PDGF-BB通过外泌体促进人骨肉瘤的血管新生

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。

PDGF-BB promotes angiogenesis of human osteosarcoma through exosomes

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.