【摘要】目的研究空肠弯曲菌(Cj)诱发实验性周围神经病的可能性和免疫学机理。方法取经两株空肠弯曲菌反复亚临床感染的、经多种丝裂原刺激的大鼠外周血单个核细胞（PBMC）上清液和新鲜血清,以未感染鼠PBMC上清液及血清作对照，分别对185只其他大鼠进行神经外膜下或静脉注射。7或14天后取坐骨神经行组织学检查。结果病变的神经原纤维呈典型的轴索变性和（或）髓鞘脱失改变, 伴巨噬细胞和淋巴细胞浸润；经神经外膜下注射者，42.5%神经原纤维病变,其中80.8%为轴索变性，对照组仅3.1％原纤维损害（P＜0.01)；经静脉注射者, 19.9%神经原纤维病变,轴索变性和髓鞘脱失分别占25.4%和74.6%, 对照组无任何病变(0/480)；两种菌型均显示相似的致病力。静脉注射PBMC上清液并不同时引起肝、肾和脑病变。经Cj感染的大鼠的PBMC上清液IgG含量明显增加。结论以Cj反复亚临床感染大鼠的上清液和血清进行神经外膜下或静脉注射，均诱发周围神经病，并对周围神经组织有免疫学特异性。细胞和体液免疫皆参与其致病过程。

Experimental peripheral neuropathy induced by Campylobacter jejuni infection

ZHU Chunni, CAI Fangcheng, ZHANG Xiaoping.

Children′s Hospital, Chongqing University of Medical Sciences, Chongqing 400014

【Abstract】ObjectiveTo induce experimental peripheral neuropathy by immunoreaction after Campylobacter jejuni (Cj) infection and explore their immune pathogenesis.MethodsPBMCs were taken from 21 Wistar rats that had been repeatedly injected with suspension of two Cj strains intraperitoneally. The supernatants or fresh sera of the infected rats or control group were respectively injected perineurally and intravenously to totally 185 other rats (experimental group), and sacrificed on the 7th day after perineural injection and the 14th day after intravenous injection. Histological examination of the sciatic nerves was performed microscopically and electron-microscopically.ResultsTypical axonal degeneration and demyelination were seen in most fibers with remarkable macrophage and lymphocyte infiltration in some severe damaged fascicles. Pathological changes were identified in 42.5% teasing fibers after perineural injection. Among them, 80.8% fibers had axonal degeneration, but in control group, only 3.1% of fibers were found damaged (P＜0.01). Pathological changes were found in 19.9% fibers after intravenous injection of PBMC supernatant, including 25.4% axonal degenerating and 74.6% demyelinating fibers. No abnormality was found in control group. Their livers, kidneys and brains of 10 rats were still normal although there were typical experimental neuropathies after intravenous injection of PBMC supernatant.ConclusionExperimental neuropathy was successfully induced by immunoreaction after Cj infection. Both cellular and humoral immunological pathogenesis were involved. Immune specificity to peripheral nerves was well shown in the pathogenic process. Axonal degeneration was much more often seen in the group injected with the PBMC supernatant perineurally than intravenously, indicating that local increase of pressure inside fascicles after perineurial injection probably plays a role in the pathogenesis of axonal degeneration.

【Key words】Campylobacter jejuniRatsPeripheral nervous system diseases

空肠弯曲菌(Campylobacter jejuni, Cj)是人类腹泻病的重要病原。近年的流行病学研究表明它还与我国部分格林-巴利综合征（Guillian-Barré syndrome, GBS)有关，但始终缺少两者关系的直接证据，更难对其致病机理作出确切说明。我们以Cj感染后大鼠外周血单个核细胞(peripheral blood mono nuclear cell,PBMC)培养上清液和血清进行静脉及坐骨神经外膜下(简称外膜下)注射, 以探讨免疫学方法诱发Cj感染后实验性周围神经病的可能性及致病机理。

材料和方法

一、材料

1. Cj混悬液配制：选用Cj-131(PEN43)和Cj-X两菌株,前者由上海市卫生防疫站提供。后者从本院1996年夏季一急性腹泻患儿粪便中分离，经形态学和生物学方法确认^［1］，马尿酸水解试验阳性。将复苏后的Cj接种于Camp-BAP培养基，置微氧气缸42℃培养24小时，无菌生理盐水洗下菌苔, 4 000 r/min离心30分钟，生理盐水洗涤沉淀物2次，弃上清液，用无菌生理盐水配制成10⁹/ml的细菌混悬液。

2. Cj反复感染大鼠PBMC的制备:选健康成年Wistar鼠26只作为PBMC供鼠, 体重232～310 g(重庆医科大学动物实验中心提供)。将其随机分为Cj-131组(10只)、Cj-X组(11只)和对照组(5只)。分别对三组动物腹腔内注入Cj-131、Cj-X细菌悬液和无菌生理盐水1 ml，每周1次，连续5周。然后经颈静脉取血3 ml，肝素抗凝。常规方法分离单个核细胞，用含10％小牛血清的RPMI 1640培养液将细胞浓度调至5×10⁶/ml, 分为3管, 分别加入植物血凝素(PHA,广州医药工业研究所)、细菌脂多糖(LPS,Sigma)和美洲商陆(PWM,Sigma)。终浓度分别为100、5和3 μg/ml。37℃、5%CO₂孵箱培养24小时后收集经PHA和LPS诱导的PBMC培养液；10天后收集PWM的培养液。无菌离心收集上清液，保存于-70℃，同时将各上清液接种于Camp-BAP培养基，微氧培养，证实无污染的可能性。

分别以Cj-131和Cj-X反复感染后的大鼠血清与两菌株悬液作细菌凝集试验，未见交叉凝集现象。

二、实验性周围神经病的诱发

1. 感染后大鼠PBMC培养上清液诱发周围神经病：(1) 经外膜下注射诱发：另选120只体重252～365 g的健康Wistar成年鼠为实验鼠。戊巴比妥钠腹腔内注射麻醉后暴露鼠坐骨神经，在解剖显微镜下，经神经外膜下分别注入经Cj反复感染大鼠的PBMC上清液40 μl，并根据提供PBMC大鼠受感染菌种，进一步将此120只实验鼠分为：①Cj-131组46只，分别注入经PHA(20只)、LPS (20只)或PWM (6只)孵育的PBMC上清液；② Cj-X组50 只，分别注入上述3种丝裂原孵育的PBMC上清液，各有22、22 和6只 ；③对照组24只，注入无Cj感染鼠的PBMC上清液，各有10、10、和4只。(2)经静脉注射诱发：再选49只体重257～344克鼠，同样分成Cj-131(18只)、Cj-X(19只)和对照(12只)3组，分别经静脉注入经上述3种丝裂原培养的PBMC上清液10^-2 ml/g体重。Cj-131组各有8、8和2只鼠，分别注入经PHA、LPS和PWM孵育的上清液；Cj-X组各有8、8和3只鼠；对照组各有5、5和2只。

2. 感染后大鼠血清诱发周围神经病：按上述方法分别以不同剂量Cj-131、Cj-X感染后和对照组的大鼠新鲜血清做实验大鼠外膜下注射：(1) Cj-131组及对照组: Cj-131感染鼠新鲜血清30 μl加健康鼠新鲜血清10 μl注入8只大鼠的一侧坐骨神经外膜下。另一侧注入40 μl正常血清作为对照。(2)Cj-X组及对照组：另选8只鼠，方法与(1)相同。

三、 组织学检查

将外膜下注射后7天、静脉注射后14天的大鼠以及Cj反复性感染的大鼠分别以多聚甲醛及戊二醛灌流固定。取坐骨神经、腰骶段脊神经节、脊神经根做半薄( 0.5 μm)和超薄(0.2 μm)切片，进行光镜及电镜观察。坐骨神经做原纤维分离。另取上述实验和对照鼠的肝、肾及大脑做常规石蜡包埋切片，镜下观察有无病理改变。

四、 PWM诱导的PBMC上清液IgG测定

采用ELISA法测定。羊抗鼠IgG购自北京天象人生物医学公司。

五、 统计学处理

均数比较用单因素方差分析和q检验。百分率作卡方检验。

结果

一、Cj感染大鼠的PBMC上清液对周围神经致病作用

1.经外膜下注射诱发的改变：原纤维分离结果示，3种丝裂原诱导的大鼠PBMC上清液外膜下注射后均导致轴索变性和 (或 )轻度髓鞘脱失,以轴索变性最突出 (表1)。两组Cj感染的轴索变性纤维总发生率（658/1 920，34.2%)和脱髓鞘纤维的总发生率(158/1 920,8.2%)与对照组(15/480,3.1%)比较差异均有非常显著意义(χ²=389、184.6 ，P均＜0.01);Cj-131与Cj-X的病变纤维发生率组间差异无显著意义(χ²=0.36, P＞0.05), 表明两菌株感染后有相似免疫学致病力。3种丝裂原中,又以PHA和PWM诱导的PBMC上清液病变原纤维发生率最高,两者间差异无显著意义(χ²=0.28, P＞0.05)，但与LPS组比较,差异均有显著意义(χ²=49.3和52.9, P＜0.01)。坐骨神经半薄切片表现与原纤维分离结果一致。光镜下原纤维数不同程度减少,或区域性严重丢失,散在或成群轴索变性,伴有继发性髓鞘塌陷,可见巨噬细胞和淋巴细胞浸润。有的切片见髓鞘异常菲薄, 符合脱髓鞘病理改变(图1，2)。

表1不同丝裂原诱导的Cj感染大鼠的PBMC上清液外膜下注射致病变神经原纤维比较