PDGF-BB通过外泌体促进人骨肉瘤的血管新生

PDGF-BB promotes angiogenesis of human osteosarcoma through exosomes

目的:探索PDGF-BB是否可以通过外泌体传递,并且验证其在人骨肉瘤中的促血管新生的功能。方法:分离多种人骨肉瘤细胞中的外泌体,WB法检测PDGF-BB在人骨肉瘤细胞及其外泌体中的表达,将骨肉瘤SJSA-1细胞来源的外泌体与HUVEC共孵育,免疫荧光和激光共聚焦扫描显微镜观察外泌体PDGF-BB进入细胞的方式;慢病毒感染法分别构建PDGF-BB过表达或敲低的SJSA-1细胞系,将其分离出的外泌体与HUVEC共孵育,微管形成实验检测其对血管新生的影响;构建SJSA-1细胞裸鼠移植瘤模型,回输过表达或敲低PDGF-BB的SJSA-1细胞来源的外泌体,观察其对肿瘤生长的影响。结果:成功分离出骨肉瘤细胞外泌体,并证实其携带大量的PDGF-BB,可通过细胞内吞进入HUVEC。成功构建PDGF-BB过表达或敲低的SJSA-1细胞系,并分离出携带相应水平PDGF-BB的外泌体,与对照组相比,PDGF-BB含量高的外泌体可以显著促进HUVEC的血管生成(P<0.01,t=13.51)和移植瘤生长(P<0.01),而PDGF-BB含量低的外泌体促血管生成能力减弱(P<0.01,t=8.226)并抑制移植瘤的生长(P<0.01)。结论:骨肉瘤细胞分泌的外泌体PDGF-BB可以直接被HUVEC摄取,诱导肿瘤血管生成,从而促进骨肉瘤生长。

Objective:To explore whether PDGF-BB can be transmitted through exosome and verify its angiogenic function in human osteosarcoma.Methods:Exosomes from a variety of human osteosarcoma cells were isolated.The expression of PDGF-BB in cells and exosomes was detected by WB.Exosomes derived from osteosarcoma SJSA-1 cells were co-incubated with HUVEC,and the pattern of exosomal PDGF-BB entering HUVEC was observed using Immunofluorescence and confocal scanning microscope.SJSA-1 cell lines with PDGF-BB over-expression or knockdown were constructed by lentiviral infection,and the exosomes derived from transfected SJSA-1 cells were isolated and incubated with HUVEC.Microtubule formation experiment was conducted to detect their effects on angiogenesis;SJSA-1 cell transplanted xenograft model was established in nude mice,and the exosomes derived from SJSA-1 cells with PDGF-BB over-expression or knockdown were infused into nude mice to observe their effects on tumor growth.Results:The exosomes derived from osteosarcoma cells were successfully isolated,in which a large amount of PDGF-BB was confirmed.The exosomes entered HUVEC by endocytosis.The SJSA-1 cell lines with PDGF-BB over-expression or knockdown were successfully constructed,and the corresponding exosomes were isolated.Compared with the control group,exosomes with high PDGF-BB content significantly promoted HUVEC angiogenesis(P<0.01,t=13.51)and tumor growth(P<0.01),while exosomes with low PDGF-BB content reduced the angiogenesis ability of HUVEC(P<0.01,t=8.226)and inhibited tumor growth(P<0.01).Conclusion:The exosomal PDGF-BB secreted by osteosarcoma cells can be directly absorbed by HUVEC and induce tumor angiogenesis,further promoting the growth of osteosarcoma.