galE变异株及亲代株空肠弯曲菌诱导动物外周神经损伤的对比研究

Comparative study on the role of parent Campylobacter jejuni and galE mutant in inducing experimental peripheral nerve damage

目的探索空肠弯曲菌(Campylobactejejuni,CJ)致外周神经损伤的关键结构,为CJ感染相关格林巴利综合征的分子模拟理论提供动物实验证据。方法将32只豚鼠随机分为亲代株组(10只)、变异株组(10只)、对照组(6只)、PBS组(6只),分别用亲代株及弗氏佐剂、galE变异株及弗氏佐剂、PBS及弗氏佐剂、单纯PBS进行全身免疫;动态检测血清细胞壁脂寡糖(LOS)IgG及神经节苷脂GM1IgG抗体水平;检查坐骨神经病理改变,包括单纤维分离、半薄切片光镜及电镜检查。结果①免疫后变异株组及亲代株组LOSIgG抗体水平均明显增高,两组间差异无统计学意义;②免疫后14、28d,亲代株组GM1IgG抗体滴度(0.661±0.290,0.984±0.025)明显高于变异株组(0.193±0.078,0.180±0.063),差异均有统计学意义;③变异株组坐骨神经单纤维病变率4.9%(98/2000),明显低于亲代株组(16%,320/2000),差异有统计学意义,后者主要为轴索变性;④亲代株组半薄切片显示以轴索变性为特征的病理改变;变异株组仅见极少数异常,两组间差异有统计学意义;⑤电镜检查证实光镜所见。结论与亲代株相比,galE变异株缺失神经节苷脂GM1样表位,不能诱导动物产生GM1抗体及周围神经损伤,支持CJ感染后格林巴利综合征发生的分子模拟理论。

Objective A comparative study on the role of Campylobacter jejuni (CJ) HB9313 and galE mutant in inducing experimental sciatic nerve damage was conducted in guinea pigs in order to explore whether CJ lipo-oligosaccharide (LOS) is critical component associated with peripheral nerve lesions and find experimental evidence for the presumption of molecular mimicry on the pathogenesis of Guillain-Barré syndromes (GBS) with CJ antecedent infection. Methods A total of 32 guinea pigs were randomly divided into four groups: parental strain group(n=10), galE mutant group(n=10), control group(n=6) and PBS group(n=6), and immunized with the whole cell antigens of CJ HB9313 with Freund's adjuvant (FA), the whole cell antigens of galE mutant (without ganglioside-like structure) with FA,PBS with FA,and PBS alone, respectively. Enzyme-linked immunosorbent assay (ELISA) was employed to detect anti-LOS and anti-ganglioside GM1 antibodies in sera of these animals, and comparative morphologic studies of pathologic changes were carried out on the sciatic nerves, including examination of teasing fibers, examination of semithin sections made from epon-embedded tissue blocks under light microscope and transmission electron microscope. Results ELISA results indicated that after immunization, the levels of anti-LOS IgG antibody were significantly elevated in animals from parental strain group and galE mutant group as compared with those before immunization (P<0.01). No statistically significant difference was found between the two groups. However, the mean optical densities (ODs) of IgG antibody against GM1 at 14 and 28 day after immunization, in parental strain group, were 0.661±0.290 and 0.984±0.025, respectively, significantly higher than those of galE mutant group, which were 0.193±0.078 and 0.180±0.063 (P<0.01). The results of morphologic examination on sciatic nerves showed that for teased-fiber study, incidence of pathologic abnormalities of teased fibers from animals of galE mutant group was 4.9% (98/2000), significantly lower than that from parental strain group, which was 16%(320/2000), characterized by predominantly axonal degeneration. The difference between them was highly significant statistically (P<0.01).Examination of semithin sections of sciatic nerves also revealed that obvious pathological changes occurred in the animals from parental strain group, while only minimal abnormalities could be seen from galE mutant group, there was a significant differences between them (P<0.01). In parental strains group, the predominant pathologicanl change was axonal degeneration with considerable variation in severity. These morphologic changes were confirmed by electron microscopy. Conclusion Compared with parental strain, galE mutant without ganglioside-like structure no longer could induce anti-GM1 antibodies, nor induce obvious immune damage of peripheral nerves in experimental guinea pigs. The results of this study provide a strong support to the hypothesis of molecular mimicry as a pathogenesis in patients with GBS following CJ antecedent infection.