关键词： 副结核杆菌；克隆病；致病菌

摘要目的探讨副结核杆菌（MP）在克隆病（CD）病因学中的作用。

方法将MP接种地鼠。诱导肉芽肿性 肠炎动物模型，然后用一部分CD肠粘膜匀浆作PCR扩增MP，另 一部分肠壁接种地鼠。

结果MP接种8月后，有80％(16/20)出现肉芽肿性改变，PCR阳性CD匀浆肠壁接种4月后，有5只出现肉芽肿性改变，与MP组的病理改变相似。PCR阴性CD匀浆组有一只出现肉芽肿性改变。

结论MP可能为部分CD的致病菌，但并非唯一的致病因素。

COMPARATIVE STUDY OF ENTERITIS INDUCED BY

MYCOBACTERIUM PARATUBERCULOSIS AND TISSUE HOMOGENATES OF CROHN'S DISEASE IN HAMSTERS

Miao Yinglei Ouyang Qin Gan Huatian

(Department of Gastroenterology,The First Affiliated Hosptal of Kun ming Medical College,Kunming 650032)

ABSTRACT AimA comparison of the macroscopical and histolog ical appearance in granulomas was made induced by Mycobacterium Paratuberculosis (MP) and Crohn's Disease(CD) tissue in hamsters,in order to investigate whether MP plays an etiologic role in CD .

Methods 40 hamsters were inoculated intraperitoneally,twenty recei ved MP,ten received dead inoculum and ten received saline.Each CD homogenated was prepared immmediately after biopsy,100μl homogenates was an alysed by PCR;and 40μl was inoculated intramurally into the cecum and terminal ileam of each hamster of 5,tissue was obtained from intestine and evaluated macroscopically nad histologically.Each hamster inoculated intr amurally was scored on the criteria for assessment of intestine damage.

Results Microscopically,16 of 20 hamsters received MP showed gra nulomatous changes,the control hamsters didn't show granulomatous enteritis.Two of five CD homogenates were positive for MP.The hamsters received PCR positive CD homogenates put on granulomatous changes in volved in 5 of 10.The correct 400bp amplification product was obtained again in the granulomatous intestinal tissue of 5 hamsters inoculated PCR positive CD homogenates when the IS900 PCR was applied.Only 1 of 15 hamsters receiv ed PCR nagative CD homogenates showed granulomatous change.The aver age score of hamsters intestinal damage induced by PCR negative CD homogenates was 3.66±1.71 and showed a marked increase(P＜0.05) as compared with those of normal gut mucosa group;but no statisically significan t difference(P＞0.05) was shown when compared with PCR positive CD homogenates group.

Conclustion MP may play an etiologic role in at least some cases of Crohn's disease.Other factor may also p lay an etiologic role in some cases of CD except MP.

KEY WORDS Mycobacterium Paratuberculosis Crohn's Disease Causal Agent

克隆病(Crohn's Disease,CD)是一种病因未明的胃肠道慢性炎性肉芽肿疾病，早期研究发现其病理改变与Johne's病相似，怀疑副结核分枝杆菌(Mycobacteriu m Paratuberculosis,MP)^［1］可能为其致病菌。最近，国内甘华田等^［2］ 利用多聚酶链反应（PCR）从部分CD的石蜡包埋组织中扩增出MP的DNA片段。不过，MP与CD的关系仍未清，为此，本文用MP感染金黄地鼠，以图建立一个肉芽肿性肠炎的动物模型；然后用 CD患者肠粘膜匀浆接种地鼠，观察接种后的病变，并与MP感染的动物模型相比较，以探讨CD与MP感染的相关性。

1材料和方法

1.1材料副结核分枝杆菌标准株P18由吉林省兽医科学研究所馈赠。12周龄、雌性健康的金黄地鼠（叙利亚种）90只购于卫生部成都生物制品研究所。CD患者5例，系华西医科大学附属一医院肠镜室的随访患者。PCR热循环仪：P-E4 80型，Perkin Elmer公司产品。DNA抽提试剂盒，上海华顺生物试剂公司产品。PCR引物：针对副结核分枝杆菌IS900序列（5′→3′）：GTTCGGGGCCGTCGCTTAGG 和GAGGTCGATCGCCCACGTGA，中国科学院上海细胞生物研究所合成。

1.2方法

1.2.1副结核分枝杆菌接种地鼠制备细菌悬液，终浓度为10mg/ml。将40只地鼠随机分为2组，实验组（20只）腹腔接种1ml细菌悬液；对照组又随机分为2组 ，各10只，分别注射1ml死菌液和1ml 0.9％的生理盐水。8月后处死作肉眼和组织学观察。

1.2.2CD肠粘膜匀浆扩增副结核分枝杆菌并接种地鼠

1.2.2.1匀浆及模板DNA的制备CD患者5例，每例取有病变肠粘膜活检组织，称重、匀浆，终浓度为0.1mg/μl。100μl匀浆于EP管中，按照DNA抽提试剂盒说明书进行。

1.2.2.2利用MPIS900引物进行扩增，取样品10μl，加入200μM的dNTP3μl,25pM的引物各2μl,Taq DNA聚合酶5μ,2μl双蒸水24μl,10×buffer 5μl。阳性对照用标准MP菌株，阴性对照用双蒸水，上PCR热循环仪，扩增条件参照Sander son^［3］的条件进行。95℃变性1min，58℃退火1min，72℃延伸3min。循环33次。为了防止假阳性和假阴性，重新用IS900引物对CD匀浆扩增一次，条件同前。PCR的所有操作按Kwok^［4］的质控标准进行。

1.2.2.3接种地鼠将CD匀浆于取材后3h内接种动物的盲肠和末段回肠近系膜肠壁处（各20μl）。每例CD匀浆接种5只地鼠，另取4只地鼠作对照（2只接种正常肠粘膜匀浆，2只接种0.9％生理盐水）。4个月后处死作肉眼和组织学观察。炎症程度评分按Tanaka^［5］的肠道损伤积分标准。

1.2.2.4将有肉芽肿性改变的动物肠道组织，用IS900引物再次扩增 ，条件同前。

1.2.3统计学分析肠道损伤积分用t检验，肉芽肿性肠炎的发病率用χ²检验。

2结果

2.1MP接种地鼠实验组中80％（16只）的地鼠的肠壁及肠系膜有上皮样细胞及朗罕氏巨细胞形成的肉芽肿性炎症（见图1），伴有淋巴细胞聚集及溃疡形成。而对照组未见肉芽肿性肠炎和溃疡形成。两组肉芽肿性肠炎的发病率有显著性差异（P＜0.05）。

图1MP接种地鼠8月后，回肠肠壁肉芽肿形成(H-E×107)

Granulomatous infiltration of the small intestine of hamsters 8 months after intraperitoneal injection of Mycobacterium paratuberculosis.(H-E,×107)

2.2PCR扩增后，有2例扩增出400bp的DNA片段，另外3例为阴性。重复扩增后，结果同前。

2.3肠粘膜匀浆接种动物PCR阳性的CD匀浆接种地鼠后，有5只肠壁出现上皮样细胞及朗罕氏巨细胞聚集形成的肉芽肿性炎症及溃疡形成，肠壁淋巴细胞浸润（见图2）。该组与副结核杆菌接种组存在异同点。PCR阴性的CD匀浆接种地鼠后，仅有1只出现上皮样细胞聚集而成的肉芽肿性炎（见图3），而对照组未见肉眼和组织学的异常改变。PCR阴性组肠道损伤平均积分为3.66±1.71，与PCR阳性组相比，无显著性差异（P＞0.05）。而高于正常肠粘膜组（P＜0 .05）。

图2利用MP IS900引物扩增CD匀浆的结果

Detection of Mycobacterium paratuberculosis DNA by IS900 polymerase Cha in Reaction(PCR) from Crohn's tissue homogenates.

Mark:PBR322-HaeⅢ1.双蒸水（阴性对照）2.CD匀浆3.MP（阳性对 照）

图3PCR阳性的克隆病肠粘膜匀浆接种地鼠肠壁4月后，肉芽肿性炎症形成

4 months later,the hamsters' terninal ileum after inoculation of crohn's tissue homogenates contained Mycobacterium paratuberculosis showing epithelioid and giantcell granulomas(H-E,×107)

2.4PCR阳性组CD匀浆接种动物后，5只出现肉芽肿性炎症，将含肉芽肿的组织再用副结核杆菌IS900引物扩增，均出现400bp的DNA片段。

3讨论