成纤维细胞系3T3细胞来源exosome对小鼠乳腺癌细胞增殖能力的影响

Effect of exosome Extracted from Fibroblast Cell Line 3T3 on Proliferation of Mouse Breast Cancer Cells

目的观察小鼠成纤维细胞系3T3来源的外泌小体(exosome)对小鼠乳腺癌细胞4T1增殖能力的影响,并探索其中可能的机制。方法 Pure Exo Exosome提取试剂盒提取3T3细胞上清液中的exosome,按照不同浓度及时间作用于4T1细胞,CCK8法检测4T1细胞的增殖能力,Brd U/PI双掺入法测定细胞DNA合成及细胞周期;免疫印迹法(Western blot)及荧光定量实时PCR(q PCR)检测人表皮生长因子受体2(epidermal growth factor receptor-2,EGFR2,也称HER2)及下游PI3K/AKT信号转导通路相关蛋白的变化。利用HER2单克隆抗体靶向药物赫赛汀(Herceptin),观察exosome是否影响4T1细胞对于Herceptin敏感度。结果 exosome处理组OD450吸光度值显著高于对照组(P<0.05),细胞增殖及细胞周期进程加快。Western blot及q PCR实验提示随着exosome浓度的增加,HER2表达逐渐升高,AKT磷酸化水平增加。而同时给予exosome可明显增加4T1细胞对Herceptin的敏感度。结论小鼠成纤维细胞系3T3来源exosome可促进小鼠乳腺癌细胞4T1增殖及周期进程,并且可能通过HER2激活其下游PI3K/AKT信号通路发挥上述作用。

Objective To investigate the effect of exosome secreted by mouse fi broblast cell line 3T3 on the proliferation of mouse breast cancer cells 4T1,and to explore the potential underlying mechanism.Methods The exosome was obtained and purifi ed by Pure Exo Exosome kit from mouse fi broblast cell line 3T3 cultural supernatant,and 4T1 cells were cultured and incubated with different doses of exosome for indicated time.Then CCK8 assay and Brd U/PI incorporation were performed to measure the proliferation and cell cycle of 4T1 cells.Western blot and q PCR were used to detect and analyze the expression of human epidermal growth factor receptor-2（HER2） and the activity of its downstream PI3K/AKT signal pathway.The monoclonal antibody of HER2,Herceptin,was used to determine whether exosome regulated the proliferation of 4T1 via HER2.Results The CCK8 assay showed at OD450 nm,the absorbency of exosome group was obvious higher than that in control group（P〈0.05）.The 4T1 cells treated by exosome also had faster proliferation and more aggressive cell cycle compared with untreated cells in control group.Meantime,exosome also increased the expression of HER2 determined by Western blot and q PCR,with elevating the expression of p-AKT.Meanwhile,exosome enhanced the sensitivity of 4T1 cells to Herceptin treatment.Conclusion The exosome extracted from mouse fi broblast cell line 3T3 could promote the proliferation and cell cycle progress of mouse breast cancer cells 4T1,and HER2 and its downstream PI3K/AKT signal pathway might play an important role in this regulation.