黑色素瘤细胞外泌体通过Prospero同源异形盒蛋白1调控肿瘤淋巴转移机制的研究  

Study on the mechanism of melanoma cell exosomes regulating tumor lymphatic metastasis through Prox-1

作　　者：曹宸 杨莹[3] 孙秋悦 严志新[1] 杨细虎[2] CAO Chen;YANG Ying;SUN Qiuyue;YAN Zhixin;YANG Xihu(Department of Burn Surgery,Affiliated Hospital of Jiangsu University,Zhenjiang,Jiangsu 212001,China;Department of Oral and Maxillofacial,Affiliated Hospital of Jiangsu University,Zhenjiang,Jiangsu 212001,China;School of Pharmacy,Changzhou University,Changzhou,Jiangsu 213164,China)

机构地区：[1]江苏大学附属医院烧伤整形科,江苏镇江212001 [2]江苏大学附属医院口腔颌面科,江苏镇江212001 [3]常州大学药学院,江苏常州213164

出　　处：《安徽医药》2023年第7期1331-1337,I0002,共8页Anhui Medical and Pharmaceutical Journal

基　　金：国家自然科学基金项目(81641174);江苏省卫生厅,江苏省青年医学人才项目(QNRC2016841);江苏大学附属医院博士人才启动基金项目(jdfyRC2020001)。

摘　　要：目的探讨黑色素瘤外泌体(melanoma exosomes,MM-ex)携带的果蝇Prospero同源异形盒蛋白1(prospero homeobox protein 1,Prox-1),在调控淋巴管内皮细胞(lymphatic endothelial cells,LECs)增殖、迁移以及生成具有转移肿瘤细胞功能淋巴管过程中的作用。方法采用慢病毒转染实验构建低表达Prox-1黑色素瘤细胞株(MM^(shProx-1))。采用试剂盒法获取MM和MM-ex^(shProx-1)的外泌体(MM-ex和MM-ex^(shProx-1))。采用免疫荧光检测、蛋白质印迹法检测、细胞增殖及细胞迁移实验,观察MM-ex及MMex^(shProx-1)对LECs生物学特性的影响。采用免疫组化(IHC)法检测2015年9月至2021年4月江苏大学附属医院收治的32例皮肤黑色素瘤病理标本中Prox-1及淋巴管内皮细胞透明质酸受体-1(lymphatic vessel endothelial hyaluronic acid receptor-1,LYVE-1)的表达情况,统计微淋巴管密度(microlymphatic vessel density,MLVD),并分析Prox-1及MLVD与黑色素瘤生物学特性的相关性。结果细胞实验:MM-ex促进淋巴管内皮细胞增殖及迁移,促进了淋巴管的成形。MM-ex^(shProx-1)对LECs无增殖及迁移的影响,对淋巴管成形无促进作用。临床研究:免疫组化的结果表明Prox-1、LYVE-1在黑色素瘤组织及肿瘤周围组织中的阳性表达率显著高于正常皮肤组织。Prox-1信号在黑色素瘤淋巴结转移组和Ⅲ期及Ⅳ期肿瘤组的表达率[87.50%(14/16)、79.17%(19/24)]明显高于无淋巴结转移组和Ⅰ期及Ⅱ期肿瘤组[37.50%(6/16)、12.50%(1/8)](均P<0.05)。LYVE-1在黑色素瘤转移组和Ⅲ期及Ⅳ期肿瘤组的表达(9.30±4.65、5.15±1.72)明显高于无淋巴结转移组和Ⅰ期及Ⅱ期肿瘤组(5.66±1.69、5.30±1.65,均P<0.05)。Prox-1、LYVE-1的表达情况均与病人的年龄、性别无关(均P>0.05)。结论MM-ex通过Prox-1促进LECs的增殖及迁移,在肿瘤淋巴管再生中起到重要作用。Prox-1、LYVE-1及MLVD与黑色素瘤淋巴转移及预后呈明显相关性,为皮肤黑色素瘤的早期淋巴转移�Objective To investigate the role of Prospero homeobox protein1(Prox-1)carried by melanoma exosomes(MM-ex)in regulating the proliferation,migration and formation of lymphatic endothelial cells(LECs).Methods The melanoma cell line with low expression of Prox-1(MM^(shProx-1))was constructed by lentivirus transfection experiment.The exosomes of MM and MM-ex^(shProx-1)(MM-ex and MM-ex^(shProx-1))were obtained by kit method.The effects of MM-ex and MM-ex^(shProx-1) on the biological characteristics of LECs were observed by immunofluorescence,Western blot,cell proliferation and cell migration.Immunohistochemical(IHC)method was used to detect the expression of Prox-1 and LYVE-1 in the pathological specimens of 32 patients with cutaneous melanoma treated in the Affiliated Hospital of Jiangsu University from September 2015 to April 2021,the microlymphatic vessel density(MLVD)was counted,and the correlation between Prox-1 and MLVD and the biological characteristics of melanoma was analyzed.Results Cell experiment:MM-ex promoted the proliferation and migration of lymphatic endothelial cells and the formation of lymphatic vessels.MM-ex^(shProx-1) had no effect on the proliferation and migration of LECs,and had no effect on lymphangioplasty.Clinical study:the expression rate of Prox-1 signal in melanoma lymph node metastasis group and advanced tumor[87.50%(14/16),79.17%(19/24)]was significantly higher than that in non-lymph node metastasis group and early tumor group[37.50%(6/16),12.50%(1/8),P<0.05].The expression of LYVE-1 in melanoma metastasis group and advanced cancer group(9.30±4.65,5.15±1.72,P<0.05)was significantly higher than that in non-lymph node metastasis group and early cancer group(5.66±1.69,5.30±1.65,P<0.05).The expressions of Prox-1 and LYVE-1 were not related to the age and gender of patients(P>0.05).Conclusions MM-ex promotes the proliferation and migration of LECs through Prox-1 and plays an important role in tumor lymphatic regeneration.Prox-1,LYVE-1 and MLVD are significantly correlated with the lymph

关 键 词：黑色素瘤 外泌体 Prospero同源异形盒蛋白1 微淋巴管密度 淋巴管内皮细胞透明质酸受体-1 淋巴转移 

分 类 号：R739.5[医药卫生—肿瘤]