CAJ | 학술논문

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、16 h和1、12 h,应用14C-氨基比林(14C-AP)摄取法测定Hp和BCF-P对组胺(1.0×10-4 mol/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2 h和16 h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,2 h抑制率为81%,16 h为94%;BCF-P作用1 h和12 h,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,1 h抑制率为24%,12 h为58%.(3)尽管与Hp孵育2 h能上调壁细胞H+-K+ATP酶α亚基mRNA表达(P<0.05),但孵育16 h则表现为下调其表达(P<0.05);BCF-P作用1 h和12 h,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.
목적 통과연구유문라간균(Hp)급기배양상청액조제단백(BCF-P)대토리체위벽세포산분비적영향,탐토Hp감염인기숙주위산분비상태개변적가능궤제.방법 토위체점막경Ⅰ형효원매소화、리심도세후획득신선분리적벽세포,병진행단기배양.제취Hp배양상청액조제단백(BCF-P)병진행진공간조농축,HeLa세포이중성홍섭취시험감정기세포공포활성.토리체벽세포여Hp급BCF-P(100μg/ml)분별공부육2、16 h화1、12 h,응용14C-안기비림(14C-AP)섭취법측정Hp화BCF-P대조알(1.0×10-4 mol/L)자격적벽세포산분비적영향;동시응용RT-PCR방법분석Hp화BCF-P대벽세포H+-K+ ATP매α아기mRNA표체적영향.결과 (1)BCF-P중함유세포공포독소(VacA),대HeLa세포표현출세포공포활성.(2)여Hp공부육2 h화16 h균능억제조알자격적벽세포산분비(P<0.05),억제작용수부육시간연장이증강,2 h억제솔위81%,16 h위94%;BCF-P작용1 h화12 h,균능억제벽세포산분비(P<0.05),억제작용야수부육시간연장이증강,1 h억제솔위24%,12 h위58%.(3)진관여Hp부육2 h능상조벽세포H+-K+ATP매α아기mRNA표체(P<0.05),단부육16 h칙표현위하조기표체(P<0.05);BCF-P작용1 h화12 h,균억제H+-K+ATP매α아기mRNA적표체(P<0.05).결론 Hp급기분비적VacA가능통과하조벽세포H+-K+ ATP매표체수평래억제조알자격적벽세포산분비.
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(100μg/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(1.0×104 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 tlle 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.