甘露糖敏感性绿脓杆菌制剂抑制肝癌上皮-间充质转化的作用

The effects of pseudomonasaeruginosa mannose sensitive hamemagglutination vaccine on epithelial-mesenchymal transition of hepatocellular carcinoma

目的 观察甘露糖敏感性绿脓杆菌制剂（PA-MSHA）对肝细胞肝癌上皮-间充质转化（EMT）的作用.方法 培养人肝癌细胞株MHCC97L及HepG2,以不同剂量的PA-MSHA作用于肝癌细胞,显微镜观察细胞形态学变化,免疫荧光技术及Western blot检测PA-MSHA作用前后肝癌细胞EMT相关基因及转录因子的表达,免疫组织化学及反转录-聚合酶链反应（RT-PCR）技术检测PA-MSHA治疗前后裸鼠模型肝癌组织中钙黏蛋白（E-cadherin）及转录因子Snail和Slug表达.结果 Western blot结果显示与对照组比较PA-MSHA治疗组肝癌细胞E-cadherin表达显著增强,而波形蛋白（Vimentin）、纤维粘连蛋白（Fibronecin）、核因子-kappa B（NF-κB）以及转录因子Snail和Slug的表达显著降低,其效应呈剂量依赖性,而外源性甘露糖显著抑制PA-MSHA对上述蛋白的调控作用.免疫荧光也显示PA-MSHA上调肝癌细胞E-cadherin表达而抑制Vimentin的表达,显微镜下细胞形态也由不规则的梭形转变为相对规则的卵圆形.免疫组织化学结果显示荷瘤裸鼠经PA-MSHA治疗后肝癌组织E-cadherin的表达显著增强,而Snail及Slug的转录则受到显著抑制（P＜0.05）.结论 PA-MSHA可以通过识别结合肝癌细胞表面的甘露糖显著抑制肝癌EMT的发生.

Objective To investigate the effects of pseudomonasaeruginosa mannose sensitive hamemagglutination vaccine （PA-MSHA） on the epithelial-mesenchymal transition （EMT） of hepatocellular carcinoma （HCC）.Methods MHCC97L and HepG2 cells were cultured and treated with different dose of PA-MSHA.After that,morphological changes of cells were observed under a light microscope.Immunofluorescence and Western blot analysis were used to evaluate the expression of EMT related genes and transcription factors.Immunohistochemistry and reverse transcription polymerase chain reaction （RT-PCR） analysis were used to evaluate the expression of E-cadherin,Snail and Slug in the tumor tissue after the mice were treated with PA-MSHA.Results Western blot and immunofluorescence analysis reveled that the expression of E-cadherin was significantly elevated while expression of Vimentin was significantly inhibited by PA-MSHA,in a dose dependent manner.Moreover,the expression of fibronectin,nuclear factor-kappa B （NF-κB）,Snail and Slug were also inhibited by PA-MSHA,but theses effects of PA-MSHA were blocked by mannose.After PA-MSHA treatment,HCC cells have altered from irregular shape to epithelial cobblestone appearance.In vivo study,PA-MSHA also significantly increased the expression of E-cadherin and inhibited the expression of Snail mRNA and Slug mRNA.Conclusion PA-MSHA can significantly inhibit EMT of HCC in a mannose-mediated manner.