【摘要】 目的研究Tie1 AS和Tie1表达变化对血肿瘤屏障通透性的影响和相关机制。方法建立体外血脑屏障和血肿瘤屏障模型,Real-time PCR检测h CMEC/D3细胞中Tie1 AS和Tie1的表达水平。转染p IRES2-EGFPTie1 AS表达载体上调体外血肿瘤屏障模型h CMEC/D3细胞中Tie1 AS的表达,Western blot检测Tie1的表达变化。将si RNA-Tie1转染人h CMEC/D3细胞并建立体外血肿瘤屏障模型,跨内皮电阻测量系统分析血肿瘤屏障通透性变化;Western blot和免疫荧光法检测h CMEC/D3细胞中紧密连接相关蛋白claudin-5、occludin和ZO-1的表达和分布变化。结果和正常脑微血管内皮细胞相比,体外血肿瘤屏障模型h CMEC/D3细胞中Tie1的表达显著增加,而Tie1 AS的表达显著降低。上调体外血肿瘤屏障模型内皮细胞中Tie1 AS的表达水平,能够显著降低Tie1的表达。下调体外血肿瘤屏障模型内皮细胞中Tie1的表达后屏障通透性显著下降,伴有claudin-5、occludin和ZO-1的表达下调以及在细胞膜上呈不连续分布。结论体外血肿瘤屏障模型内皮细胞中低表达的Tie1 AS能够负性调控Tie1的表达,进而通过调节紧密连接相关蛋白的表达和分布影响屏障的通透性。更多还原

【Abstract】 Objective To investigate the effects and underlying mechanism on blood-tumor barrier(BTB) permeability regulated by Tie1 AS and Tie1. Methods After establishing the blood-brain barrier model and BTB model in vitro, the expressions of Tie1 AS and Tie1 in h CMEC/D3 cells were detected by Real-time PCR. The p IRES2-EGFP-Tie1 AS vector was transfected into the h CMEC/D3 cells of BTB model in vitro to upregulate the expression of Tie1 AS, and the expression of Tie1 was detected by Western blot. Moreover, the si RNA-Tie1 was transfected into the h CMEC/D3 cells, and the transendothelial electrical resistance of BTB was analyzed. Meanwhile, the expression and distribution of tight junction associated proteins claudin-5, occludin and ZO-1 were detected by Western blot and immunofluorescence staining. Results Compared with normal cerebral microvascular endothelial cells, the expression level of Tie1 in the h CMEC/D3 cells of BTB model in vitro increased significantly, while the expression level of Tie1 AS decreased significantly. The upregulation of the Tie1 AS in the h CMEC/D3 cells of BTB model in vitro reduced the expression level of Tie1 markedly. The downregulation of Tie1 in the h CMEC/D3 cells of BTB model in vitro resulted in a significant decrease of the BTB permeability, accompanied with the reduced expression levels of claudin-5, occludin and ZO-1, as well as the discontinuous distribution on the membrane. Conclusion The low expressed Tie1 AS in the h CMEC/D3 cells of BTB model in vitro regulates the expression of Tie1 negatively, which affected the BTB permeability by regulating the expression and distribution of tight junction associated proteins.更多还原