摘要
目的 探讨Penner 19空肠弯曲菌毒素 (CJT)对细胞毒性的影响。方法 (1)应用CHO及Vero细胞检测CJT毒性效应 ,以及CJT抗血清和GM1中和 /抑制作用 ;(2 )ELISA检测CJT结合GM1、CHO细胞受体 ,评价CJT抗血清及GM1中和或抑制作用。结果 (1)CJT对CHO和Vero细胞均具毒性 ,效应最低CJT量均为 3 12 5mg/L ,细胞变形率分别为 (5 7 3± 5 6 ) %及 (5 8 6± 5 2 ) % ,变形细胞中死亡率分别为 (5 3 8± 6 2 ) %及 (37 2± 6 9) %。对照组细胞变形率分别为 (8 6± 2 1) %及 (7 3±2 4 ) % ,显著低于CJT组 (P <0 0 1)。 (2 )与GM1呈阳性反应的CJT最低量为 1 5 6 3mg/L ,吸光度差值(P -N)为 0 2 4± 0 0 1(P >0 2 0 )。 (3)CJT抗血清可中和CJT毒性效应 ,其 1∶8及 1∶1倍稀释时 ,细胞变形率分别 <5 0 %和 <10 % ,与对照组比较差异有显著意义 (P <0 0 1)。 (4)GM1由 0 0 1增至 0 0 8μg/10 0 μl时 ,CJT致细胞形变率逐渐下降 ,继续增加GM1剂量 (0 0 8～ 0 6 4 μg/10 0 μl) ,细胞变形率却维持不变 ,且始终大于 5 0 %。 (5 )CJT结合CHO于作用后 3min最明显。CJT抗血清及GM1均可导致CJT结合CHO细胞能力减低。CJT抗血清完全抑制 ;而GM1部分抑制。结论 CJT对细胞可产生形态及致死?
Objectives Penner′s serotype 19 of Campylobacter jejuni(Pen 19 CJ) has been identified as the most frequent infectious agent associated with Guillain Barre syndrome (GBS) The exact pathogenic mechanism remains unclear It has been hypothesized that several factors may be involved in pathogenesis of GBS Among them, toxin of CJ(CJT) could be one of the most important virulence factors. In the present study, it was to be explored that the cytotoxic effect of the exotoxin of Pen 19 CJ. Methods (1)Toxin assay : Chinese hamster ovary(CHO) and African green monkey kidney (Vero) cells were used for the detection of the cytotoxic activity of the CJT All samples were tested in serial dilutions with working volumes of 100 μl per well Incidence of morphological alteration (IMA) of more than 50% of the cells in a well was considered as a positive response (2)GM 1 ELISA: The CJT reacted with ELISA plate coated by GM 1 in an enzyme linked immunosorbent assay(ELISA). A 1∶500 dilution of crude rat anti CJT antiserum was the first antibody, followed by horseradish peroxidase conjugated monoclonal goat anti rat immunoglobulin G antiserum(1∶4 000) as the second antibody The samples were regarded as positive(CJT containing) when an A 490 nm of ≥0 20 above the negative control(uninoculated medium) value was obtained. (3)Neutralization / inhibition assays: Twofold serial dilutions of 100 μl CJT antiserum(1∶1-1∶64) and GM 1 solution with different concentration (0 01-0 64 μg/100 μl) were incubated at 37 ℃ for 30 min , with equal volumes containing 2 times the minimum amounts of CJT (6 25 μg/ml for both) that gave positive response in the CHO and Vero assays respectively ,and then the mixtures were added in cell culture IMA≤50% was regarded as positve Normal serum as negative controls (4)Binding specificity : An ELISA was used to calculate the binding capability of CJT to CHO receptors Adherence blocking studies were done by using the preincubation of CJT wi
出处
《中华儿科杂志》
CAS
CSCD
北大核心
2002年第5期283-287,共5页
Chinese Journal of Pediatrics
基金
国家自然科学基金资助项目 (3 9770 783 )
