小鼠黑色素瘤细胞外泌体促进成纤维细胞表达Rac1蛋白

Mouse melanoma cell exosomes promote expression of Rac1 protein in fibroblasts

目的:探讨小鼠黑色素瘤细胞外泌体对成纤维细胞表达Ras相关C3肉毒毒素底物1(Ras-related C3 botulinum toxin substrate 1,Rac1)蛋白的影响。方法:超高速离心法分离小鼠黑色素瘤B16-F10细胞外泌体,电镜负染色观察外泌体的形态,纳米颗粒跟踪分析(nanoparticle tracking analysis,NTA)测定外泌体的粒径分布,Western blot鉴定外泌体标志蛋白肿瘤易感基因101(tumor susceptibility gene 101,Tsg101)和酪氨酸酶相关蛋白2(tyrosinase-related protein 2,Tyrp2)的表达;激光共聚焦显微镜观察在共培养过程中小鼠胚胎成纤维细胞(mouse embryonic fibroblast,MEF)摄取外泌体的过程;免疫细胞化学染色和Western blot实验分析MEF表达Rac1蛋白的情况。结果:B16-F10细胞外泌体在电镜下呈典型茶托状形态,NTA测得其粒径范围在141~255 nm,Western blot测出其标志蛋白Tsg101和Tyrp2。激光共聚焦显微镜观察结果显示,与共培养0 h相比,共培养12 h的MEF内有少量的外泌体,且共培养24和36 h的MEF内聚集了大量的外泌体。Western blot结果表明,与共培养0 h相比,共培养24和36 h的MEF中Rac1蛋白表达水平显著增加(P<0. 01)。免疫细胞化学染色结果表明,与共培养0 h相比,共培养12、24和36 h的MEF中Rac1蛋白阳性表达水平显著增加(P<0. 05或P<0. 01)。结论:小鼠黑色素瘤细胞外泌体可促进成纤维细胞表达Rac1蛋白。

AIM:To investigate the effect of exosomes secreted by mouse melanoma cells on the expression of Ras-related C3 botulinum toxin substrate 1(Rac1)protein in fibroblasts.METHODS:Ultracentrifugation was adopted to separete exosomes secreted by mouse melanoma B16-F10 cells. The morphological structure of exosomes was observed by negative-staining electron microscopy. The size distribution of exosomes was determined by nanoparticle tracking analysis(NTA). The exosomal markers,tumor susceptibility gene 101(Tsg101)and tyrosinase-related protein 2(Tyrp2),were identified by Western blot. Laser confocal microscopy was used to observe the process that mouse embryonic fibroblasts(MEF)took in exosomes during co-culture. Immunocytochemical staining and Western blot were used to detect the expression of Rac1 protein in MEF.RESULTS:B16-F10 cell exosomes showed a typical tea tray-like structure,with a size range of 141~255 nm,and expressed protein markers Tsg101 and Tyrp2. The results of laser confocal microscopy showed that compared with co-culture at 0 h,a small number of exosomes appeared in the MEF at 12 h,and a large number of exosomes accumulated in the MEF after co-cultured for 24 and 36 h. Western blot analysis showed that compared with co-culture at 0 h,the expression of Rac1 protein in the MEF was significantly increased at 24 h and 36 h of co-culture(P<0. 01). The results of immunocytochemical staining showed that compared with co-culture at 0 h,the positive expression level of Rac1 in the MEF cells was significantly increased at 12 h,24 h and 36 h of co-culture(P<0. 05 or P<0. 01).CONCLUSION:Intake of exosomes secreted by mouse melanoma cells promotes the expression of Rac1 protein in fibroblasts.