圆球形幽门螺杆菌生物学性状的研究

Study on biological characteristics of coccoid form Helicobacter pylori

目的 通过改变幽门螺杆菌 (Hp)体外培养的温度条件 ,诱导Hp从螺旋形转变为圆球形 ,研究圆球形Hp生物学性状并阐明其致病性。方法 取临床患儿胃黏膜进行Hp分离培养、鉴定和纯培养 ,随后洗脱纯种Hp进行液体培养。根据不同的温度条件 ,分为 3组 :①常温组 :液体培养温度 3 7℃、微需氧、pH 7、震荡( 12 0r/min)并加小牛血清 ,获得的细菌为正常弯曲形Hp ;②高温组 :液体培养温度 40℃ ,其余条件同常温组 ;③低温组 :液体培养温度 2 5℃ ,余条件同常温组。研究培养获得细菌的形态及其超微结构 ,检测圆球形Hp的尿素酶活性和尿素酶基因。结果 常温液体培养的Hp呈螺旋形 ,高温或低温液体培养的细菌呈圆球形。圆球形Hp光镜下为圆球形或粗短杆状 ,电镜下可分 2类 ,一类细胞体积较大 ,细胞膜完整 ,胞质疏松 ,鞭毛脱落 ;另一类细胞较小 ,有完整的细胞膜且有鞭毛。圆球形Hp尿素酶活性为阴性 ,411bp尿素酶A基因的PCR为阳性。 结论 圆球形Hp是适应外部不良条件的一种生存方式 ,完整的细胞结构是维持细胞新陈代谢的基础 。

Objective Coccoid form Helicobacter pylori (H. pylori) is now believed to play a role in the transformation of H. pylori and in relapses after antibiotic therapy, however its biological characteristics remain controversial. In this study, we induced morphological conversion of H. pylori from spiral to coccoid form by different culture tempertures and studied its biological characteristics and pathogenetic mechanism. Methods The H. pylori strain uesd in this study was isolated from gastric antrum of patients. The cells were grown in solid culture (Campylobacter agar medium, 10%vol/vol sheep blood and Skirrow's supplement) at 37℃ under microaerophilic conditions (5% O 2, 7 % CO 2, 8% H 2, 80% N 2). Subsequent assay and subculture were performed. Pure culture was harvested and suspended in liquid medium (brain heart infusion, 10% calf serum, Skirrow's supplement). Liquid culture was divided into three groups in different terms of culture tempertures: ① normothermic group: it was supplemented with calf serum at 37℃ under microaerophilic conditions, pH 7 and agitation (120 r/min) to obtain spiral shaped bacterium; ② hyperthermic group: the culture temperature was 40℃, and the other conditions were the same as those of the normothermic group; ③ hypothermic group: culture temperature 25℃ and the other conditions were the same as those of the normothermic group. We observed the morphology and ultrastructure by light microscopy and elctronmicroscopy. Detection of the activities of urease was taken by rapid urease reagent and the gene variety of urease A (411 bp UreA, ureA 1 5′ GCCAATGGTAAATTAGTT 3′ UreA 2 5′ CTCCTTAATTGTTTTTAC 3′) by PCR. Results The coccoid H. pylori were transformed from spiral form by hyperthermia and hypothermia. Coccoid forms were observed under light microscopy. Under elctronmicroscopy, some coccoid H. pylori exhibited as small cells with intact cellular membranes and flagella, others presented as large cells without flagella. The urease reaction was negative for coccoid forms. 411 bp UreA in coccoid H. pylori were shown to be positive by PCR. Conclusion Extremely high or low temperatures during culture may be considered as a necessary environment for H. pylori transformation. The morphological conversion of H. pylori from spiral to coccoid form was the expression of adpataion to an unsuitable environment. Intact cellular structures may be necessary for its metablism. Flagella and urease genes may be prerequisit for its pathogenetic mechanism.