硒对幽门螺杆菌空泡毒素及其重组影响

Effects of selenium on VacA and recombinanted VacA of Helicobacter pylori

目的研究硒(Se)对幽门螺杆菌(Hp)空泡毒素(VacA)及重组VacA的影响。方法采用盐析、透析、柱层析、浓缩、抽滤等方法提纯幽门螺杆菌分泌的空泡毒素。通过琼脂糖凝胶电泳、基因序列分析鉴定重组载体,重组VacA产物经Ni-TEDTM蛋白纯化系统纯化,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、及免疫印迹(Western blot)鉴定,间接酶联免疫吸附(ELISA)法检测其抗原特异性。进行细胞培养、细胞爬片;采用分光光度计、倒置显微镜、透射电镜及流式细胞仪等方法检测硒对幽门螺杆菌分泌的VacA及重组毒素蛋白功能的影响。结果12 mmol/L硒即可使幽门螺杆菌形态发生球形变,鞭毛及菌毛出现脱落,溶血作用减弱,硒对VacA空泡毒作用无直接影响(P>0.05);硒通过抑制Hp生长减少VacA产生量从而使空泡毒作用减弱;通过抑制表达VacA的大肠埃希菌生长,减少重组VacA表达量;能增强重组VacA对人胃上皮癌细胞株(SGC-7901)生长抑制作用,使细胞分裂指数、集落形成率、S期细胞数量降低,G1期细胞总数增加;使表达重组VacA的大肠埃希菌产生脂溶性砖红色色素,并随硒浓度增加颜色加深。结论硒能抑制Hp生长,改变Hp的形态及结构;可使重组VacA表达量减少;能增强重组VacA对SGC-7901细胞的生长抑制及诱导细胞凋亡作用;可使表达重组VacA的大肠埃希菌产生脂溶性砖红色色素;不能直接抑制VacA的空泡毒作用。

Objective To study the effects of selenium on the vacolating cytotoxin（VacA） and the recombinanted VacA of Helicobacter pylori（Hp）. Methods The VacA of Hp was purified by salting out, dialysis, gel filtration and concentration. The pET32a - vacA - E. coli BL21 （DE3） bacteria were cultivated and the recombinanted plasmids were analysed by restriction endonuclease . The recombinanted VacA protein was purified by Ni - TEDTM kit and identified by SDS - PAGE and Western blot.Different concentrations of VacA, recombinanted VacA and selenium（Se） were added into the SGC- 7901 cells, then the changes of the cell cycle of SGC - 7901 were observed by spectrophoto meter, inverted microssope, transmission electron microscope and Flow Cytometry. Results The configuration of Hp was changed by Se （ 12 mmol/1）. The proliferation inhibition of recombinanted VacA on the SGC - 7901 cells was strengthened by Se. The percentage of G1 phase cells was increased and of the S phase cells was decreased by Se. Brick red appeared after Se was added into the E. coll. Conclusion Se can inhibit the proliferation of Hp and decrease the production of VacA, but it fails to inhibit the cellular vacolation effect of VacA. Se can improve the inhibitive effects of the recombinant VacA on the SGC- 7901 and decrease the VacA expression by inhibiting the Hp proliferation. But Se fails to reduce the cellular vacolation of VacA directly.