中文摘要：

目的：研究微小RNA（microRNA,miRNA）miR-200b是否介导血肿瘤屏障（blood-tumor barrier,BTB）通透性增加。方法：测量跨内皮阻抗值（TEER）、辣根过氧化物酶（HRP）渗漏量,分析血脑屏障（bloodbrain barrier,BBB）和BTB通透性的改变,应用Real-time PCR检测血脑屏障BBB和BTB大鼠脑微血管内皮细胞（rat cerebral microvascular endothelial cell RCMEC,ECs）miR-200b的表达,应用miR-200b agomir和miR-200b antagomir分别转染GECs（ECs和U87脑胶质瘤细胞共培养的细胞）后分析血肿瘤屏障的通透性改变;应用Western blot检测紧密连接相关蛋白ZO-1（zonula occludens-1）的表达;应用免疫荧光方法观察GECs紧密连接相关蛋白ZO-1和丝状肌动蛋白（filamentous actin,F-actin）结构和分布的改变。结果：与BBB相比,BTB的TEER值显著降低,HRP显著升高;与BBB的ECs相比,GECs内源性miR-200b表达显著降低;miR-200b agomir和miR-200b antagomir成功转染到GECs中;miR-200b agomir组TEER值显著升高,HRP流量显著降低,以及ZO-1表达显著升高,抑制ZO-1由内皮细胞的边缘向细胞质转移,抑制丝状肌动蛋白由细胞膜边缘向细胞中央区分布,F-actin分布于细胞边缘明显增加,应力纤维形成明显减少;miR-200b antagomir结果与miR-200b agomir实验结果相反。结论：MiR-200b介导BTB通透性增加。

英文摘要：

Objective： To investigate whether the mechanism of microRNA（ miRNA） miR- 200 b nediated an increase in blood- tumor barrier（ BTB）. Methods： HRP flux and TEER assays revealed blood- brain barrier（ BBB）and BTB permeability. Endogenous expression of miR- 200 b was detected by Real- time PCR in rat cerebral microvascular endothelial cell RCMEC（ ECs） of BBB and BTB. miR- 200 b agomir and miR- 200b antagomir were transfected into GECs（ ECs with U87 glioma cells co- culturing）,respectively. The protein expression level of ZO- 1 was detected by Western blot. The stress fiber formation and distribution of filamentous actin（ F- actin） and ZO- 1 were assessed by immunofluorescence microscopy. Results： Compared with BBB,the value of TEER significantly decreased in BTB,and HRP flow significantly increased in BTB,which resulted to an increase in BTB permeability. The endogenous expression of miR- 200b in ECs significantly decreased in BTB compared to BBB. miR- 200b agomir and miR- 200 b antagomir were successfully transfected into GECs. Overexpression of miR- 200 b inhibited endothelial leakage and restored normal transendothelial electric resistance values. Simultaneously,overexpression of miR- 200b inhibited a decrease in the protein expression level of ZO- 1. In addition,overexpression of miR- 200b inhibited the relocation of ZO- 1 from cellular borders into the cytoplasm as well as the production of stress fiber formation in GECs of the BTB. miR- 200 b silencing produced opposite results as that obtained from that of the miR- 200b over-expression group. Conclusion： miR- 200 b mediated an increase in BTB permeability.