裸鼹鼠皮肤成纤维细胞外泌体的分离及鉴定

Isolation and Identification of Exosomes from Skin Fibroblasts of Naked Mole Rats

目的分离培养裸鼹鼠皮肤成纤维细胞并对其进行鉴定,探讨裸鼹鼠皮肤成纤维细胞是否具有分泌外泌体的能力。方法无菌取新生裸鼹鼠背部皮肤,剪碎成1~2 mm2大小,将组织块平铺于细胞培养皿中,待贴壁后以低糖DMEM(含20%胎牛血清)培养液进行培养,胰酶消化法对传代培养细胞进行纯化;通过细胞免疫荧光、蛋白质印迹法检测波形蛋白表达情况,对成纤维细胞进行鉴定;待细胞生长至融合度约80%时,改换低糖DMEM(含20%去除外泌体的胎牛血清)培养48 h后收集上清液,超高速离心法分离外泌体,透射电镜观察形态,纳米流式分析颗粒直径,蛋白质印迹法检测外泌体相关特异性蛋白表达情况,进行外泌体鉴定。结果新生裸鼹鼠背部皮肤组织块培养30 h后可见少量细胞自组织块游离出,培养5 d后可见大量细胞自组织块游离生长,细胞呈细长梭形或不规则多角形,放射状生长。细胞免疫荧光及蛋白质印迹法检测均证实分离细胞表达波形蛋白。超高速离心法离心细胞上清液,透射电镜下可观察到膜状杯托样外泌体,纳米流式分析粒径在46.75~206.75 nm,蛋白质印迹法检测显示其对外泌体特异性蛋白CD63、肿瘤易感基因101(tumor susceptibility gene,TSG101)和热休克蛋白70(heat shock protein 70,Hsp70)具有特异性。结论分离培养的细胞具有成纤维细胞的形态学特征,并表达成纤维细胞的特征分子波形蛋白。裸鼹鼠皮肤成纤维细胞具有分泌外泌体的能力,为研究裸鼹鼠外泌体所携带的生物活性分子及其生物学功能提供了基础。

Objective To identify skin fibroblasts isolated and cultured from naked mole rats, and explore whether the skin fibroblasts have the ability to secrete exosomes. Methods The back skin samples of germ-free newborn naked mole rats were collected and were cut into pieces to a size of 1-2 mm2. The samples were spread evenly over the plate, cultured in low-sugar DMEM(containing20% fetal bovine serum) after cell adhesion, and treated by trypsin digestion for purification. The vimentin expressions were detected by immunofluorescence and Western blotting. When the cells were grown to about 80% confluency, they were moved to low-sugar DMEM(containing 20% fetal bovine serum without exosomes) and cultured for 48 h. Next the exosomes in supernatant were seperated by ultra-high-speed centrifuge, and determined vesicular structures under transmission electron microscope, particle size by nano flow cytometry, and specific proteins related to exosomes by Western blotting, respectively. Results A small amount of cells were seen free from the tissues after culturing for 30 h, and a large number of cells after 5 d. The cells were elongated spindle or irregular polygonal, growing radially. Immunofluorescence and Western blotting analysis confirmed that the skin fibroblasts expressed vimentin. Saucer-like membranous exosomes were observed under transmission electron microscopy, with particles size between 46.75-206.75 nm. Western blotting showed that the proteins specific to exosomes included the lysosmal membrane associated glycoprotein 3(CD63), tumor susceptibility gene 101(TSG101) and heat shock protein 70(Hsp70).Conclusion The cells isolated from naked mole rats have typical fibroblast morphology and express actually vimentin. The skin fibroblasts are successfully isolated from the naked mole rats, and can secrete the exosomes, which provides a basis for studying the bioactive molecules carried by naked mole rat exosomes and their biological functions.