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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: To investigate the pathway of extracellular vesicle extraction by ultracentrifugation and to explore the communication pathway between C17.2 extracellular vesicles of mouse neural stem cells and 3T3-fGFP cells of mouse embryonic fibroblasts. Methods Exosomes (EX) were obtained by ultracentrifugation and Total Exosome Isolation Kit respectively. Morphology was compared between the two samples by transmission electron microscopy and atomic force microscopy. SDS-PAGE and Western blot were compared biochemically for the two samples, respectively; extracellular vesicles were obtained by dye-labeled ultracentrifugation; the labeled extracellular vesicles were co-incubated with 3T3-fGFP cells for extracellular vesicle uptake by 3T3-fGFP cells The main pathway of uptake of extracellular extracellular vesicles by 3T3-fGFP cells was screened through pharmacological experiments. Results C17.2 cells grew well with no obvious morphological differentiation. Under TEM, vesicles with circular bilayer membrane structure and “cup-shaped” shape were observed in both samples. The diameter distribution was mostly concentrated within 300 nm, and the average diameter of extracellular vesicles was found to be larger than that of exosomes . Atomic force microscopy results showed that the height distribution of the two samples were mostly concentrated within 2 ~ 20 nm, the diameter distribution was mostly concentrated within 300 nm, and the average height and average diameter of the exosomes were larger than that of the extracellular vesicles. SDS-PAGE results showed that the protein content of the two samples, expression abundance close. The expression intensity of HSP70 was similar in the same amount of sample, but the expression of CD63, CD9 and CD81 were stronger in extracellular vesicles. Ingestion experiments showed that extracellular vesicles of C17.2 cells could be internalized by 3T3-fGFP cells, encapsulated in vesicles, transported to the area around the nucleus, and the uptake increased with time (P <0.05). Clathrin-mediated endocytosis was the main pathway for uptake of extracellular vesicles of C17.2 cells by 3T3-f GFP cells through pharmacological experiments. Conclusion The exosomes of mouse neural stem cells may participate in the communication between neural stem cells and 3T3-fGFP cells mainly through clathrin-mediated pathway.