Penner19空肠弯曲菌毒素的细胞毒性作用研究

Cytotoxic effects of the exotoxin produced by penner′s 19 serotype of Campylobacter jejuni

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 with serial concentrations of CJT antiserum and GM 1 respectively(as designed above) ,and normal serum as negative controls Results (1) CJT produced the cytotoxic activities in CHO and Vero cells ,its minimal responsible concentrations for toxic activities in the cells were both 3 125 mg/L. IMA of both cells were (57 3±5 6)% and (58 6±5 2)%,in which, the incidences of cell death was (53 8±6 2)% and (37 2±6 9)% respectively The rate of cell degeneration of CJT was significantly different to controls[IMA (8 6±2 1)% and (7 3±2 4)%, respectively, P <0 01] (2)An optical absorbance of CJT at 490 nm in ELISA was 0 24±0 01 above the value of controls (while the minimal responsible concentration of CJT was 1 563 mg/L) (3)The cytotoxic effects of CJT to CHO and Vero cells were neutralized by preincubation with CJT antiserum diluted 1∶8 (IMA≤50%),and with the serum dilution of 1∶1(IMA≤10%), but the IMA were more than 50% in controls(normal serum) There were significant differences between CJT antiserum and controls( P <0 05) (4) The rate of cell degeneration of CJT in CHO and Vero cells reduced gradually by preincubated with GM 1 (0 01-0 08 μg/100 μl),although the IMA of all was still more than 50% However, it was not changed since the concentration of GM 1 continually increased from 0 08 to 0 64 μg/100 μl (5)During incubation, the binding capability of CJT to CHO cells was maximal after 3 h assessed by ELISA, and the capability to host cell was completely inhibited by prior treatment with anti CJT serum, but did not inhibited further with the larger dosage after the concentration of GM 1 had been reached at 0 08 μg/100 μl Conclusions These results suggest that the CJT produced by Pen 19 CJ may induce the morphologic and cytolethal changes in CHO and Vero cells However the toxin was binding to CHO cells via some other receptors on cell membranes besides GM 1 in its pathogenetic process