航天医学与医学工程

目的 探讨低氧条件下肝癌细胞Hep G2和Huh7来源的外泌体（EXO）中mi R-181b-5p对血管内皮细胞增殖、凋亡、迁移和成管能力的调控作用及分子机制。方法 用超速离心法分离肝癌细胞外泌体，分为常氧外泌体组（Hep G2-EXO组和Huh7-EXO组）和低氧外泌体组（Hyp-HepG2-EXO组和Hyp-Huh7-EXO组，以1%氧气浓度培养24 h进行低氧处理）。通过电镜观察外泌体形态，动态光散射法分析粒径分布，Western blot检测外泌体标志蛋白CD9、CD81、CD63的表达；RT-q PCR检测mi R-181b-5p的表达水平。将人脐静脉内皮细胞（HUVECs）分为Control组（无处理）、Hep G2-EXO组（常氧外泌体处理）、Hyp-Hep G2-EXO组（低氧外泌体处理）、Hyp-Hep G2-EXO+inhibitor-NC组（低氧外泌体加阴性对照抑制剂处理）、Hyp-HepG2-EXO+miR-181b-5p-inhibitor组（低氧外泌体加miR-181b-5p抑制剂处理），以同样的方式设置Huh7对应组别。采用CCK-8法检测HUVECs增殖率，流式细胞术检测HUVECs凋亡率，Transwell实验检测HUVECs迁移细胞数，小管形成实验检测HUVECs成管长度和分支点数，Western blot检测β-联蛋白、Twist1蛋白、基质金属蛋白酶-9(MMP-9)、血管生成素1(Ang-1)的表达。结果 Hep G2-EXO、Huh7-EXO、Hyp-Hep G2-EXO和Hyp-Huh7-EXO的粒径范围分别为60～170 nm、62～163 nm、52～小管形成183 nm和73～188 nm,Hep G2-EXO、Huh7-EXO、Hyp-Hep G2-EXO和Hyp-Huh7-EXO中CD9、CD81、CD63蛋白均呈阳性表达，而Hep G2、Huh7、Hyp-Hep G2、Hyp-Huh7细胞中CD9、CD81、CD63蛋白的表达明显低于各自对应的外泌体组（均P<0.05）。与Hep G2-EXO组相比，Hyp-Hep G2-EXO组mi R-181b-5p表达显著上调（P<0.05），与Huh7-EXO组相比，Hyp-Huh7-EXO组mi R-181b-5p表达同样上调（P<0.05）。与Control组相比，Hep G2-EXO组HUVECs增殖率、迁移细胞数、成管长度和分支点数均显著增加，β-联蛋白、Twist1、MMP-9、Ang-1的蛋白表达水平升高，细胞凋亡率显著降低（均P<0.05）；与Hep G2-EXO组比较，Hyp-Hep G2-EXO组HUVECs增殖率、迁移细胞数、成管长度和分支点数均分别增加，β-联蛋白、Twist1、MMP-9、Ang-1的蛋白表达水平升高，凋亡率降低（均P<0.05）；与Hyp-Hep G2-EXO组相比，Hyp-Hep G2-EXO+miR-181b-5p-inhibitor组HUVECs增殖率、迁移细胞数、成管长度和分支点数显著减少，β-联蛋白、Twist1、MMP-9、Ang-1的蛋白表达水平降低，凋亡率升高（均P<0.05）；与Hyp-HepG2-EXO+inhibitor-NC组相比，Hyp-HepG2-EXO+miR-181b-5p-inhibitor组HUVECs增殖率、迁移细胞数、成管长度和分支点数显著减少，β-联蛋白、Twist1、MMP-9、Ang-1的蛋白表达水平降低，凋亡率升高（均P<0.05）。Huh7各处理组的指标变化趋势与Hep G2组相同。结论 低氧环境上调肝癌细胞外泌体mi R-181b-5p的表达，mi R-181b-5p通过促进血管内皮细胞增殖、迁移、成管能力和抑制凋亡，促进肿瘤血管的生成，mi R-181b-5p抑制剂可逆转此效应，表明mi R-181b-5p是低氧驱动肿瘤血管生成的关键调控因子。

Objective To investigate the regulatory function and molecular mechanisms of microRNA-181b-5p(miR-181b-5p) derived from hepatocellular carcinoma(HepG2 and Huh7) cell exosomes(EXO) under hypoxic conditions on the proliferation, apoptosis, migration, and tube formation capabilities of vascular endothelial cells. Methods Exosomes from hepatocellular carcinoma cells under normoxia(HepG2-EXO and Huh7-EXO) and hypoxia(Hyp-HepG2-EXO and Hyp-Huh7-EXO) were isolated via ultracentrifugation. Hypoxic treatment was performed by culturing cells at 1% oxygen concentration for 24 h. Exosome morphology was observed via electron microscopy, particle size distribution was analyzed via dynamic light scattering, and exosomal marker proteins(CD9, CD81, CD63) were detected via Western blot. The expression level of miR-181b-5p was measured via quantitative real-time PCR(RT-qPCR). Human umbilical vein endothelial cells were divided into the control group(no treatment), HepG2-EXO group(normoxic exosome treatment), Hyp-HepG2-EXO group(hypoxic exosome treatment), Hyp-HepG2-EXO+inhibitor-NC group(hypoxic exosomes + negative control inhibitor treatment), and Hyp-HepG2-EXO+miR-181b-5p-inhibitor group(hypoxic exosomes + miR-181b-5p inhibitor treatment). Huh7 corresponding groups were set up similarly. Cell proliferation rates were assessed using the cell counting Kit-8(CCK-8) assay, apoptosis rates were detected via flow cytometry, migration was evaluated via Transwell assay, tube formation length and branch points were measured via Matrigel assay, and Western blot was used to detect the expression of β-catenin, Twist1, matrix metalloproteinase-9(MMP-9), and angiopoietin-1(Ang-1). Results The particle size ranges of HepG2-EXO, Huh7-EXO, Hyp-HepG2-EXO and Hyp-Huh7-EXO were 60–170 nm, 62–163 nm, 52–183 nm and 73–188 nm, respectively. CD9, CD81 and CD63 proteins were positively expressed in HepG2-EXO, Huh7-EXO, Hyp-HepG2-EXO and Hyp-Huh7-EXO, whereas their expression was lower in cellular fractions compared to exosomal fractions(all P<0.05). Compared to the HepG2-EXO group, miR-181b-5p expression was significantly upregulated in the Hyp-HepG2-EXO group(all P<0.05). Similarly, it was upregulated in Hyp-Huh7-EXO compared to Huh7-EXO(all P<0.05). Compared to the control group, the HepG2-EXO or Huh7-EXO groups showed significantly increased proliferation rates, migrated cell numbers, tube lengths, branch points, and the protein expression levels of β-catenin, Twist1, MMP-9 and Ang-1, along with decreased apoptosis rates(all P<0.05). Compared to HepG2-EXO or Huh7-EXO groups, the Hyp-HepG2-EXO or Hyp-Huh7-EXO groups showed further increases in proliferation rates, migration, tube formation, branching, and the protein expression levels of β-catenin, Twist1, MMP-9 and Ang-1, with reduced apoptosis(all P<0.05). In contrast, the Hyp-HepG2-EXO+miR-181b-5p-inhibitor or Hyp-Huh7-EXO+miR-181b-5p-inhibitor groups showed significantly decreased proliferation, migration, tube formation, branching, and the protein expression levels of β-catenin, Twist1, MMP-9 and Ang-1, along with increased apoptosis compared to Hyp-HepG2-EXO or Hyp-Huh7-EXO groups(all P<0.05). Similar changes were observed when comparing Hyp-HepG2-EXO+miR-181b-5p-inhibitor or Hyp-Huh7-EXO+miR-181b-5p-inhibitor groups to their respective inhibitor-negative control(NC) groups(all P<0.05). Conclusion Hypoxia upregulates exosomal miR-181b-5p expression in hepatocellular carcinoma cells. miR-181b-5p enhances tumor angiogenesis by promoting endothelial cell proliferation, migration and tube formation while inhibiting apoptosis. The miR-181b-5p inhibitor reverses this effect, indicating that miR-181b-5p is a key regulator of hypoxia-driven tumor angiogenesis.