乳腺癌细胞外泌体激活肝星状细胞转变为肿瘤相关成纤维细胞

Breast cancer cell exosomes activate hepatic stellate cells to transform into tumor-associated fibroblasts

目的研究乳腺癌细胞外泌体对肝星状细胞的作用。方法通过超速差速离心法提取乳腺癌细胞外泌体,通过透射电子显微镜、粒径分析软件和蛋白印迹分析等实验鉴定外泌体的形态结构、粒径分布及标志蛋白CD63的表达。通过活细胞荧光显微镜观察荧光标记后外泌体被肝星状细胞的摄取情况。通过蛋白印迹分析研究经乳腺癌细胞外泌体处理后的肝星状细胞中α-平滑肌肌动蛋白的表达水平。结果透射电子显微镜显示乳腺癌细胞外泌体为类圆形双层膜的囊泡样结构;粒径分析显示外泌体大小分布范围为30~200 nm;蛋白印迹分析显示外泌体表面标志蛋白CD63为阳性。通过活细胞荧光显微镜观察发现PKH67标记的外泌体能够被DAPI标记的肝星状细胞摄取。蛋白印迹分析结果显示经乳腺癌细胞外泌体处理过的肝星状细胞中α-SMA的蛋白表达水平较对照组显著提高。结论肝星状细胞与乳腺癌细胞外泌体共孵育后能够被激活为肿瘤相关成纤维细胞。

Objective To study the effect of supernatant exosomes of breast cancer cells on hepatic stellate cells.Methods Exosomes were extracted from supernatant of breast cancer cells by hypervelocity differential centrifugation.The morphology,size,particle size distribution and expression of CD63 were identified by transmission electron microscopy,particle size analysis software and western blot analysis.The uptake of fluorescent-labeled exosomes by hepatic stellate cells was observed by living cell fluorescence microscope.The expression level ofα-smooth muscle actin(α-SMA)in hepatic stellate cells treated with supernatant exosomes of breast cancer cells was studied by western blot analysis.Results The exosomes of supernatant of breast cancer cells showed round vesicle-like structure by transmission electron microscope.Particle size analysis showed that the size distribution range of exosomes was 30~200 nm.Western blot analysis showed that the exosome surface marker protein CD63 was positive,and PKH67 labeled exosomes can be ingested by DAPI labeled hepatic stellate cells by living cell fluorescence microscopy.The expression level ofα-SMA in hepatic stellate cells treated with supernatant exosomes of breast cancer cells was significantly higher than that of the control group.Conclusion Hepatic stellate cells can be activated as tumor-associated fibroblasts after co-incubation with supernatant exosomes of breast cancer cells.