海尔曼螺杆菌的流行病学研究和致病性的活体评价

发布时间：2018-06-18 17:11

本文选题：H.heilmannii + 培养　；参考：《南方医科大学》2008年硕士论文

【摘要】：背景、目的: 海尔曼螺杆菌(Helicobacter heilmannii,H.heilmannii),是除幽门螺杆菌(Helicobacter pylori,H.pylori)以外的唯一能够在人胃定植的螺杆菌属细菌,被视为胃炎的可能病原体。H.heilmannii感染与胃多种病变相关:急慢性胃炎、消化性溃疡、胃癌和胃B细胞粘膜相关淋巴组织淋巴瘤,也有引起食管疾病的报道,其临床意义值得重视。与国外相比,我国有较高的H.heilmannii感染率(欧美0.25%～0.95%,国内1.78%～7.32%)。随着H.pylori的发现获得诺贝尔奖,H.pylori与慢性胃病的关系已得到广泛认同,抗菌治疗己成为常规。在大规模除菌治疗多年后的今天,H.heilmannii的流行情况发生了什么变化却不得而知。至今为止,H.heilmannii仍不能在体外培养成功,快速诊断还是主要依靠切片染色的形态学方法。目前国外研究大多集中于流行病学调查,个案报道以及分子鉴定,国内自94年后的研究一直处于空白,H.heilmannii的致病机制尚不明确,目前世界上尚未建立稳定传代的H.heilmannii菌株。因此,构建H.heilmannii能持久定植的实验动物模型、建立体外培养的稳定方法就显得尤为重要,成为深入研究的必要手段和必然途径。此次研究的目的:了解目前国内H.heilmannii的流行情况;体外培养H.heilmannii;建立H.heilmannii长期感染小鼠的实验动物模型;初步研究其致病机制。材料和方法: 1.收集南方医院消化科2005-2006年行上消化道电子胃镜活检的2339例患者以及1994年1-4月819例患者的临床资料、内镜资料、病理标本。 2.筛检H.heilmannii感染患者:对2007年1-6月行上消化道电子内镜检查的703例胃镜受检者行胃粘膜涂片染色,镜下根据H.heilmannii典型形态筛选阳性患者,获取H.heilmannii感染患者的胃粘膜活检标本并在小鼠体内传代保存。 3.在多种培养基和多种气体条件以及不同温度条件下对成功定植H.heilmannii的鼠胃粘膜组织进行细菌的分离培养。 4.建立H.heilmannii长期感染小鼠模型:4～6周龄健康雄性BALB/c小鼠、雌性C57BL/6小鼠、雄性裸鼠各36只(SPF),分别随机分为实验组(n=30)和正常对照组(n=6)。实验组灌注H.heilmannii匀浆液0.2ml/只,并于灌胃后第4、8、12、24、36周分别处死6只。正常对照组分别灌注无菌布氏肉汤0.2ml/只,于实验第4、12、36周(裸鼠为4、8、12周)分别随机处死2只。观察细菌定植情况和组织病理变化。 5.半定量RT-PCR检测SPF级Balb/c小鼠、C57BL/6小鼠和裸鼠胃粘膜的IFN-γ、IL-1β、IL-4、IL-10mRNA表达水平差异,初步探索其致病机制。结果: 1.从2339例胃镜活检标本中检出7例H.heilmannii感染标本,总检出率为0.30%,与国外同期报道相符,但低于90年代检出率0.98%(P=0.0331)。 2.703例急慢性胃病患者中筛选出1例女性患者,并取其胃粘膜在小鼠体内传代保存,在不同条件下进行培养尝试,目前未获成功。 3.成功建立了BALB/c小鼠、C57BL/6小鼠、裸鼠H.heilmannii长期定植的动物模型。三种小鼠的定植量随时间的延长都有增加的趋势,BALB/c小鼠有显著性差异(Mann-Whitney U秩和检验,P＜0.05)。随着周期延长,BALB/c小鼠的慢性炎症评分在8周时最高,随后下降,而后期再次增高(P=0.042)。而C57BL/6小鼠持续较高,各时间点没有显著差异(P=0.877)。裸鼠未见明显慢性炎症,但第4-12周全程维持着明显的中性粒细胞浸润(Mann-Whitney U秩和检验,P＞0.05)。BALB/c小鼠急性活动性炎症评分在12周最高,36周最低(Mann-Whitney U秩和检验,P＜0.05):C57BL/6小鼠活动性炎症评分最低,但8周以后趋于平稳(Mann-Whitney U秩和检验,P＞0.05)。第24周后,实验组免疫健全小鼠开始出现轻度萎缩、肠化和淋巴滤泡的形成,到第36周83%的受染BALB/c小鼠出现淋巴滤泡,明显破坏粘膜肌层,甚至粘膜全层。对照组三种小鼠均未见明显感染和组织学改变。 4.感染初期,免疫健全小鼠胃粘膜的IFN-γ、IL-1β、IL-4、IL-10mRNA均较对照组明显升高(P＜0.05),IL-1β在第4周即达高峰,而IFN-γ、IL-4、IL-10在随后的第8至12周有最大表达。免疫健全小鼠的炎症因子表达趋势相似,但C57BL/6小鼠的IFN-γ表达量在12周后变化不大,而BALB/c小鼠明显下调,且第36周C57BL/6小鼠的IL-4低于对照组(P＜0.05)。裸鼠各细胞因子也较对照组稍有增高,除IL-1β、IL-4mRNA外(P＜0.05),IFN-γ、IL-10组间相比均无显著性差异(P＞0.05)。BALB/c小鼠的慢性炎症程度与IL-1β、定植程度分别相关(rs=0.489,P=0.005;rs=0.446,P=0.012),C57BL/6小鼠慢性胃炎程度与IFN-γ相关(rs=0.552,P=0.001)。结论: 1.现阶段H.heilmannii在上消化道疾病中检出率较90年代降低,大规模抗生素的使用可能是其原因;检出病种多见于萎缩性胃炎,提示H.heilmannii是萎缩性胃炎的一种重要致病菌。 2.成功建立了H.heilmannii BALB/c小鼠和C57BL/6小鼠体内长期定植,为研究H.heilmannii的致病机制提供了研究模型。 3.T淋巴细胞代表的细胞免疫在H.heilmannii感染中占主要地位,体液免疫也起到了一定作用。 4.不同品系小鼠对H.heilmannii感染的免疫机制不同,C57BL/6小鼠对H.heilmannii感染呈Th1型免疫占优势,而BALB/c小鼠为Th1/Th2型混合免疫反应。
[Abstract]:Background and Purpose :

Helicobacter pylori ( H . heillosis ) is a possible pathogen other than Helicobacter pylori ( H . pylori ) . H . heillosis infection is associated with gastric lesions : acute and chronic gastritis , peptic ulcer , gastric cancer and gastric B cell lymphoma . It also has the clinical significance . Compared with foreign countries , China has higher H . heillosis infection rate ( 0.25 % ~ 0.95 % in Europe and America , and 1.78 % ~ 7.32 % in China ) . With the discovery of H . pylori , the relationship between H . pylori and chronic gastric disease has been widely accepted , and the antibiotic therapy has become routine .

The aim of this study was to understand the prevalence of H . heiloride in China , and to establish an experimental animal model of H . heiloride in vitro .

Materials and methods :

1 . The clinical data , endoscopic data and pathological specimens were collected from 2339 patients in the digestive department of Southern Hospital from 2005 to 2006 , and 819 cases from January to April 1994 .

2 . Screening of patients with H . heillosis infection : The gastric mucosa smear staining was performed on 703 gastroscopic subjects who underwent endoscopy of upper gastrointestinal tract from January to June 2007 , and the positive patients were screened under the microscope according to the typical morphology of H . heiloride , and the specimens of gastric mucosa biopsy specimens from H . heiloride infected patients were obtained and stored in mice .

3 . Isolation and culture of bacteria from gastric mucosal tissues of murine gastric mucosa successfully implanted with H . heiloride under a variety of media and a variety of gas conditions , as well as under different temperature conditions .

4 . The mice model of H . heiloride long - term infection was established : BALB / c mice , female C57BL / 6 mice and 36 male nude mice were randomly divided into two groups : experimental group ( n = 30 ) and normal control group ( n = 6 ) . The experimental groups were randomly divided into experimental group ( n = 30 ) and normal control group ( n = 6 ) .

5 . Semi - quantitative RT - PCR was used to detect IFN - 纬 , IL - 1尾 , IL - 4 and IL - 10mRNA expression in SPF Balb / c mice , C57BL / 6 mice and nude mice .

Results :

1 . 7 cases of H . heillosis infection were detected from 2339 cases of gastroscopic biopsy , the total detection rate was 0.30 % , which was consistent with the report of the same period abroad , but the detection rate was 0.98 % in the 1990s ( P = 0.0331 ) .

2.703 patients with acute and chronic gastric diseases were screened out of 1 female patients and their gastric mucosa was passaged in mice , and the culture was attempted under different conditions .

3 . BALB / c mice , C57BL / 6 mice , nude mice H . heiloride were successfully established . The chronic inflammatory score of BALB / c mice was the highest at 8 weeks with the prolonged cycle ( P = 0.042 ) , while in C57BL / 6 mice , there was no significant difference at all time points ( P = 0.877 ) , but the whole course of 4 - 12 weeks maintained a significant neutrophil infiltration ( Mann - Whitney U rank sum test , P > 0.05 ) . The acute active inflammatory score of BALB / c mice was the highest at 12 weeks and the lowest in 36 weeks ( Mann - Whitney U rank and test , P < 0.05 ) . The active inflammatory score of C57BL / 6 mice was lowest , but was stable after 8 weeks ( Mann - Whitney U rank and test , P > 0.05 ) . At the 24th week , the mice in the experimental group began to develop mild atrophy , intestinal and lymphatic follicles , and the lymphatic follicles appeared in 83 % of the infected BALB / c mice at the 36th week , which significantly destroyed the mucosal muscularis and even the whole mucosa . None of the three mice in the control group showed significant infection and histological changes .

The levels of IFN - 纬 , IL - 1尾 , IL - 4 , IL - 10 mRNA in BALB / c mice were significantly higher than those in control group ( P < 0.05 ) . The levels of IFN - 纬 and IL - 4 in C57BL / 6 mice were significantly lower than those in control group ( P > 0.05 ) .

Conclusion :

1 . The detection rate of H . heiloride in upper gastrointestinal tract is lower than that in 1990 ' s , and the use of large - scale antibiotics may be the cause ; the detection of diseases is mostly seen in atrophic gastritis , which suggests that H . heiloride is an important pathogen of atrophic gastritis .

2 . The long - term colonization of H . heiloride BALB / c mice and C57BL / 6 mice was successfully established , which provided a research model for studying the pathogenesis of H . heillosis .

3 . The cellular immunity of T lymphocytes plays a role in H . heillosis infection , and humoral immunity plays a certain role .

4 . The immune mechanism of different strain mice to H . heillosis infection was different . C57BL / 6 mice had Th1 type immune dominance for H . heillosis infection , while BALB / c mice were Th1 / Th2 type mixed immune response .
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R516;R181.3

【参考文献】