军事医学
軍事醫學
군사의학
BULLETIN OF THE ACADEMY OF MILITARY MEDICAL SCIENCES
2014年
9期
714-718,744
,共6页
常辉%左钱飞%敬海明%邹全明%兰春慧%陈东风
常輝%左錢飛%敬海明%鄒全明%蘭春慧%陳東風
상휘%좌전비%경해명%추전명%란춘혜%진동풍
螺杆菌,幽门%细胞空泡毒素%蛋白纯化%细胞凋亡%胃肿瘤
螺桿菌,幽門%細胞空泡毒素%蛋白純化%細胞凋亡%胃腫瘤
라간균,유문%세포공포독소%단백순화%세포조망%위종류
Helicobacter pylori%vacuolating cytotoxin%protein purification%apoptosis%stomach neoplasms
目的：分离纯化幽门螺杆菌分泌和重组表达的细胞空泡毒素抗原（ VacA）蛋白，并评价其致细胞空泡效应及致细胞凋亡效应。方法分别从幽门螺杆菌ATCC26695菌株培养上清和重组表达VacA蛋白的pQE30-VacA-E．coliM15基因工程菌中分离纯化VacA蛋白，经酸化后，以不同终浓度（5，10 ng／ml）分别与人胃腺癌AGS细胞共孵24 h，观察致空泡效应，并通过流式细胞术检测细胞凋亡。结果成功分离纯化出幽门螺杆菌分泌和重组表达的VacA蛋白；幽门螺杆菌分泌的VacA蛋白能显著引起AGS细胞的空泡样改变及凋亡（P＜0．01），而重组表达的VacA蛋白致细胞空泡样改变及凋亡不显著（ P＞0．05）。结论幽门螺杆菌分泌的VacA蛋白有良好的空泡毒性及致凋亡效应，而重组表达的VacA蛋白无致空泡及凋亡效应，幽门螺杆菌分泌的VacA蛋白可用于VacA作用机制的研究。
目的：分離純化幽門螺桿菌分泌和重組錶達的細胞空泡毒素抗原（ VacA）蛋白，併評價其緻細胞空泡效應及緻細胞凋亡效應。方法分彆從幽門螺桿菌ATCC26695菌株培養上清和重組錶達VacA蛋白的pQE30-VacA-E．coliM15基因工程菌中分離純化VacA蛋白，經痠化後，以不同終濃度（5，10 ng／ml）分彆與人胃腺癌AGS細胞共孵24 h，觀察緻空泡效應，併通過流式細胞術檢測細胞凋亡。結果成功分離純化齣幽門螺桿菌分泌和重組錶達的VacA蛋白；幽門螺桿菌分泌的VacA蛋白能顯著引起AGS細胞的空泡樣改變及凋亡（P＜0．01），而重組錶達的VacA蛋白緻細胞空泡樣改變及凋亡不顯著（ P＞0．05）。結論幽門螺桿菌分泌的VacA蛋白有良好的空泡毒性及緻凋亡效應，而重組錶達的VacA蛋白無緻空泡及凋亡效應，幽門螺桿菌分泌的VacA蛋白可用于VacA作用機製的研究。
목적：분리순화유문라간균분비화중조표체적세포공포독소항원（ VacA）단백，병평개기치세포공포효응급치세포조망효응。방법분별종유문라간균ATCC26695균주배양상청화중조표체VacA단백적pQE30-VacA-E．coliM15기인공정균중분리순화VacA단백，경산화후，이불동종농도（5，10 ng／ml）분별여인위선암AGS세포공부24 h，관찰치공포효응，병통과류식세포술검측세포조망。결과성공분리순화출유문라간균분비화중조표체적VacA단백；유문라간균분비적VacA단백능현저인기AGS세포적공포양개변급조망（P＜0．01），이중조표체적VacA단백치세포공포양개변급조망불현저（ P＞0．05）。결론유문라간균분비적VacA단백유량호적공포독성급치조망효응，이중조표체적VacA단백무치공포급조망효응，유문라간균분비적VacA단백가용우VacA작용궤제적연구。
Objective To isolate and purify VacA protein secreted by Helicobacter pylori or recombinant VacA , and to investigate the effect of VacA-induced cell vacuolar change and apoptosis .Methods VacA proteins were separated and pu-rified from the culture supernatant of H.pylori ( ATCC26695 ) or from the split products of genetically engineered bacteria (pQE30-VacA-E.coli M15) expressing recombinant VacA.The VacA protein obtained was acidified and then incubated with AGS cells for 24 h at different final concentrations of 5 and 10 ng/ml before the vacuolar change and apoptosis of AGS cells were detected via microscopy and flow cytometry assay , respectively .Results H.pylori-secreted VacA and recombi-nant VacA were successfully separated and purified .The H.pylori-secreted VacA significantly induced the vacuolar change and apoptosis of AGS cells (P<0.01) while the recombinant VacA did not.Conclusion H.pylori-secreted VacA protein can effectively induce cell vacuolar change and apoptosis, but recombinant VacA can not, suggesting that the purified VacA protein secreted by H.pylori can be used to explore VacA-induced pathogenesis.
