幽门螺杆菌空泡毒素活性检测方法的比较

Comparison of two methods in detection of vacuolating toxin activity of Helicobacter pylori

目的 分别采用细胞计数法及中性红摄入法 (NRU)检测幽门螺杆菌 (Hp)空泡毒素活性 ,探讨NRU检测空泡毒素活性的应用价值。方法 采用Hp液体培养上清浓缩液作为粗制VacA毒素 ,经倍比稀释(1∶16 0～ 1∶2 )后与胃癌SGC 790 1细胞共同孵育 2 4h。用细胞计数法及NRU同时评价VacA的空泡毒素活性 ,比较两种方法检测VacA空泡毒素活性的差异。结果 经中性红染色后 ,胃癌细胞吸收大量染料 ,空泡更加明显。NRU对空泡毒素活性的判断结果与细胞计数法基本符合 ,细胞计数法显示 1∶2 0～ 1∶2滴度样本均呈阳性结果 ,NRU于 1∶2 0～ 1∶5滴度亦得到阳性结果 ,1∶2滴度组因细胞大量死亡得到“假阴性”结果 (A550 =0 .0 6±0 .0 4 )。 1∶4 0滴度组细胞计数法判断为阴性 ,但NRU检测阳性 (A550 =0 .12± 0 .0 4 ,P <0 .0 5 )。结论 NRU检测空泡毒素活性具有简便、快速的特点 ,有助于对空泡毒素活性的定量评价 ,敏感性高于细胞计数法 。

Objective To investigate the value of neutral red uptake assay(NRU) in detecting the vacuolating activity of VacA.Methods The concentrated supernatant of Helicobacter pylori was used as VacA toxin. Diluted VacA (1∶160～1∶2) was incubated with SGC 7901 cells for 24 h. Then the percentage of vacuolating cells was determined with succedent NRU. The difference between these two methods was also compared.Results After the dyeing vacuole showed more conspicous distinction, the results of NRU were mostly in accordance with that by calculating the percentage of vacuolated cells. Groups of 1∶20～1∶2 dilution were judged as positive by counting the cells. Such result could also be derived from NRU in 1∶20～1∶5 dilution( P <0.01). A false negative result was obtained in dilution 1∶2 by NRU because of death of the cells( A 550 =0.06±0.04). NRU detected a positive result ( A 550 =0.12±0.04, P <0.05)in group 1∶40 whose percentage of vacuolation was 0.8%±0.3% when a negative result was obtained by counting.Conclusion NRU is a simple, rapid and sensitive method of detecting vacuolating activity, and can help quantify the toxicity, such method can be used in the detection of VacA activity in vitro.