IDO基因转染对小鼠Lewis肺癌细胞侵袭和转移的影响研究

Effects of IDO gene transfection on the invasion and metastasis of mouse Lewis lung cancer cell

目的探讨吲哚胺-2,3-双加氧酶(indoleamine-2,3-dioxygenase,IDO)基因转染对小鼠Lewis肺癌细胞侵袭和转移能力的影响,并对其机制进行初步探讨。方法通过基因转染技术,利用阳离子脂质体将含有人全长IDO基因序列的真核表达质粒pEGFP-IDO转染到小鼠Lewis细胞,G418筛选获得Lewis-IDO细胞,设置空白对照组(Lewis细胞)及空质粒转染组(Lewis-EGFP细胞),观察细胞形态学改变,利用transwell侵袭小室检测3种细胞侵袭能力,将3种细胞分别种植到C57/LB小鼠,检测不同细胞种植瘤和转移灶的形成、小鼠外周血中调节性T细胞(Regulatory T cell,Treg)的增殖。结果与Lewis-EGFP细胞和Lewis细胞相比,Lewis-IDO细胞贴壁比例增加,形态变为长梭形,伪足明显增多延长,前二者形态相似;Lewis-IDO细胞侵袭能力增强,破坏matrigel基质蛋白胶层进入下室内的细胞数明显增多,Lewis-IDO细胞、Lewis-EGFP细胞及Lewis细胞分别为237.4±22.1、166.0±14.2和185.6±19.5,前者与后二者比较差异具有统计学意义(P<0.05);种植Lewis-IDO、Lewis-EGFP及Lewis细胞的小鼠发生肝肺转移的小鼠数量及转移灶分别为12只44处、5只7处及4只7处,前者与后二者比较具有统计学意义(P=0.01);流式细胞术分析,种植Lewis-IDO细胞的小鼠外周血中Treg细胞比例明显高于Lewis-EGFP细胞组和Lewis细胞组,其比例分别为(13.7±4.5)%、(8.9±3.3)%和(9.2±3.4)%,前者与后二者比较差异具有统计学意义(P<0.05)。结论IDO基因转染能改变Lewis细胞的培养形态,可以明显增强Lewis细胞的侵袭能力和转移能力,其机制可能与IDO对Lewis细胞形态学改变及诱导Treg细胞的增殖有关。

Objective To investigate the effects of IDO gene transfection on the invasion and metastasis of mouse Lewis lung cancer cell line and to explore its possible mechanisms. Methods Recombinant eukaryotic expression vector pEGFP-IDO was transfected into mouse Lewis lung cancer cell line by cationic liposome Lipofectimine 2000. By G418 selection, RT-PCR and Western blotting analysis, Lewis-IDO cells was obtained. The parental Lewis cell and Lewis cell transfected with blank plasmid pEGFP （Lewis-EGFP） was used as control group. The changes of adhesion ability to the culture utensil, the capability of invasion in vitro and metastasis in C57/BL mouse of Lewis-IDO, Lewis-EGFP and Lewis cells were studied respectively. The Regulatory T cells （Treg） in the peripheral blood（PB） of mouse were measured. Results Compared with the Lewis-EGFP and Lewis cells,Lewis-IDO cells became longer and had more filopodia. By transwell chamber system, the capability of invasion of Lewis-IDO cells was significantly increased. The quantity of the Lewis-IDO cells breaking through the matrigel increased significantly（P〈0.05）. The capability of metastasis to lung and liver of Lewis-IDO cell was significantly higher than that of Lewis-EGFP cells or Lewis cells（P= 0.01） The Treg cells in PB of the mouse transplanted with Lewis-IDO cells were statistically higher than that from the mouse transplanted with Lewis-EGFP cells or Lewis cells（P〈0.05）. Conclusion The tranfection of IDO gene could increase the Lewis cell＇s capability of invasion and metastasis by inducing the morphological changes and the proliferation of Treg cells.