外泌体对鼻咽癌细胞耐药的作用研究

Effect of exosomes on drug resistance in nasopharyngeal carcinoma cells

目的探讨外泌体在鼻咽癌细胞顺铂耐药机制中的作用研究。方法以鼻咽癌细胞CNE-1为基础,利用浓度梯度递增与大剂量药物冲击相结合的方法建立鼻咽癌耐药细胞系,进行形态学观察,CCK-8法检测其耐药指数,免疫印迹法(Western blot)检测耐药蛋白的表达三种方法以验证耐药细胞系的建立。利用Transwell小室将耐药细胞与敏感细胞共培养,通过CCK8检测共培养后细胞的耐药性,Western blot检测耐药蛋白的变化;利用差速离心法提取细胞外泌体,通过透射电子显微镜观察外泌体形态、纳米颗粒跟踪分析(NTA)法检测外泌体的分子大小,Western blot检测外泌体特征蛋白TSG101、CD9、CD81的表达三种方法相结合对提取的外泌体加以验证。利用PKH67对外泌体进行荧光标记染色,并与敏感细胞共培养,通过共聚焦显微镜观察外泌体被细胞摄取的过程。结果建立的鼻咽癌耐药细胞系CNE-1/DDP,耐药指数为5.15;显微镜下观察可见敏感细胞呈现规则的圆形,耐药细胞呈不规则的梭形或多边形状态,耐药蛋白MVP、P-gp蛋白高表达。共培养后的敏感细胞耐药指数为1.98,形态学变化尚不明显。透射电镜观察外泌体形态为40~150nm类圆形双层膜结构,粒径分析显示外泌体密度为2.2×10^(11)个/毫升,直径集中分布于50~150nm之间,其同时表达TSG101、CD9、CD81三种蛋白,证实我们提取外泌体是正确的。共聚焦显微镜下观察到PKH67绿色荧光蛋白标记的外泌体,可被CNE-1细胞摄取。结论鼻咽癌耐药细胞通过分泌的外泌体可导致敏感细胞耐药。

Objective To investigate the role of exosomes in the mechanism of cisplatin resistance in nasopharyngeal carcinoma cells.Methods Based on CNE-1cells,the nasopharyngeal carcinoma cell line was established with the method of increasing concentration gradient combined with the shock of high-dose drug.Morphology of the cell line was observed.CCK-8method was adopted to detect the index of drug resistance and Western blot to test the three expression ways of drug resistance protein in order to verify the establishment of cell lines with drug resistance.Drug-resistant cells were co-cultured with sensitive cells using Transwell.CCK8was used to detect the drug resistance of co-cultured cells,and Western blot to monitor the changes of drug-resistant proteins.Exosomes were extracted by differential centrifugation.The transmission electron microscopy(TEM)was employed to observe the extracted exosomes’morphology;nanoparticle tracking analysis(NTA)to measure the molecular size;Western blot to detect the expressions of the characteristic proteins TSG101,CD9and CD81in exosomes.The extracted exosomes were verified by the combination of the above three methods.The exosomes were stained with PKH67and co-cultured with sensitive cells.The process of cells taking up exosomes was observed by confocal microscopy.Results The established drug-resistant cell line CNE-1/DDP in nasopharyngeal carcinoma had a drug resistance index of 5.15;under the microscope,sensitive cells showed regular round shape,while drug-resistant cells had irregular spindle shapes or polygons in terms of morphology.Drug-resistant proteins MVP and P-gp were highly expressed.After co-culture,the drug resistance index of sensitive cells was 1.98,with unconspicuous morphological changes.The morphology of exosomes under transmission electron microscopy was a structure of quasi-circular double-layer membrane of 40~150nm.Particle size analysis showed that the density of exosomes was 2.2×10^(11)/ml,and the diameters were concentrated between 50~150nm.The exosomes expressed three types of protein(TSG101,CD9and CD81)at the same time,which confirmed that the extraction of exosomes was feasible.The exosomes labeled with green fluorescent protein PKH67were observed under confocal microscopy and could be taken up by CNE-1cells.Conclusion Nasopharyngeal carcinoma cells with drug resistence can lead to drug-resistance in sensitive cells by secreting exosomes.