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

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。

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.