目的 通过诱导空肠弯曲菌进入活的不可培养(VBNC)状态建立细胞学模型,用VeroE6细胞和HeLa细胞检测空肠弯曲菌产细胞膨胀性肠毒素(cytotoxic distendingtoxin,CDT)能力的改变,并在RNA水平上进一步验证毒素的表达。方法 采用4℃冷藏及-70℃冷冻的方法诱导空肠弯曲菌进入VBNC状态,利用细胞总数计算、活细胞染色计数及可培养细胞计数间接确证VBNC状态细菌的存在,细胞对于VBNC状态下的细菌仍然敏感,并用反转录-聚合酶链反应(RT-PCR)证实空肠弯曲菌毒素基因的表达。结果 3种不同状态的细胞计数结果表明存活细胞进入了VBNC状态,细菌经4℃冷藏及-70℃冷冻后数量减少约1个数量级,在冷冻前,细菌对细胞毒性较强,72h时70%以上细胞凋亡,冷冻后的VBNC状态细菌对VeroE6和HeLa细胞的生长仍有影响,72h时约有40%细胞进入凋亡状态。结论 低温、贫养条件下空肠弯曲菌可进入VBNC状态,用常规传统的羊血平板培养基仅能使不到10%的空肠弯曲菌复苏生长,从而可以检测,在VBNC状态时,空肠弯曲菌产毒素能力改变较大,远远低于正常状态,但仍具备产毒素的能力,也从RNA水平实验得到证实。

Objective The existence of a viable but non-culturable(VBNC)state has been described for Campylobacter jejuni.This state can be induced and detected using established VeroE6,HeLa cell lines.Methods VBNC C.jejuni cell were induced by low temperature and starvation.Sensitization test of established HeLa,Vero cells model to CDT of viable but non-culturable Campylobacter jejuni showed that the cell was still sensitive,and the result was further confirmed by RT-PCR test.The experiment found that VBNC C.jejuni cell still had the virulence to cell cultures although it decreased.Total and active cells were counted by conventional culture method and after staining with 49,6 diamino-2-phenylindole(DAPI).Induced VBNC cell were obtained by freezed at-70 ℃.Results The rest VBNC cell were confirmed by different state cell count.C.jejuni decreased about one magnitude after refrigeration.About 70% cell can be induced into apotosis before refrigeration and only 40% cell die after C.jejuni refrigeration.Conclusion Low temperature and nutrient poor conditions can induce cell enter into VBNC state.Remarkable change of C.jejuni cell to produce virulence can be observed,and we confirmed this further at RNA level.VBNC C.jejuni cell can still produce virulence although the quantity decreased remarkably. if(document.getElementById('ChDivSummary').innerHTML!=""){CutSpan('ChDivSummary',500);DisplaySpanDiv('ChDivSummary');ClearSummaryOnLoad('SummaryLinkChID','SummaryLinkEnID');}else{CutSpan('EnDivSummary',1000);DisplaySpanDiv('EnDivSummary');ClearSummaryOnLoad('SummaryLinkEnID','SummaryLinkChID');}