内皮祖细胞外泌体抑制紫杉醇诱导的血管内皮细胞焦亡

目的：探索内皮祖细胞外泌体（EPCs-Exo）对紫杉醇诱导的内皮细胞焦亡的影响及其可能机制。方法：培养原代骨髓间充质干细胞（BMSCs）和内皮祖细胞（EPCs）并鉴定。提取BMSCs和EPCs培养液中的外泌体，使用透射电镜观察外泌体形态并检测CD63和CD9的表达鉴定外泌体。分别用BMSCs-Exo和EPCs-Exo干预经紫杉醇诱导的内皮细胞焦亡模型，光镜下观察细胞形态；Western blot检测外泌体标志蛋白CD63、CD9和细胞焦亡相关蛋白NLRP-3、IL-18、GSDMD-N的表达情况；电镜下观察细胞超微结构的变化。基因芯片检测两组外泌体中差异的非编码RNA，RT-qPCR检测lncRNA FGD5-AS1表达情况。结果：大鼠骨髓细胞中分离培养分化出BMSCs和EPCs，免疫荧光法证实BMSCs中CD90 和CD44阳性，EPCs中CD34 和VEGFR2阳性。相比于对照组，紫杉醇组细胞膜被破坏，细胞内线粒体间距离增加，细胞内纤维排列紊乱，细胞中NLRP-3、IL-18、GSDMD-N等细胞焦亡蛋白表达增加（P ＜0.05）；相比于紫杉醇组，紫杉醇+EPCs-Exo组内皮细胞中细胞膜较完整，细胞系损伤较轻，NLRP-3、IL-18、GSDMD-N表达减少（P ＜0.05）；紫杉醇组与紫杉醇+BMSCs-Exo组内皮细胞形态和焦亡蛋白的表达差异无统计学意义。芯片及RT-PCR结果显示相比于BMSCs-Exo，lncRNA FGD5-AS1在EPCs-Exo中表达增加（P ＜0.05）。结论：EPCs外泌体抑制紫杉醇诱导的血管内皮细胞焦亡。

EPCs-Exo inhibit paclitaxel-induced pyrolysis of vascular endothelial cells

Objective: To explore the effect of endothelial progenitor cell exosomes (EPCs-Exo) on paclitaxel-induced endothelial cell pyrolysis and its possible mechanism. Methods: The primary bone marrow mesenchymal stem cells (BMSCs) and ECPs were cultured and identified. The exosomes in the cultured medium of BMSCs and ECPs were extracted, and the morphology of the exosomes was observed by transmission electron microscope. The expression of CD63 and CD9 was detected by western blot to identify the exosomes. BMSCs-Exo and EPCs-Exo were used to intervene the endothelial cell pyrolysis model induced by paclitaxel, and the cell morphology was observed by microscope; Western blot was used to detect the exosomal marker proteins CD63,CD9 and pyrolysis-related proteins NLRP-3, IL-18, GSDMD-N. The ultrastructure change of the cells were observed under electron microscope. RNA sequencing was used to detect the difference of non-coding RNA in the two groups of exosomes, and qRT-PCR was performed to detect the expression of LncRNA FGD5-AS1 in each group. Results: BMSCs and EPCs were isolated, cultured and differentiated from rat bone marrow cells. The positiveness of CD90, CD44 in BMSCs and CD34, VEGFR2 in EPCs were confirmed by immunofluorescence.Compared with the control group, the cell membrane of the paclitaxel group was destroyed, the distance between mitochondria in the cell increased, the arrangement of intracellular fibers was disordered, and the expression of pyrooptosis proteins such as NLRP-3, IL-18, GSDMD-N was increased (P<0.05). Compared with the paclitaxel group, the cell membrane of the paclitaxel+EPCs-Exo group was more complete, the cell line damage was mild, and the expression of NLRP-3, IL-18, and GSDMD-N was reduced (P<0.05). The microarray and RT-PCR results showed that compared with BMSCs-Exo, the expression of LncRNA FGD5-AS1 in EPCs-Exo increased (P<0.05). Conclusion: EPCs-Exo inhibited paclitaxel-induced pyrolysis of vascular endothelial cells.