RhoA在内皮-单核细胞激活多肽-Ⅱ增强血肿瘤屏障通透性中的作用

Role of signaling molecule RhoA in the endothelial monocyte-activating polypeptide-II-induced enhancement in blood-tumor barrier permeability

目的探索信号分子RhoA在内皮-单核细胞激活多肽-II（endothelial monocyte activating polypeptide-II, EMAP-II ）增强血肿瘤屏障（blood—tumor barrier，BTB）通透性中的作用。方法采集出生3～5d的Wistar胎鼠的大脑皮质，应用酶消化法及葡聚糖离心法获得脑微血管段后，接种于培养皿中进行脑微血管内皮细胞（brain microvascular endothelial cells, BMECs）原代培养；将BMECs与c6脑胶质瘤细胞共培养，构建体外BTB模型；共培养后的BMECs随机分成3组（每组6例）：对照组、EMAP—II组和C3 exoenzyme＋EMAP—II组。测定跨内皮阻抗值和辣根过氧化物酶流量评估各组BTB通透性变化情况；Western blot法检测BMECs上紧密连接相关蛋白ZO-1的表达水平；免疫荧光法检测ZO—1和细胞骨架蛋白F—actin在BMECs上的分布与表达。结果与对照组相比较，EMAP—II组BTB通透性显著增高，ZO-1和F—actin在BMECs上的表达水平显著降低，应力纤维形成明显增多；EMAP—II的上述作用受到RhoA抑制剂C3 exoenzyme预处理的显著抑制。结论信号分子RhoA在EMAP—II增强BTB通透性中发挥着重要的作用。

Objective To investigate the role of RhoA in the endothelial monocyte-activating polypeptide-II （EMAP-II）-induced enhancement in the blood-tumor barrier（BTB） permeability. Methods Relatively pure cerebral microvessel fragments were obtained from the cortex of 3-5 days old Wistar rats by using careful dissection, enzyme digestion and dextran centrifugation. Then, these fragments were seeded on dishes and cultured primarily. BTB models were constructed by co-cultivation of rat brain microvascular endothelial cells （BMECs） with C6 glioma cells in vitro. Confluent monolayers of co-cultured BMECs were divided randomly into 3 groups （n=6 for each）： control, EMAP-II and C3 exoenzyme ＋ EMAP-II groups. Transendothelial electric resistance values and horseradish peroxidase flux were measured to evaluate changes in the BTB permeability. The expression level of tight junction-related protein ZO-1 on BMECs was measured by western blot assay. Immunofluorescence was used to identify the expression and distribution of ZO-1 and actin-cytoskeleton protein F-actin on BMECs. Restdts Compared with control group, the BTB permeability of EMAP-II group was increased significantly, the expression levels of ZO-1 and F-actin on BMECs were significantly decreased, accompanied with marked increase in the stress fibers formation. These above-mentioned effects of EMAP-II were significantly inhibited by pretreatment with C3 exoenzyme, the inhibitor of RhoA. Conclusion Signaling molecule RhoA plays an important role in EMAP-II-induced increase in the BTB permeability.