Purification and characterization of cytotoxin produced by H.phlori, and mechanisms for the occurrence of gastroduodenal diseases by H.pylori infection.

为了研究幽门螺杆菌感染与胃十二指肠疾病发生的关系，从患有这些疾病的患者中分离出了幽门螺杆菌。胃溃疡、十二指肠溃疡和萎缩性胃炎患者幽门螺杆菌的检出率分别为83%、85%和75%。而急性浅表性胃炎和粘膜下肿瘤患者的阳性率分别为25%和33%。快速尿素酶试验对培养法的敏感性、特异性、阳性预测值和阴性预测值分别为76%、85%、91%和63%，是一种有用的筛查方法。定量培养结果表明，尿素酶试验阳性反应需要5000 ~ 10000 CFU的根癌线虫，近一半的根癌线虫产生细胞毒素（CT），导致培养细胞空泡化。结果表明，CT在70 ℃时不耐热，分子量大于20 kDa的蛋白质毒素。用50%硫酸铵沉淀CT后，用Sephacryl S300、苯基Superose、Q Sepharose FF和Mono Q柱纯化CT。然而，通过这些方法不能获得高度纯化的CT。使用RK-13检测CT的阳性率（兔肾），FL（人羊膜），Vero（猴肾），BHK-21（仓鼠肾）和HeLa RK-13细胞株对人胃癌细胞的粘附率分别为73、61、27、27和21%，表明RK-13细胞株是一个很好的指示细胞。（MKN 45，KATO III）细胞和人肠（Int-407）细胞。H.pylori straius对MKN 45、KATO III和Int-407细胞的平均粘附率分别为76.6%、42.7%和15.1%。这些结果表明，幽门螺杆菌对胃细胞，特别是MKN 45细胞，而不是肠细胞具有亲和力。幽门螺杆菌的粘附活性和CT产生之间无显著相关性。

To examine association of Helicobacter pylori infection with the occurrence of gastroduodenal diseases, isolation of H.phlori from patients with the diseases was carried out. Isolation rates of H.pylori from the patients with gastric ulcer, duodenal ulcer and atrophic gastritis were 83,85 and 75%, respectively. In contrast, those from the patients with acute superficial gastritis and submucosal tumour were 25 and 33%, respectively. Sensitivity, specificity, positive^- and negative^-predictive values of rapid urease test of biopsy specimens to cultivation method were 76,85,91 and 63%, respectively, indicating that the rapid urease test is an useful screening test. By quantitative culture, it was shown that 5,000 - 10,000 CFU of H.phlori were required for positive reaction in the urease test.Nearly half strains of H.phlori produce cytotoxin (CT) which induces vacuolation in cultured cells. It was demonstrated that CT was heat-labile at 70ﾟC and proteinous toxin with molecular weight of more than 20kDa. After precipitation of CT with 50% ammonium sulphate, purification of CT was tried using Sephacryl S300, phenylsuperose, Q Sepharose FF and Mono Q columns. However, highly purified CT was not obtained by these procedures. Positive rates for detection of CT using RK-13 (rabbit kidney), FL (human amnion), Vero (monkey kidney), BHK-21 (hamster kidney) and HeLa (human uterine cancer) cells were 73,61,27,27 and 21%, respectively, indicating that RK-13 cell line is a good indicator.Adherence activity of H.phlori strains to human gastric cancer (MKN45, KATO III) cells and human intestine (Int-407) cells was analyzed by flowcytometer. Average adherence rates of H.pylori straius to MKN 45, KATO III and Int-407 cells were 76.6,42.7 and 15.1%, respectively. These results suggest that H.pylori has an affinity to gastric cells, particularly MKN 45 cells, rather than instinal cells. No significant correlation between adherence activity of and CT production by H.pylori was observed.