claudin-5通过调节血脑屏障通透性参与肿瘤脑转移过程的分子机制研究

Background. A key step in brain metastasis (BM) is the interaction and penetration of the Blood-brain barrier (BBB) by cancer cells. Tight junction (TJ) of brain vascular endothelium, part of the BBB, is critical barrier which the cancer cells have to overcome in order to penetrate and initiate brain metastasis. The transmembrane protein claudin-5 is a key TJ protein in endothelial cells. However, its the molecular mechanisms in regulation BBB in the formation of brain metastases are poorly understood . In the present study, we aimed to investigate the pattern of expression of this molecule in regulation the tight junction of brain vascular endothelial cell and try to identify the role of claudin-5 in the regulation of BBB permeability during the brain metastatic process. Materials and methods. The Claudin-5 gene was highly expressed in human brain vascular endothelial cell, hCMEC/D3 cell line and was virtually negative in the non-brain vascular endothelial cells, HECV. Claudin-5 was either knocked down using ribozyme technology in hCMEC/D3 or over-expressed in HECV cells . Lung cancer cell line A549/SKEMS1 was added to observe the interaction between the cancer cell and brain vascular endothelial cell. o Cells: human brain vascular endothelial cell, hCMEC/D3 cell line non-brain vascular endothelial cells, HECV. Lung cancer cell line A549 Lung cancer cell line SKEMS1 o Transfection with plasmids containing either the expression cassette or ribozyme to creat over-expression/knockdown of claudin-5 where applicable. o Successful transfection were measured using RT-PCR,Western Blotting and immunofluorescence. o Function assays: transendothelial electrical resistance (TEER) Paracellular permeability（PCP） o Cell invasion assay: seed hCMEC/D3 to Marigel layer-incubation- A549 /SKEMS1 cells were aliquoted into each insert -the invading cells were identified using fluorescent microscope o Cell growth assay: a crystal violet colorimetric assay o ECIS based cell migration and micromotion analyses o Cell migration were measured using an electric wounding assay o Micromotion were determined and calculated using the cell modelling of ECIS o Barrier functions were determined over a range of frequencies The present study portrays an interesting role for Claudin-5 in keeping the the barrier functions of the brain vascular endothelial cells and blocking cancer cells from transpassing. Claudin-5 through the integrity of blood brain barrier, reduces tumor cell penetration, which play an important role in controlling brain metastases.

脑转移瘤是最为致命的颅内肿瘤，随着生存时间的延长，脑转移瘤发生率逐渐增高。原发肿瘤转移进入颅内必须经过血脑屏障,因此研究调节血脑屏障通透性的分子机制非常重要。我们既往发现Claudin-5作为血管内皮细胞间紧密连接的关键蛋白在调节BBB通透性方面发挥重要作用。本研究首先对野生型人源脑血管内皮细胞系hCMEC/D3上claudin-5的表达水平进行测定，并通过生长、粘附、侵袭、细胞间电阻等细胞功能实验观察野生型hCMEC/D3的表型。再通过质粒转染建立稳定claudin-5低/高表达的细胞系，并重新通过细胞功能试验观察细胞表型的变化。随后用肺癌细胞系A549/SKEMS1侵袭由Marigel和hCMEC/D3共同建立的血脑屏障模型，观察不同Claudine5表达水平D3细胞系通透性的变化。最后研究CL-5过/低表达血管内皮细胞中相关信号分子的变化，确认CL-5影响血脑屏障通透性的信号通道。

项目背景：脑转移瘤是最为致命的颅内肿瘤。原发肿瘤转移进入颅内必须经过血脑屏障,因此研究调节血脑屏障通透性的分子机制非常重要。我们既往发现Claudin-5作为血管内皮细胞间紧密连接的关键蛋白在调节BBB通透性方面发挥重要作用。..主要研究内容：.1.过表达和敲降CLDN5表达水平的hCMEC/D3细胞模型的建立：①掌握本实验所需细胞系的特点；②进行CLDN5引物的设计及慢病毒的包装；③构建了CLDN5的干扰序列并获取了转染细胞；④构建了CLDN5过表达载体及成熟的稳转D3细胞系；⑤对CLDN5表达量进行了测定。.2.CLDN5对D3细胞增殖、迁移、细胞间电阻及血脑屏障通透性的影响：应用不同手段进行细胞功能实验；建立了单层BBB及抗肺癌细胞侵袭BBB模型。.3.CLDN5调节血脑屏障通透性和肺癌脑转移的作用机制研究：①通过对miRNA、mRNA、lncRNA进行微芯片和计算分析；②对过表达和对照组进行差异分析；③构建CLDN5的lncRNA-mRNA共表达网络；④对基因功能和通路分析。.4.对转移瘤患者的标本进行CLDN5表达测定，对临床预后进行随访。.重要结果：.1.本实验获得了一个CLDN5过表达插入位点、2个干扰位点。成功的进行了病毒包装并荧光检测且进行表达量验证。.2.CLDN5表达量与D3细胞的增殖、细胞间电阻及屏障功能呈正相关；D3细胞的迁移呈负相关。.3.对所有样本进行了分层聚类分析；CLDN-5诱导D3细胞中RNA表达模式发生显著改变；GO分析发现CLDN5参与通路及细胞功能；.4.获得了转移瘤及瘤周组织的CLDN5表达及临床资料。.关键数据：.微芯片检测发现其中1378个上调，307个下调；明确了41个miRNA、954个lncRNA和222个在CLDN5过表达组中具有显着差异表达的mRNA，构建了lncRNA-mRNA共表达网络；DEGs参与了83个显著功能，差异基因参与了48个富集相关通路；确定了8种基因功能，7个信号通路。在相互作用网络中的10个mRNA的GO分析和Pathway分析来预测作为ceRNA的27个lncRNA的生物学作用。.科学意义：通过研究不同CLDN5表达水平的D3细胞系对肺癌细胞屏障功能的变化，明确CLDN5在改变BBB通透性中的作用机制及相关基因、通路的相互作用，为脑转移瘤的研究提供一定的理论支持。