南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (08): 1065-1071.doi: 10.12122/j.issn.1673-4254.2020.08.01

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肝素酶通过诱导血管内皮细胞凋亡促进肝癌细胞跨内皮迁移

陈晓鹏，叶 蕊，戴大飞，伍玉海，俞远林，程 斌   

  • 出版日期:2020-08-20 发布日期:2020-08-20
  • 基金资助:

Heparanase promotes trans-endothelial migration of hepatocarcinoma cells by inducing apoptosis of microvascular endothelial cells

  

  • Online:2020-08-20 Published:2020-08-20

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摘要： 目的 探索肝癌肝素酶（HPSE）对微血管内皮细胞（MVECs）凋亡及跨细胞迁移的影响。方法 用qRT-PCR和Western blot法从HepG2，BEL-7402和HCCLM3三种肝癌细胞中筛选高表达HPSE的肝癌细胞；构建含有HPSE之RNA干扰序列的慢病毒载体（LV-HPSE -RNAi-1249-2, RNAi组），用含有阴性对照序列的慢病毒载体（LV-HPSE-Control, Control组）作为对照，分别感染肝癌细胞，并验证感染效率。用人脐静脉血管内皮细胞（HUVECs）代表肝癌MVECs，用Transwell小室行肝癌细胞跨内皮迁移试验；肝癌细胞和HUVECs细胞非接触共培养24 h，应用CCK-8法检测内皮细胞存活率；Annexin V-FITC/PI双染法流式细胞仪检测细胞凋亡情况，电镜观察细胞形态。肝癌细胞腹腔注射法制作裸鼠肝癌模型，观察肝癌MVECs凋亡及癌细胞跨内皮成瘤情况。结果 从3种肝癌细胞中筛选出高表达HPSE的HCCLM3肝癌细胞。慢病毒载体分别感染HCCLM3细胞72 h后，荧光显微镜显示细胞感染效率达到70%以上，qRT-PCR和Western blot 法检测RNAi组肝素酶 mRNA和蛋白表达下降（P<0.05）。RNAi组肝癌细胞跨内皮迁移率为0.39，显著低于Control组的0.62（P<0.01）。HCCLM3细胞与HUVECs非接触共培养，RNAi 组内皮细胞成活率为1.14，显著高于Control组的0.77（P<0.05）；RNAi组凋亡指数3.94±1.51明显低于Control组（12.53±0.55） （P<0.05）。透射电镜示RNAi组细胞形态大致正常；Control组内皮细胞体积变小、变形，胞膜有发泡现象；染色质浓缩、边缘化，部分分割成块状，并见凋亡小体。实验裸鼠全部成活，Control组6只肝组织中均出现GFP标记的肿瘤细胞，镜下成瘤率100% （6/6），显著高于RNAi组成瘤率（33.3%，2/6）（P<0.05）。光镜下Control组肝组织内可见肝癌细胞，部分血管内皮细胞坏死，胞浆可见空泡，核质浓缩；RNAi组肝组织内罕见肝癌细胞，内皮细胞基本正常。结论 肝癌肝素酶可能通过诱导血管内皮细胞凋亡而促进癌细胞转移。

Abstract: Objective To explore the effect of heparanase (HPSE) on apoptosis of microvascular endothelial cells (MVECs) and trans-endothelial migration of hepatocellular carcinoma (HCC) cells. Methods A HCC cell line with high HPSE expression was selected by real-time quantitative PCR (qRT-PCR) and Western blotting and transefected with a lentiviral vector containing an interfering RNA sequence of HPSE. Transwell migration assay was performed to detect the trans-endothelial migration (TEM) rate of the transfected HCC cells across human umbilical vein endothelial cells (HUVECs). In a Transwell indirect co-culture system, the effect of HPSE silencing in the HCC cells was determined on apoptosis of HUVECs in vitro. A nude mouse model of HCC was used to verify the effect of HPSE on apoptosis of MVECs and liver metastasis of the tumor. Results HCCLM3 cell line highly expressing HPSE was selected for the experiment. Transfection of the HCC cells with the lentiviral vector for HPSE interference the HCC cells resulted in significantly lowered TEM rate as compared with the cells transfected with the control vector (P<0.01). In the indirect co-culture system, the survival rate of HUVECs co-cultured with HCCLM3 cells with HPSE interference was significantly higher and their apoptotic index was significantly lower than those in the control group (P<0.05). Ultrastructural observation showed no obvious apoptosis of HUVECs co-cultured with HCCLM3 cells with HPSE interference but revealed obvious apoptotic changes in the control group. In the animal experiment, the tumor formation rate in the liver was 100% (6/6) in the control group, significantly higher than that in RNAi group (33.3%, 2/6) (P< 0.05). Under optical microscope, necrosis and apoptosis of the MVECs was detected in the liver of the control mice, while the endothelial cells remained almost intact in RNAi group. Conclusion HPSE promotes the metastasis of HCC cells by inducing apoptosis of MVECs.