中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2010年
1期
60-65
,共6页
谢旦立%王波%丁卉%郑丽%楼永良%严杰
謝旦立%王波%丁卉%鄭麗%樓永良%嚴傑
사단립%왕파%정훼%정려%루영량%엄걸
创伤弧菌%溶细胞素%人Caco-2细胞%IL-8
創傷弧菌%溶細胞素%人Caco-2細胞%IL-8
창상호균%용세포소%인Caco-2세포%IL-8
Vibrio vulnificus%Cytolysin%Human Caco-2 cells%IL-8
目的 研究创伤弧菌溶细胞素融合蛋白(rVvhA)对人肠上皮细胞(human intestinalepithelial cell,Caco-2)IL-8基因表达的影响.方法 IPTG诱导、表达、纯化、复性及Western blot鉴定rVvhA表达和纯化情况;CCK-8法检测rVvhA对Caco-2的细胞毒性作用;RT-PCR检测rVvhA诱导Caco-2细胞IL-8基因的表达情况;ELISA检测Caco-2细胞培养上清IL-8的分泌情况.结果 Ni~(2+)-NTA亲和层析柱对rVvhA进行纯化后纯度可达95%以上;CCK-8结果显示rVvhA活性蛋白显著降低了Caco-2细胞的存活率,有效浓度为1.5 HU/ml(P<0.05);RT-PCR结果显示,0.6 HU/ml rVvhA30 minllp可诱导Caco-2细胞IL-8 mRNA基因表达上调;ELISA结果显示,Caco-2细胞经rVvhA作用后,培养液上清中IL-8多肽的表达时相在4 h.RT-PCR与ELISA结果皆显示,IL-8基因的转录及IL-8表达皆具有时间-剂量依赖性.结论 rVvhA在转录水平上能诱导人Caco-2细胞IL-8 mRNA表达,促进IL-8的合成,在创伤弧菌引起的过度炎症反应和败血症的发生、发展中可能具有重要意义.
目的 研究創傷弧菌溶細胞素融閤蛋白(rVvhA)對人腸上皮細胞(human intestinalepithelial cell,Caco-2)IL-8基因錶達的影響.方法 IPTG誘導、錶達、純化、複性及Western blot鑒定rVvhA錶達和純化情況;CCK-8法檢測rVvhA對Caco-2的細胞毒性作用;RT-PCR檢測rVvhA誘導Caco-2細胞IL-8基因的錶達情況;ELISA檢測Caco-2細胞培養上清IL-8的分泌情況.結果 Ni~(2+)-NTA親和層析柱對rVvhA進行純化後純度可達95%以上;CCK-8結果顯示rVvhA活性蛋白顯著降低瞭Caco-2細胞的存活率,有效濃度為1.5 HU/ml(P<0.05);RT-PCR結果顯示,0.6 HU/ml rVvhA30 minllp可誘導Caco-2細胞IL-8 mRNA基因錶達上調;ELISA結果顯示,Caco-2細胞經rVvhA作用後,培養液上清中IL-8多肽的錶達時相在4 h.RT-PCR與ELISA結果皆顯示,IL-8基因的轉錄及IL-8錶達皆具有時間-劑量依賴性.結論 rVvhA在轉錄水平上能誘導人Caco-2細胞IL-8 mRNA錶達,促進IL-8的閤成,在創傷弧菌引起的過度炎癥反應和敗血癥的髮生、髮展中可能具有重要意義.
목적 연구창상호균용세포소융합단백(rVvhA)대인장상피세포(human intestinalepithelial cell,Caco-2)IL-8기인표체적영향.방법 IPTG유도、표체、순화、복성급Western blot감정rVvhA표체화순화정황;CCK-8법검측rVvhA대Caco-2적세포독성작용;RT-PCR검측rVvhA유도Caco-2세포IL-8기인적표체정황;ELISA검측Caco-2세포배양상청IL-8적분비정황.결과 Ni~(2+)-NTA친화층석주대rVvhA진행순화후순도가체95%이상;CCK-8결과현시rVvhA활성단백현저강저료Caco-2세포적존활솔,유효농도위1.5 HU/ml(P<0.05);RT-PCR결과현시,0.6 HU/ml rVvhA30 minllp가유도Caco-2세포IL-8 mRNA기인표체상조;ELISA결과현시,Caco-2세포경rVvhA작용후,배양액상청중IL-8다태적표체시상재4 h.RT-PCR여ELISA결과개현시,IL-8기인적전록급IL-8표체개구유시간-제량의뢰성.결론 rVvhA재전록수평상능유도인Caco-2세포IL-8 mRNA표체,촉진IL-8적합성,재창상호균인기적과도염증반응화패혈증적발생、발전중가능구유중요의의.
Objective To investigate the effect of VvhA recombinant protein to the expression of IL-8 in human intestinal epithelial Caco-2 cells. Methods The VvhA recombinant protein(rVvhA) was ex-pressed by prokaryotic expression vector pET-28a (+)-whA in E. coli BL21 (DE3) , purified by Ni~(2+)-NTA affinity chromatography refoldod by stepwise deliquation together with dialysis methods and identified by Western blot. The cytotoxic of rVvhA to human Caco-2 cells was measured by CCK-8. The transcription of IL-8 mRNA in human Caco-2 cells induced by rVvhA was determined by RT-PCR, and the expression of IL-8 in human Caco-2 cells induced by rVvhA was determined by ELISA assay. Results rVvhA was purified with high purity up to 95%. The viability of human Caco-2 cells treated with 1.5 HU/ml rVvhA was inhibi-ted significantly (P < 0.05). The rVvhA can induce human Caco-2 cells to increase the transcription and expression of IL-8 in dose- and time-dependent manner. The transcription of IL-8 gene in human Caco-2 cells treated with 0.6 HU/ml rVvhA in 30 min can be up-regulated significantly, and the expression of IL-8 in human Caco-2 cells treated with rVvhA in 4 h can be increased significantly. Conclusion rVvhA has cy-totoxic to human Caco-2 and can increase the expression of IL-8, it might play a major role in the inflamma-tory reaction of rVvhA-exposed cells.
