摘要
背景:外泌体可以向周围环境释放RNA、蛋白质等信号分子,影响肿瘤发展,而血管生成素2在口腔鳞癌组织中表达显著升高且血管生成素2过表达与肿瘤的淋巴管生成、微血管密度增加及患者不良预后等密切相关,但口腔鳞状细胞癌细胞来源外泌体是否含有血管生成素2并影响肿瘤发展及血管生成尚不清楚。目的:探讨口腔鳞状细胞癌细胞来源外泌体调控血管生成的潜在机制。方法:①通过CCK-8、Transwell实验观察Cal-27、Scc-25细胞培养上清对人脐静脉内皮细胞增殖、迁移的影响;②采用外泌体提取试剂盒提取Cal-27、Scc-25细胞培养上清中的外泌体并鉴定;③免疫荧光实验检测人脐静脉内皮细胞是否可以摄取Cal-27、Scc-25外泌体;④Western blot检测Cal-27、Scc-25细胞及外泌体中血管生成素2的表达;⑤用不同质量浓度(25,50μg/mL)的Cal-27、Scc-25细胞外泌体干预人脐静脉内皮细胞,通过Western blot、CCK-8、Transwell、成管实验检测人脐静脉内皮细胞中血管生成素2及CD34的表达以及对人脐静脉内皮细胞增殖、迁移、成管的影响;⑥慢病毒转染构建过表达、敲低血管生成素2的Cal-27、Scc-25细胞及外泌体,qRT-PCR和Western blot检测转染效率,Western blot、CCK-8、Transwell、成管实验检测过表达、敲低Ang-2的Cal-27、Scc-25外泌体对人脐静脉内皮细胞的调控作用;⑦5周龄BALB/cA雌性小鼠20只,随机分为4组:对照组、Cal-27外泌体组、过表达血管生成素2的Cal-27外泌体组、敲低血管生成素2的Cal-27外泌体组,每组5只。小鼠皮下荷瘤Cal-27细胞,待肿瘤体积长至50 mm^(3),荷瘤后第10天时,按分组瘤周注射外泌体,对照组注射等量的PBS,2 d/次,第30天收取肿瘤组织进行苏木精-伊红染色、Ki67和CD31免疫组化染色。结果与结论:①与对照组相比,Cal-27、Scc-25细胞培养上清促进了人脐静脉内皮细胞增殖、迁移(P<0.05);②人脐静脉内皮细胞可以摄取Cal-27、Scc-25外泌体;③Cal-27、Scc-25细胞及外泌体中均含有血管生成素2,随着外泌体浓度增加,人脐静脉内皮细胞中血管生成素2及CD34蛋白表达水平增高,人脐静脉内皮细胞增殖、迁移和管形成增强(P<0.05);④过表达血管生成素2的Cal-27、Scc-25外泌体进一步促进了人脐静脉内皮细胞的增殖、迁移和管形成(P<0.05);敲低血管生成素2的Cal-27、Scc-25外泌体抑制了人脐静脉内皮细胞的增殖、迁移和管形成(P<0.05);⑤动物实验显示:与对照组相比,Cal-27外泌体组瘤体积增大不明显,但Ki67和CD31表达水平升高;过表达血管生成素2的Cal-27外泌体组瘤体积显著增大,Ki67和CD31表达水平明显升高;敲低血管生成素2的Cal-27外泌体组瘤体积和Ki67、CD31表达水平均降低(P<0.05)。结果表明:口腔鳞状细胞癌细胞来源外泌体通过递送血管生成素2参与肿瘤血管生成,影响肿瘤发展。
BACKGROUND:Exosomes can release RNA,proteins and other signaling molecules to the surrounding environment to influence tumor development.Angiopoietin-2 expression is significantly increased in oral squamous carcinoma tissues and angiopoietin-2 overexpression is closely associated with tumor lymphangiogenesis,increased microvessel density,and poor prognosis of the patients.However,it is not clear whether exosomes of oral squamous cell carcinoma origin contain angiopoietin-2 and influence tumor development and angiogenesis.OBJECTIVE:To investigate the potential mechanism of exosomes regulating angiogenesis in oral squamous cell carcinoma.METHODS:(1)The effects of Cal-27 and Scc-25 cell supernatants on the proliferation and migration of human umbilical vein endothelial cells were observed by CCK8 and Transwell assays.(2)The exosomes in the supernatants of Cal-27 and Scc-25 cells were extracted and identified using an exosome extraction kit.(3)Immunofluorescence assay was used to detect whether human umbilical vein endothelial cells could take up Cal-27 and Scc-25 exosomes.(4)Western blot assay was used to detect the expression of angiopoietin-2 in Cal-27 and Scc-25 cells and exosomes.(5)Cal-27 and Scc-25 cell exosomes at different mass concentrations(25 and 50μg/mL)were used to intervene in human umbilical vein endothelial cells.Western blot assay,CCK-8 assay,Transwell,and tube formation experiments were used to detect the expression of angiopoietin-2 and CD34 in human umbilical vein endothelial cells and their effects on the proliferation,migration,and tube formation of human umbilical vein endothelial cells.(6)Lentivirus transfection was used to construct Cal-27 and Scc-25 cells and exosomes with overexpression and knockdown of angiopoietin-2.qRT-PCR and western blot assay were used to detect the transfection efficiency.Western blot assay,CCK-8 assay,Transwell,and tube formation experiments were used to detect the regulatory effects of Cal-27 and Scc-25 exosomes with overexpression and knockdown of angiopoietin-2 on human umbilical vein endothelial cells.(7)Twenty 5-week-old BALB/cA female mice were randomly divided into four groups:control group,Cal-27 exosome group,Cal-27 exosome group with angiopoietin-2 overexpression,and Cal-27 exosome group with angiopoietin-2 knockdown,with five mice in each group.Mice were subcutaneously implanted with Cal-27 cells.When the tumor volume grew to 50 mm^(3),exosomes were injected peritumorally on day 10 after tumor implantation according to the group assignment.The control group was injected with an equal amount of PBS every 2 days.On day 30,tumor tissues were collected for hematoxylin-eosin staining,Ki67 and CD31 immunohistochemical staining.RESULTS AND CONCLUSION:(1)Compared with the control group,the supernatants of Cal-27 and Scc-25 cells promoted the proliferation and migration of human umbilical vein endothelial cells(P<0.05).(2)Human umbilical vein endothelial cells could take up Cal-27 and Scc-25 exosomes.(3)Angiopoietin-2 was contained in Cal-27 and Scc-25 cells and exosomes.With the increase of exosome concentration,the expression levels of angiopoietin-2 and CD34 proteins in human umbilical vein endothelial cells increased,and the proliferation,migration,and tube formation of human umbilical vein endothelial cells were enhanced(P<0.05).(4)Cal-27 and Scc-25 exosomes overexpressing angiopoietin-2 further promoted the proliferation,migration,and tube formation of human umbilical vein endothelial cells(P<0.05).Cal-27 and Scc-25 exosomes with knockdown of angiopoietin-2 inhibited the proliferation,migration,and tube formation of human umbilical vein endothelial cells(P<0.05).(5)Animal experiments showed that compared with the control group,the tumor volume of the Cal-27 exosome group did not increase significantly,but the expression levels of Ki67 and CD31 increased.The tumor volume of the Cal-27 exosome group with angiopoietin-2 overexpression increased significantly,and the expression levels of Ki67 and CD31 increased significantly.The tumor volume and the expression levels of Ki67 and CD31 in the Cal-27 exosome group with angiopoietin-2 knockdown decreased(P<0.05).These results indicate that oral squamous cell carcinoma-derived exosomes participate in tumor angiogenesis by delivering angiopoietin-2 and affect tumor development.
作者
韩腾
马洪
杨若仪
罗祎
李超
Han Teng;Ma Hong;Yang Ruoyi;Luo Yi;Li Chao(Department of Oral and Maxillofacial Surgery,School of Stomatology,Guizhou Medical University,and Affiliated Stomatological Hospital of Guizhou Medical University,Guiyang 550001,Guizhou Province,China;School of Clinical Medicine,Chengdu University of Traditional Chinese Medicine,Chengdu 610075,Sichuan Province,China;Department of Thyroid-Oral and Maxillofacial Head and Neck Surgery,Sichuan Cancer Clinical Medical Research Center,Sichuan Cancer Hospital·Research Institute,Sichuan Cancer Prevention and Control Center,Tumor Hospital Affiliated to University of Electronic Science and Technology of China,Chengdu 610041,Sichuan Province,China)
出处
《中国组织工程研究》
北大核心
2026年第7期1755-1767,共13页
Chinese Journal of Tissue Engineering Research
基金
四川省自然科学基金项目(2023NSFSC1501,2024ZYD0051,2024YFHZ0215),项目负责人:李超
口腔疾病防治全国重点实验室开放课题(SKLOD2024OF02),项目负责人:李超。
