幽门螺杆菌及其培养上清液粗提蛋白对兔离体胃壁细胞酸分泌的影响

The effects of H. priori and its crude extracted proteins on isolated rabbit parietal cells acid secretion

目的通过研究幽门螺杆菌（Hp）及其培养上清液粗提蛋白（BCF-P）对兔离体胃壁细胞酸分泌的影响，探讨Hp感染引起宿主胃酸分泌状态改变的可能机制。方法兔胃体黏膜经Ⅰ型胶原酶消化、离心淘洗后获得新鲜分离的壁细胞，并进行短期培养。提取Hp培养上清液粗提蛋白（BCF-P）并进行真空干燥浓缩，HeLa细胞以中性红摄取试验鉴定其细胞空泡活性。兔离体壁细胞与Hp及BCF-P（100μg／ml）分别共孵育2、16h和1、12h，应用^14C-氨基比林（^14C-AP）摄取法测定Hp和BCF-P对组胺（1．0×10^-4mol／L）刺激的壁细胞酸分泌的影响；同时应用RT-PCR方法分析Hp和BCF-P对壁细胞H^＋-K^＋ATP酶α亚基mRNA表达的影响。结果（1）BCF-P中含有细胞空泡毒素（VacA），对HeLa细胞表现出细胞空泡活性。（2）与Hp共孵育2h和16h均能抑制组胺刺激的壁细胞酸分泌（P〈0．05），抑制作用随孵育时间延长而增强，2h抑制率为81％，16h为94％；BCF-P作用1h和12h，均能抑制壁细胞酸分泌（P〈0．05），抑制作用也随孵育时间延长而增强，1h抑制率为24％，12h为58％。（3）尽管与Hp孵育2h能上调壁细胞H^＋-K^＋ATP酶α亚基mRNA表达（P〈0．05），但孵育16h则表现为下调其表达（P〈0．05）；BCF-P作用1h和12h，均抑制H^＋-K^＋ATP酶α亚基mRNA的表达（P〈0．05）。结论Hp及其分泌的VacA可能通过下调壁细胞H^＋-K^＋ATP酶表达水平来抑制组胺刺激的壁细胞酸分泌。

Objective To explore the effects of H. pylori and crude extracted proteins secreted by H. pylori （broth culture filtrate protein, BCF-P） on acid secretion from isolated rabbit parietal cells. Methods Parietal cells from rabbit gastric mucosa were isolated and enriched with digestion and elutriation. H. pylori （NCTC 11637, CagA^＋ VacA^＋ ） were grown in liquid broth culture and BCF-P was precipitated with ammonium sulfate. The vacuolation activity of BCF-P was evaluated with neutral red dye uptake test in HeLa cell. Isolated parietal cells were incubated with H. pylori （ bacteria/cell = 100：1 ） for 2 h and 16 h,or BCF-P （100 μg/ml） for 1 h and 12 h. Acid secretion from parietal cells was studied using ^14C-aminopyrine （^14C-AP） accumulation indirectly and H^＋-K^＋ ATPase α subunit mRNA expression was assessed using RT-PCR. Results （ 1 ） BCF-P containing vacuolating cytotoxin （VacA） with vacuolation activity on HeLa cells had positive result on neutral red uptake test. （2） The basal expression of H^＋-K^＋ ATPase α subunit mRNA could be detected in isolated parietal cells and ^14C-AP accumulation was significantly increased in response to the stimulation of histamine with different concentrations for 30 min （P 〈0.05）. These results indicated that the isolated parietal cells retain relative intact acid secretion function. （3）The histamine （1.0 × 10^-4 mol/L） stimulated acid secretion was inhibited sustainedly in response to H. pylori by 81% at 2 h and by 94% at 16 h （P 〈 0. 05）. However, H^＋-K^＋ ATPase α subunit mRNA expression was up-regulated in the acute period （2 h） and was down-regulated in the chronic period （ 16 h） by H. pylori（ P 〈 0.05 ）. （4） BCF-P significantly inhibited the histamine-stimulated acid secretion by 24% at 1 h and by 58% at 12 h （P〈0. 05）, and this inhibition was accompanied by the down-regulated expression of H^＋-K^＋ ATPase α subunit mRNA. Conclusions Intact H. pylori and VacA secreted by H. pylori could directly inhibit histamine-stimulated acid secretion from parietal cells and this inhibition may be mediated by the down-regulated H^＋-K^＋ ATPase expression.