected. According to whether or not having acute ischemic cerebrovascular disease, all the patients were divided into symptomatic group and asymptomatic group. Hp-CagA-IgG antibody in blood and carotid atherosclerotic plaque were performed by Enzyme Linked Immunosorbent Assay (ELISA) and blood serum hs-CRP level was detected by immunonephelometry.
Result 1.Compared with asymptomatic group, the density of blood serum Hp-CagA-IgG antibody was obviously high in symptoms group and had statistics significance(P<0.05). 2.Comparison with no symptoms group, level of hs-CRP was obviously high in symptomatic group and had statistics significance(P<0.05). 3.Comparison with no symptoms group, the density of Hp-CagA-IgG antibody in carotid atherosclerotic plaque was obviously high in symptoms group and had statistics significance(P<0.05).
Conclusion Through strengthening the whole body and the partial inflammatory reaction and the immune response, Severe Hp infection possibly raises the contents of Hp-CagA-IgG antibody in blood and carotid atherosclerotic plaque as well as the blood serum hs-CRP to aggravates the carotid artery pathological change and affect carotid atherosclerotic plaque stability, which cause acute ischemic cerebrovascular disease.
PART FOUR
Establishment of a carotid arteriosclerosis model by transvenous injection of Hp CagA+ strain
Objective To investigate possible mechanism between Hp CagA+ strain infection and carotid arteriosclerosis with a carotid arteriosclerosis model, which is established on New Zealand white rabbit by intravenous infusion of Hp CagA+ strain.
Method A total of 48 healthy male New Zealand white rabbits were equally randomized into a control group of 6 and an experimental group of 42. Ordinary feed was feed in control group, and high fat feed was feed in experimental group. Blood fats, carotid intima-media thickness (IMT), macrography, histopathologic study were examinated respectively after 6th week to determine a hyperlipemia rabbit model establishment. The 36 hyperlipemia rabbit were equally randomized into high fat diet group, Hp infection group and Hp treatment group, which were feed with high fat feed. The rabbits in Hp infection group and Hp treatment group were firstly injected Hp CagA+ strain, the Hp treatment group simultaneously was feed the anti-Hp drugs, the high fat diet group was substitutes the SS1 vaccine by the isometric physiological saline to make similar process. When 8th week ( first infected 2 weeks later), Hp CagA+ strain was given again by the same way. And 6 rabbits were chooesed randomly from each group. Blood fats, blood serum hs-CRP, blood plasma YKL-40, Hp-CagA-IgG antibody as well as carotid IMT were detected by the way mentioned previously. Hp CagA DNA was detected by PCR method in carotid atherosclerotic plaque. The same indexes of all the surplus animals were detected on at 12th week.
Result 1.Hyperlipemia rabbit model were successful established when at 6th week. The blood fats did not obviously change (P>0.05) at 8th week, 12th week. 2.When at 8th week, TC, TG, LDL-C, hs-CRP, YKL-40 were obviously higher in Hp infection group compared with high fat diet group and Hp treatment group(P<0.05), at the same time HDL-C was low and carotid IMT was obvious accumulation(P<0.05). Fatty streak was seen. Thickening of arterial wall and multi-layer lipid-laden macrophages were seen under the mirror. They were more different from that of high fat diet group and Hp treatment group (P<0.05). Compared with high fat diet group, blood fats, hs-CRP, YKL-40, carotid artery IMT in Hp treatment group werenot obvious change (P>0.05) and extent of atherosclerosis was consistent. 3.TC, TG, LDL-C, hs-CRP, YKL-40 were obviously higher in Hp infection group at 12th week than that of high fat diet group and Hp treatment group at 8th week(P<0.05), at the same time HDL-C was low (P<0.05), and extent of atherosclerosis was severe. Compared with the same indexes of 8th week, blood fats, hs-CRP, YKL-40 werenot obvious change in high fat diet group and in Hp treatment group (P>0.05), extent of atherosclerosis was severe. Compared with high fat diet group, blood fats, hs-CRP, YKL-40, extent of atherosclerosis in Hp treatment group werenot obvious change (P>0.05).
Conclusion 1.Carotid arteriosclerosis model can be successfully established by intravenous injection of Helicobacter pylori on hyperlipidemia rabbit. 2.Hp CagA+ strain caused abnormal metabolism of blood fat. 3.Blood serum hs-CRP and blood plasma YKL-40 were elevated after Hp CagA+ strain infection. 4.The Hp CagA+ strain infection may promote and aggravate carotid arteriosclerosis through enhancement of inflammaory and immune response.
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[1]张贞耀,陈达光,陈玲,等.颈总动脉内径扩大与内膜-中层厚度是高血压合并动脉粥样硬化的两个标志.中国循环杂志, 1996, 11(6): 337-341.
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