舌癌细胞exosome中致癌因素的细胞间转移研究

Study of the Intercellular Transfer of the Oncogenic Factors by exosome from Tca8113

目的观察人舌癌细胞Tca8113来源exosome与人正常口腔黏膜上皮细胞间进行致癌因素的细胞间转移，探索正常口腔黏膜上皮细胞是否是舌癌细胞exosome的靶细胞。方法构建含有EGFRvIII绿色荧光蛋白表达载体，用脂质体转染入舌癌细胞Tca8113，倒置荧光显微镜下观察转染情况；采用超滤离心结合蔗糖密度梯度超速离心的方法从已转染舌癌细胞的培养上清液中分离出exosome，在透射电子显微镜下观察确认；将ex-osome与人正常口腔黏膜上皮细胞共培养，倒置荧光显微镜下观察人正常口腔黏膜上皮细胞是否能摄取exo-some。结果重组质粒pEGFP-N1-EGFRvIU转染的舌癌细胞在倒置荧光显微镜下呈现绿色荧光；透射电子显微镜下观察exosome具有特征性的盘状结构，由双层膜构成，直径在30-100nm之间；exosome与口腔黏膜上皮细胞共培养2h后，黏膜上皮细胞荧光显微镜下观察到绿色荧光。结论成功分离人舌癌细胞exosome，人正常口腔黏膜上皮细胞是人舌癌细胞exosome的靶细胞，exosome能进行致癌因素的细胞间转移。

Objective To study the intercellular transfer of the oncogenic factors by exosome derived from TcaS113 tmnor cells and to explore whether the exosomes are able to target oral mucosa epithelial cell. Methods Firstly eukaryotic expression vector of pEGFP-N1- EGFRv m was constructed and recombinant plasmid was transfected intoT- ca8113 instantly. Tca8113 transfected -derived exosomes were isolated and purified from the culture supernatants by seri- al ultracentrifugation and sugar density uhracentrifugation. Exosomes were observed with transmission electronic micro- scope. Incubated exosome with oral mucosa epithelial cell to observe whether green signal exosomes could be uptaken by the cells. Results Some Tca8113 transfected could be seen bright green signal under the fluorescence microscope. The exosomes were exhibited heterogenous round or elliptic microvesicles ranging from 30nm to 100nm in diameter with intact membrane. The HOK cell incubated with exosome eoule seen green signal. Conclusion This study successfully isolate exosomes of Tca8113 and prove that oral mucosa epithelial cells are the biological targets of such exosomes. Exosome can contribute to a horizontal propagation of oncogenic factors among cells.