EFFECT OF UREASE ON HELA-CELL VACUOLATION INDUCED BY HELICOBACTER-PYLORI CYTOTOXIN
EFFECT OF UREASE ON HELA-CELL VACUOLATION INDUCED BY HELICOBACTER-PYLORI CYTOTOXIN
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DOI：
10.1128/iai.59.4.1264-1270.1991
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发表时间：
1991-04-01
影响因子：
3.1
通讯作者：
BLASER, MJ
中科院分区：
文献类型：
--
作者：
COVER, TL;PURYEAR, W;BLASER, MJ
50 - 60%幽门螺杆菌菌株的浓缩肉汤培养上清液在体外诱导真核细胞空泡化。 基于细胞内空泡快速摄取中性红染料，建立了细胞空泡化的定量测定方法。 可见空泡化的HeLa细胞的中性红染料摄取（NRU）显着大于nonvacuolated细胞。 通过使用快速NRU测定，我们试图确定H。pylori细胞毒素、尿素酶和氨在HeLa细胞空泡形成中的作用。 在含有氯化铵或硫酸铵的培养基中孵育的HeLa细胞的NRU显著大于在单独的培养基中孵育的细胞。 此外，铵盐还能增强H. pylori上清液。 通过向细胞培养基中加入尿素来增强由刀豆尿素酶诱导的NRU;这表明尿素酶介导的NRU通过产生氨而发生。 乙酰异羟肟酸阻断了刀豆脲酶和尿素诱导的NRU，但不能阻断H. pylori上清液。 不产尿素酶的H. pylori突变株诱导的NRU与尿素酶阳性亲本株相同。 这些结果表明，H.幽门螺杆菌上清液中的氨的产生并不只是由尿素酶活性介导的，还可能是由尿素酶介导的氨产生增强的。
Concentrated broth culture supernatants from 50 to 60% of Helicobacter pylori strains induce eukaryotic cell vacuolation in vitro. A quantitative assay for cell vacuolation was developed on the basis of the rapid uptake of neutral red dye into intracellular vacuoles. The neutral red dye uptake (NRU) of visibly vacuolated HeLa cells was significantly greater than that of nonvacuolated cells. By using the rapid NRU assay, we sought to determine the roles of H. pylori cytotoxin, urease, and ammonia in the vacuolation of HeLa cells. The NRU of HeLa cells incubated in medium containing ammonium chloride or ammonium sulfate was significantly greater than that of cells incubated in medium alone. In addition, ammonium salts augmented the NRU induced by H. pylori supernatants. The NRU induced by jack bean urease was augmented by the addition of urea to cell culture medium; this suggests that urease-mediated NRU occurs via the generation of ammonia. Acetohydroxamic acid blocked the NRU induced by jack bean urease and urea but failed to block the uptake induced by H. pylori supernatants. Supernatant from a non-urease-producing H. pylori mutant strain induced NRU identical to that of the urease-positive parental strain. These observations indicate that the vacuolating activity in H. pylori supernatants is not mediated solely by urease activity but that it may be potentiated by urease-mediated ammonia production.