癌源性外泌体对乳腺癌MCF-7细胞生物学行为的影响

目的 探究乳腺癌细胞来源的外泌体对乳腺癌MCF-7细胞生物学行为的影响。方法 使用超速离心法对人乳腺癌MCF-7细胞培养液中的外泌体进行提取和纯化,通过透射电子显微镜观察外泌体的形态和大小,Western blot实验鉴定外泌体的标志蛋白。以外泌体与MCF-7细胞共培养48 h后的细胞为实验组,以未处理的MCF-7细胞为对照组。用MTT法、克隆形成实验、细胞划痕实验、细胞侵袭实验、流式细胞术实验和Western blot实验检测乳腺癌源性外泌体对MCF-7细胞增殖、克隆形成、迁移、侵袭、凋亡及上皮-间充质转化(Epithelial-mesenchymal transition,EMT)能力的影响。结果 透射电子显微镜下外泌体的形状为典型的杯托状或者凹的半球状结构,直径40~110 nm;Western blot实验结果显示外泌体标志性蛋白CD9及CD81均明显表达;MTT和克隆形成实验结果显示,实验组细胞增殖及克隆形成能力较对照组明显增强(P＜0.05);划痕实验和侵袭实验结果显示,与对照组相比,实验组细胞迁移及侵袭能力明显增强(P＜0.001);流式细胞术分析显示,在外泌体刺激的乳腺癌细胞中,凋亡细胞的百分比显著低于对照组(P＜0.05);Western blot结果显示,乳腺癌来源的外泌体增加了N-cadherin和Vimentin的表达,降低了E-cadherin表达,促进了乳腺癌EMT过程(P＜0.05)。结论 乳腺癌细胞来源的外泌体能增强肿瘤细胞的细胞增殖、克隆形成、迁移和侵袭能力,促进乳腺癌EMT过程,并抑制肿瘤细胞凋亡。表明肿瘤来源外泌体可能作为细胞间信号转导的介质,在促进肿瘤生长和转移的信息传递方面发挥重要作用。

Effects of cancer-derived exosomes on the biological beharior of breast cancer MCF-7 cells

Objective The aim of this study was to investigate the effect of breast cancer cell-derived exosomes on the biological behavior of breast cancer MCF-7 cells. Methods Exosomes in human breast cancer MCF-7 cell culture medium were extracted and purified by ultracentrifugation,the morphology and size of exosomes were observed by transmission electron microscopy,and the marker protein of exosomes was identified by Western blot experiments.MCF-7 cells co-cultured with exosomes for 48 h were used as the experimental group,and untreated MCF-7 cells were used as the control group.The effects of breast cancer-derived exosomes on MCF-7 cell proliferation,clonal formation,migration,invasion,apoptosis,and epithelial-mesenchymal transition(EMT) were investigated by MTT,clonal formation,cell scratch experiment,cell invasion experiment,flow cytometry experiment and Western blot experiment. Results The shape of exosomes under transmission electron microscopy is a typical cup-tray or concave hemispherical structure,with a diameter of 40-110 nm;The results of Western blot showed that the exosomes′ signature proteins CD9 and CD81 were clearly expressed.The results of MTT and clonal formation experiments showed that the cell proliferation and clonal formation ability of the experimental group were significantly enhanced compared with the control group(P＜0.05);The results of the scratch experiment and invasion experiment showed that compared with the control group,the cell migration and invasion ability of the experimental group was significantly enhanced(P＜0.001);Flow cytometry analysis showed that the percentage of apoptotic cells in exosome-stimulated breast cancer cells was significantly lower than that in the control group(P＜0.05);Western blot results showed that breast cancer-derived exosomes increase the expression of N-cadherin and Vimentin,decrease E-cadherin expression,and promote the EMT process in breast cancer(P＜0.05). Conclusion Exosomes derived from breast cancer cells can enhance the cell proliferation,clonal formation,migration and invasion ability of tumor cells,promote the EMT process of breast cancer,and inhibit apoptosis of tumor cells.It shows that tumor-derived exosomes can be used as mediators for cell-to-cell signal transduction and play an important role in promoting information transmission of tumor growth and metastasis.