本文選題：H.heilmannii + 培養(yǎng)　； 參考：《南方醫(yī)科大學(xué)》2008年碩士論文

【摘要】：背景、目的: 海爾曼螺桿菌(Helicobacter heilmannii,H.heilmannii),是除幽門螺桿菌(Helicobacter pylori,H.pylori)以外的唯一能夠在人胃定植的螺桿菌屬細(xì)菌,被視為胃炎的可能病原體。H.heilmannii感染與胃多種病變相關(guān):急慢性胃炎、消化性潰瘍、胃癌和胃B細(xì)胞粘膜相關(guān)淋巴組織淋巴瘤,也有引起食管疾病的報道,其臨床意義值得重視。與國外相比,我國有較高的H.heilmannii感染率(歐美0.25%～0.95%,國內(nèi)1.78%～7.32%)。隨著H.pylori的發(fā)現(xiàn)獲得諾貝爾獎,H.pylori與慢性胃病的關(guān)系已得到廣泛認(rèn)同,抗菌治療己成為常規(guī)。在大規(guī)模除菌治療多年后的今天,H.heilmannii的流行情況發(fā)生了什么變化卻不得而知。至今為止,H.heilmannii仍不能在體外培養(yǎng)成功,快速診斷還是主要依靠切片染色的形態(tài)學(xué)方法。目前國外研究大多集中于流行病學(xué)調(diào)查,個案報道以及分子鑒定,國內(nèi)自94年后的研究一直處于空白,H.heilmannii的致病機制尚不明確,目前世界上尚未建立穩(wěn)定傳代的H.heilmannii菌株。因此,構(gòu)建H.heilmannii能持久定植的實驗動物模型、建立體外培養(yǎng)的穩(wěn)定方法就顯得尤為重要,成為深入研究的必要手段和必然途徑。 此次研究的目的:了解目前國內(nèi)H.heilmannii的流行情況;體外培養(yǎng)H.heilmannii;建立H.heilmannii長期感染小鼠的實驗動物模型;初步研究其致病機制。 材料和方法: 1.收集南方醫(yī)院消化科2005-2006年行上消化道電子胃鏡活檢的2339例患者以及1994年1-4月819例患者的臨床資料、內(nèi)鏡資料、病理標(biāo)本。 2.篩檢H.heilmannii感染患者:對2007年1-6月行上消化道電子內(nèi)鏡檢查的703例胃鏡受檢者行胃粘膜涂片染色,鏡下根據(jù)H.heilmannii典型形態(tài)篩選陽性患者,獲取H.heilmannii感染患者的胃粘膜活檢標(biāo)本并在小鼠體內(nèi)傳代保存。 3.在多種培養(yǎng)基和多種氣體條件以及不同溫度條件下對成功定植H.heilmannii的鼠胃粘膜組織進(jìn)行細(xì)菌的分離培養(yǎng)。 4.建立H.heilmannii長期感染小鼠模型:4～6周齡健康雄性BALB/c小鼠、雌性C57BL/6小鼠、雄性裸鼠各36只(SPF),分別隨機分為實驗組(n=30)和正常對照組(n=6)。實驗組灌注H.heilmannii勻漿液0.2ml/只,并于灌胃后第4、8、12、24、36周分別處死6只。正常對照組分別灌注無菌布氏肉湯0.2ml/只,于實驗第4、12、36周(裸鼠為4、8、12周)分別隨機處死2只。觀察細(xì)菌定植情況和組織病理變化。 5.半定量RT-PCR檢測SPF級Balb/c小鼠、C57BL/6小鼠和裸鼠胃粘膜的IFN-γ、IL-1β、IL-4、IL-10mRNA表達(dá)水平差異,初步探索其致病機制。 結(jié)果: 1.從2339例胃鏡活檢標(biāo)本中檢出7例H.heilmannii感染標(biāo)本,總檢出率為0.30%,與國外同期報道相符,但低于90年代檢出率0.98%(P=0.0331)。 2.703例急慢性胃病患者中篩選出1例女性患者,并取其胃粘膜在小鼠體內(nèi)傳代保存,在不同條件下進(jìn)行培養(yǎng)嘗試,目前未獲成功。 3.成功建立了BALB/c小鼠、C57BL/6小鼠、裸鼠H.heilmannii長期定植的動物模型。三種小鼠的定植量隨時間的延長都有增加的趨勢,BALB/c小鼠有顯著性差異(Mann-Whitney U秩和檢驗,P＜0.05)。隨著周期延長,BALB/c小鼠的慢性炎癥評分在8周時最高,隨后下降,而后期再次增高(P=0.042)。而C57BL/6小鼠持續(xù)較高,各時間點沒有顯著差異(P=0.877)。裸鼠未見明顯慢性炎癥,但第4-12周全程維持著明顯的中性粒細(xì)胞浸潤(Mann-Whitney U秩和檢驗,P＞0.05)。BALB/c小鼠急性活動性炎癥評分在12周最高,36周最低(Mann-Whitney U秩和檢驗,P＜0.05):C57BL/6小鼠活動性炎癥評分最低,但8周以后趨于平穩(wěn)(Mann-Whitney U秩和檢驗,P＞0.05)。第24周后,實驗組免疫健全小鼠開始出現(xiàn)輕度萎縮、腸化和淋巴濾泡的形成,到第36周83%的受染BALB/c小鼠出現(xiàn)淋巴濾泡,明顯破壞粘膜肌層,甚至粘膜全層。對照組三種小鼠均未見明顯感染和組織學(xué)改變。 4.感染初期,免疫健全小鼠胃粘膜的IFN-γ、IL-1β、IL-4、IL-10mRNA均較對照組明顯升高(P＜0.05),IL-1β在第4周即達(dá)高峰,而IFN-γ、IL-4、IL-10在隨后的第8至12周有最大表達(dá)。免疫健全小鼠的炎癥因子表達(dá)趨勢相似,但C57BL/6小鼠的IFN-γ表達(dá)量在12周后變化不大,而BALB/c小鼠明顯下調(diào),且第36周C57BL/6小鼠的IL-4低于對照組(P＜0.05)。裸鼠各細(xì)胞因子也較對照組稍有增高,除IL-1β、IL-4mRNA外(P＜0.05),IFN-γ、IL-10組間相比均無顯著性差異(P＞0.05)。BALB/c小鼠的慢性炎癥程度與IL-1β、定植程度分別相關(guān)(rs=0.489,P=0.005;rs=0.446,P=0.012),C57BL/6小鼠慢性胃炎程度與IFN-γ相關(guān)(rs=0.552,P=0.001)。 結(jié)論: 1.現(xiàn)階段H.heilmannii在上消化道疾病中檢出率較90年代降低,大規(guī)�？股氐氖褂每赡苁瞧湓�;檢出病種多見于萎縮性胃炎,提示H.heilmannii是萎縮性胃炎的一種重要致病菌。 2.成功建立了H.heilmannii BALB/c小鼠和C57BL/6小鼠體內(nèi)長期定植,為研究H.heilmannii的致病機制提供了研究模型。 3.T淋巴細(xì)胞代表的細(xì)胞免疫在H.heilmannii感染中占主要地位,體液免疫也起到了一定作用。 4.不同品系小鼠對H.heilmannii感染的免疫機制不同,C57BL/6小鼠對H.heilmannii感染呈Th1型免疫占優(yōu)勢,而BALB/c小鼠為Th1/Th2型混合免疫反應(yīng)。
[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 - 緯 , 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 - 緯 , 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 - 緯 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 .
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R516;R181.3

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