C57BL/6小鼠幽门螺杆菌感染动物模型的建立

摘要

目的: 建立幽门螺杆菌(H. pylori)小鼠感染模型.

方法: 以C₅₇BL/6小鼠为实验动物, 经口感染接种H. pylori悉尼株(SS1), 置层流柜中饲养. 用HE染色、石碳酸-碱性品红染色、免疫组织化学染色、尿素酶实验、细菌培养等方法检测接种小鼠, 并用PCR、基因测序方法对胃组织细菌、胃组织分离培养细菌DNA进行分析.

结果: 小鼠胃组织分离培养的细菌与接种细菌形态及特性相同. 胃窦隐窝可观察到细菌定植, 胃黏膜下层可见炎症细胞浸润. 胃组织DNA中成功扩增出H. pylori 16SrRNA及cagA基因的目的片段, 基因测序结果显示与接种菌株序列完全一致.

结论: H. pylori SS1经口接种C₅₇BL/6小鼠2 mo后可成功在胃内定植并导致成慢性胃炎.

关键词: N/A

Establishment of a mouse model colonized by Helicobacter pylori

Xue-Fei Tian, Xue-Gong Fan, Yan Zhang, Yan Huang

Xue-Fei Tian, Xue-Gong Fan, Yan Zhang, Yan Huang, Department of Infectious Diseases, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China

Supported by: the National Natural Science Foundation of China, No. 30271171, Teacher Foundation of the National Ministry of Education, No.2000-65, Nature Scientific Foundation of Hunan Province, No. 01jjy2114 and the Scientific Research Foundation of Health Bureau of Hunan Province No. 00022..

Correspondence to: Dr. Xue-Gong Fan, Department of Infectious Diseases, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China. xgfan@hotmail.com

Received: February 11, 2004
Revised: February 20, 2004
Accepted: February 24, 2004
Published online: June 15, 2004

Abstract

AIM: To establish a mouse model of H. pylori infection.

METHODS: C₅₇BL/6 strain mouse, used as an experiment animal, was inoculated orally H. pylori SS1 strain and fed in laminar flow cabinet. Inoculated mouse examined by HE stain, carbolic acid- basic fuchsin stain, immunohistochemical stain, urease test and bacteria cultivation. Both bacteria DNA from gastric tissue and H. pylori isolated from stomach were analyzed by PCR and gene sequencing.

RESULTS: The bacteria isolated from stomach had the identical shape and character compared with inoculated bacteria. Bacteria colonizing in antrun crypt and infiltration of inflammatory cells in gastric submucosa were observed. H. pylori 16SrRNA and cagA gene were amplificated successfully from gastric tissue DNA, gene sequencing results showed the complete concordance.

CONCLUSION: After inoculated into C₅₇BL/6 mouse 2 months, H. pylori SS1 can colonize successfully the mouse stomach and cause chronic gastritis.

Key Words: N/A

0 引言

研究者一直在探索与人类病理变化类似的幽门螺杆菌(H. pylori)感染动物模型, 1987年首次用限菌小猪建立实验性H. pylori感染模型以来, 已有猕猴、小鼠、蒙古沙鼠、大鼠、猪、犬、猫等多种动物应用于模型[1-9]. 虽然与人类感染H. pylori的病理变化有一定差距, 但在H. pylori致病机制、疫苗筛选、新药疗效考察等研究中, 动物模型仍发挥了重要作用. 目前国内应用较多的模型为沙土鼠感染模型, 而小鼠动物模型尚不成熟, 为此, 我们采用C₅₇BL/6小鼠为研究对象进行了H. pylori的感染模型制作.

1 材料和方法

1.1 材料

14只C₅₇BL/6小鼠(SPF), 6-8周龄, ♂, 体质25-30 g. H. pylori使用悉尼株(SS1). 兔抗H. pylori多克隆抗体购自丹麦Dako公司, SABC试剂盒购自武汉博士德生物工程有限公司, 蛋白酶K购自德国Merck公司, PCR相关试剂为大连宝生物工程有限公司产品. PCR以接种的H. pylori悉尼株为阳性对照细菌, 阴性对照细菌为购自中国预防医学科学院流行病学微生物研究所的空肠弯曲菌(Lior4887519), 阴性对照为灭菌双蒸水.

1.2 方法

H. pylori培养按我研究组建立的H. pylori培养方法进行[10]. 造模方法参照Lee et al[11]方法: 将C₅₇BL/6小鼠完全随机分为模型组与正常对照组, 接种前禁食24 h. H. pylori培养72 h收获, 刮取菌落, 灭菌0.01 mmoL/L PBS混悬, 浓度3×108CFU/L, 0.5 mL/只灌胃接种, 隔日1次, 共3次. 对照组给予等量灭菌PBS. 接种后将动物置层流柜中饲养, 2 mo后处死. 样本获取及检测方法: 取全胃沿胃大弯剪开, 生理盐水漂洗去除胃内容物, 一半胃组织40 g/L甲醛固定, 石蜡包埋切片, 病理切片包括HE染色, 免疫组织化学染色及石碳酸-碱性品红染色 [12]. H. pylori的定植水平等级评分标准参照迟晶方法制订[13]. 免疫组织化学染色一抗采用兔抗H. pylori多克隆抗体, SABC法, DAB显色, 苏木素轻度复染. 一半胃组织用于细菌培养以及PCR检测, 组织及细菌DNA提取参照彭小宁等方法[14]. PCR引物设计[15]: 16SrRNA基因上游引物5'-GTC ATG ACG GGT ATC C-3', 下游引物: 5'-GAT TTT ACC CCT ACA CCA-3', 扩增出400 bp片段, 55 ℃退火1 min 30 s, 72 ℃延伸2 min; cagA基因上游引物5'-ATA ATG CTA AAT TAG ACA ACT TGA GCG A-3', 下游引物: 5'-TTA GAA TAA TCA ACA AAC ATC ACG CCA T-3', 扩增出297 bp片段, 55 ℃退火1 min, 72 ℃延伸90 s; 16SrRNA, cagA PCR产物纯化后基因测序, 大连宝生物工程技术有限公司完成.

2 结果

2.1 胃黏膜细菌培养

从胃黏膜分离培养的H. pylori在培养平板上可见清晰的针尖样菌落, 尿素酶试纸检测呈强阳性, 革兰染色阴性, 形态与原始接种细菌无差别, 光镜下均为弯曲杆状(图1A), 免疫组织化学染色结果菌体为棕黄色(图1B).

图1 小鼠胃黏膜分离培养H. pylori ×1 500, A: 红色弯曲杆状(Gram); B: 深棕色呈弯曲杆状(SABC).

2.2 定植与分布

在8 wk H. pylori胃贲门部(1.17±0.41), 胃体部(0.17±0.41)及胃窦部位(2.33±0.52)都可观察到细菌的定植, 以胃窦部位的黏膜腺体观察到的定植细菌最多(P<0.05), 大量细菌分布于胃窦隐窝, 胃体部相对少, 仅见少量细菌散在分布, 有的动物甚至未观察到. 在胃腺体表面及上层黏液中可见大量细菌(图2A), 油镜下观察呈清晰的S状或螺旋状(图2B), 与人类感染类似, 光镜下可见一些细菌紧密黏附于小鼠胃上皮细胞. 细菌免疫组织化学结果进一步证实了定植细菌为H. pylori, 菌体染色为棕黄色或深棕色(图2C).

图2 胃黏膜上可见大量H. pylori . A: 石炭酸-碱性品红染色 ×600; B: S形或螺旋形, 石炭酸-碱性品红染色 ×1 500; C: SABC ×600.

2.3 胃黏膜病理改变

小鼠经过细菌接种2 mo后均出现与人类类似的慢性活动性胃炎的病理改变, 出现炎症细胞浸润. 胃黏膜的完整性与连续性受到破坏, 黏膜下层可见大量淋巴细胞浸润并形成淋巴滤泡, 中间夹杂少量中性粒细胞浸润(图3A-D).

图3 小鼠胃黏膜(HE). A: 连续性破坏 ×300; B: 黏膜下层出现淋巴细胞浸润为主的炎症反应 ×600; C: 出现淋巴滤泡 ×600; D:正常 ×300.

2.4 PCR及测序结果

在7例感染小鼠胃组织DNA标本中全部扩增出400 bp (16S rRNA)与297 bp (cagA)目的片段, 对照细菌及阴性对照未扩增出条带. 取2个阳性标本与接种H. pylori SS1株的PCR 产物进行序列分析, 用16SrRNA、cagA引物进行正向及反向测序, 结果与接种H. pylori SS1株进行比较, 发现具有100% 的同源性, 证实成功定植的细菌与原始接种的H. pylori SS1属于同一株细菌(图4, 5).

图4 PCR 扩增H. pylori 16SrRNA基因. M: Mr标准; 1: 阴性对照; 2: 阳性对照细菌(SS1); 3, 4, 5: 阳性组织; 6: 阴性对照细菌(Lior48 87519).

图5 PCR 扩增H. pylori cagA基因. M: Mr标准; 1: 阳性对照; 2: 阳性对照细菌(SS1); 3, 4, 5: 阳性组织; 6: 阴性对照细菌(Lior48 87519).

3 讨论

迄今已有许多动物被用来接种H. pylori期望制作出类似于人类细菌感染的动物模型. 1997年Lee et al[11] 鉴定的H. pylori 悉尼株(SS1)目前已被认为是理想的动物模型接种菌株, 经口接种沙土鼠后, 8 mo可出现炎症细胞浸润、黏膜腺体的异型性增生、肠上皮化生、黏膜萎缩等病理改变[16]. 我们也采用SS1株经口接种, 结果表明C₅₇BL/6小鼠的易感性好, 成功率高, 在小鼠胃内定植具有部位选择性, 以胃窦部最多, 这与人类H. pylori感染胃内定植部位类似. 与人类感染相同, 细菌分布在胃黏膜上皮细胞表层黏液及腺窝内, 甚至侵入上皮细胞间或黏膜固有层内. 此前, 我们曾以临床分离株及SS1株接种过中国昆明种小白鼠, 因定植效果太差而放弃. 小鼠接种2 mo后虽然未出现溃疡及萎缩性病理改变, 但出现以淋巴细胞浸润为主的慢性炎症反应, 且出现淋巴滤泡, 而淋巴滤泡是MALT淋巴瘤的重要病理学改变之一[17-21]. 已有研究表明, 人MALT淋巴瘤的发生与H. pylori感染具有相关性[22-27], 增多的淋巴细胞以B淋巴细胞为主, 这与H. pylori感染后T淋巴细胞活化引起B淋巴细胞增生有关[28-30]. 为了确证定植细菌来源于经口接种而非外源污染所致, 我们将小鼠胃组织内成功定植的细菌与接种细菌DNA进行PCR扩增并行测序对比分析, 结果显示成功定植的细菌来源于原始接种菌株, 表明了模型的可靠性. 该动物模型与其他模型相比较, 定植成功率更高, 并且价格便宜, 容易饲养, 具有良好的推广应用前景.

备注

$[Footnotes]

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