CagA对幽门螺杆菌诱导胃黏膜上皮细胞分泌IL-8水平的影响

The role of CagA on H.pylori-induced IL-8 production from gastric epithelial cells

目的:利用小分子干扰RNA(siRNA)探讨CagA在幽门螺杆菌诱导胃黏膜上皮细胞分泌IL-8中的作用。方法:设计5条siRNA,采用电穿孔法转入CagA+幽门螺杆菌NCTC11637,与胃黏膜上皮细胞共培养后测定IL-8水平,并以RT-PCR、Western blotting法检测穿孔前、后不同时点细菌的CagA表达。采用CagA-株NCTC11639作对照。结果:CagA+NCTC11637诱导胃黏膜上皮细胞分泌IL-8水平明显高于CagA-NCTC11639[(1200·00±32·51)ng/Lvs(100·00±8·58)ng/L](P<0·01)。siRNAⅢ转化的幽门螺杆菌诱导胃黏膜上皮细胞分泌IL-8水平显著低于转化前[(400·00±17·35)ng/Lvs(1200·00±32·51)ng/L](P<0·05);siRNAⅢ转化的幽门螺杆菌CagA mRNA水平显著低于转化前,穿孔后6h mRNA水平最低为31·3%(0·270/0·861),抑制率为68·7%。Western blotting显示siRNAⅢ组CagA蛋白水平低于穿孔前(P<0·01);其余4条siRNA未见显著变化。结论:CagA在幽门螺杆菌诱导胃黏膜上皮细胞分泌IL-8中起重要作用,小分子干扰RNA可能通过抑制CagA基因表达而减少幽门螺杆菌诱导胃黏膜上皮细胞IL-8的分泌。

AIM： To investigate the role of CagA and the effect of small intefference RNA （ siRNA） on the release oflL - 8 in H. pylori- infected gastric epithelial cells. METHODS： siRNA were transferred into CagA positive strain H. pylori NCTC 11637 by using electroporation. The CagA positive strain NCTC 11637 and the CagA negative strain NCTC 11639 were cocultured with gastric epithelial cells and the level of IL - 8 in the supernatant was measure by ELISA. RT- PCR and Westrn blotting were performed to detect CagA expression. RESULTS： The level of IL- 8 induced by CagA positive strain NCTC 11637 was higher than the level induced by CagA negative strain Ncrc 11639 [ （ 1 200.00 ± 32.51 ） ng/L vs （ 100.00 ± 8.58） ng/L, P 〈 0.011. Treatment with siRNAⅢ resulted in a decrease in IL - 8 production by 2/3 [ from （ 1 200.00 ± 32.51 ） ng/L to （400.00 ± 17.35） ng/L, P〈0.05]. In siRNA V group, it reduced a little （P〉0.05）. Meanwhile, CagA mRNA decreased significantly in siRNAⅢ group. The levels of CagA mRNA at 6, 12, and 24 h after electroporation were 31.3% （0.270/0.861）, 57.6% （0.496/ 0.861）, and 73.9% （0.637/0.861） of the control levels, respectively. Inhibition rate by siRNAm was 68.7%. The Western blotting result in siRNAⅢ group showed that the level of CagA protein degraded to 30.7% （0.4/1.3） at 12 h after electroporation. In siRNA V group, the expression of CagA mRNA at 6 h is suppressed by 23.1% （ P 〈 0.05）. There was no significant difference between the expression level of CagA mRNA at 12 h, 2 h after treatment and before treatment. The other specific siRNA had no effect on CagA mRNA expression. CONCLUSION： CagA is involved in the release of IL - 8 by H. pylori - infected gastric epithelial cells. Small interference RNA can influence the IL- 8 release by inhibiting the expression of CagA in H. pylori.