3T3-fGFP细胞摄取神经干细胞胞外泌囊泡的途径

Communication channel of 3T3-fGFP cells by uptake of mouse neural progenitor cell derived exosomes

目的以超速离心方案提取胞外囊泡的鉴定为基础,初步探求小鼠神经干细胞C17.2胞外囊泡与小鼠胚胎成纤维细胞3T3-fGFP细胞之间的通讯交流通路。方法分别用超速离心法获得胞外囊泡（EV）和Total Exosome Isolation Kit试剂盒获得外泌体（EXO）;用透射电镜和原子力显微镜分别对两种样品进行形态学比较;用SDS-PAGE和Western blot分别对两种样品进行生物化学比较;用染料标记超速离心法获得胞外囊泡;将标记的胞外囊泡与3T3-fGFP细胞共孵育,对3T3-fGFP细胞摄取胞外囊泡进行研究;通过药理实验筛选3T3-fGFP细胞摄取胞外囊泡的主要通路。结果 C17.2细胞生长良好,形态规则未出现明显分化现象。在透射电镜下两种样品均观察到圆形双层膜结构和＂杯状＂外形的囊泡,直径分布多集中在300 nm以内,并且发现胞外囊泡的平均直径大于外泌体。原子力显微镜的结果显示,两种样品的高度分布多集中在2~20 nm以内,直径分布多集中在300 nm以内,并且外泌体的平均高度和平均直径均大于胞外囊泡。SDS-PAGE结果显示两种样品所含蛋白种类、表达丰度接近。在相同上样量下HSP70的表达强度两者接近,但CD63、CD9和CD81 3种蛋白在胞外囊泡中表达更强。摄取实验表明C17.2细胞胞外囊泡可以被3T3-fGFP细胞内化,被包裹在囊泡内,转运到细胞核周围的区域,并且摄取数量随着时间延长而增加（P〈0.05）。通过药理实验确定网格蛋白（Clathrin）介导的内吞通路为3T3-f GFP细胞摄取C17.2细胞胞外囊泡的主要通路。结论小鼠神经干细胞外泌体可能主要经网格蛋白介导的通路参与了神经干细胞与3T3-fGFP细胞的通讯。

Objective Based on the identification of extracellular vesicles（ EVs） isolated by ultracentrifugation,to investigate the communication channel and mechanism of mouse 3T3-fGFP fibroblast cells by uptake of mouse neural progenitor cells（ m NPCs） derived exosomes（ EXOs）. Methods EVs and EXOs from m NPC C17. 2 culture supernatants were purified by ultracentrifugation and total exosome isolation kit,and visualized with a transmission electron microscope（ TEM） and an atomic force microscope（ AFM）.Biochemical identification of the EVs and EXOs was performed by SDS-PAGE electrophoresis and Western blotting. EVs were labeled with dyes,and incubated with 3T3-fGFP cells to study the cellular uptake.Pharmacological experiments were carried out to determine the main pathway. Results C17. 2 cells did not show obvious differentiation. Under the TEM,membrane vesicle samples with bilayer membrane structure and cup shape were observed,and the diameter was about 300 nm. The average diameter of EV samples was greater than that of EXO samples. AFM showed that the height of the samples was more concentrated within10- 20 nm,and the lateral diameter was about 300 nm. Both the average height and the average diameter of the EXO samples were bigger than those of the EV samples. SDS-PAGE results showed that the 2 kinds of samples contained a variety of proteins,with similar expression intensity. Under condition of the same sample content,HSP70 expression intensity was close,but CD63,CD9 and CD81 expression was more abundant in the EV samples than in the EXO samples. EVs were internalized by 3T3-fGFP cells,trapped in the vesicles,and transported to perinuclear region,and the number was increased along with time（ P 〈 0. 05）. Clathrinmediated endocytosis was the main pathway for the vesicle uptake. Conclusion m NPC-derived EXOs involve in the communication between NPCs and 3T3-fGFP cells via clathrin-mediated endocytosis.