信号分子PKC调控内皮-单核细胞激活多肽Ⅱ增强血肿瘤屏障通透性机制的研究

Mechanisms of Signaling Molecule PKC in the Endothelial Monocyte-activating Polypeptide-Ⅱ-induced Increasing of Blood-tumor Barrier Permeability

目的探索信号分子蛋白激酶C（PKC）参与内皮-单核细胞激活多肽Ⅱ（EMAP-Ⅱ）增强血肿瘤屏障（BTB）通透性过程的相关机制。方法采集出生3～5d的Wistar胎鼠大脑皮质，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（BMEC）原代培养；将BMEC与C6脑胶质瘤细胞共培养，构建体外BTB模型；共培养后的BMEC随机分成3组：对照组、EMAP-Ⅱ组和H7＋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通透性的变化，Westernblot法和免疫荧光法检测紧密连接相关蛋白claudin-5在各组BMEC上的表达水平变化；共培养后的BMEC被随机分成5组：EMAP-Ⅱ0，0．5，1，2，4h组，Westernblot法检测EMAP-Ⅱ作用不同时间点时BMEC膜上PKC各亚型的蛋白表达变化。结果与对照组比较，EMAP-Ⅱ组BTB的通透性显著增高（P=0．002），BMEC上claudin-5的表达水平显著降低（P=0．005），EMAP-Ⅱ的上述作用受到PKC抑制剂H7预处理的显著抑制（P=0．036）；EMAP-Ⅱ作用0．5h时，BMEC膜上PKC-α和PKC-β的蛋白表达水平显著增加，1h时达到峰值，其后表达水平逐渐下降，4h时恢复至未用药水平。BMEC膜上PKC-ζ的蛋白表达水平于O．5h时显著增加，其后表达水平逐渐下降，2h时恢复至未用药水平。结论信号分子PKC参与EMAP-Ⅱ增强BTB通透性的过程，其机制可能与BMEC膜上PKC-α、-β和-ζ的表达水平上调有关。

Objective To investigate the mechanisms of signaling molecule protein kinase C （PKC） in endothelial monocyte-activating polypeptide -Ⅱ （EMAP- Ⅱ）-induced increase of blood-tumor barrier （BTB） permeability. Methods Relatively pure cerebral microvessel fragments were ob- tained from the cortex of 3-5 days old wistar rats through carefully dissection, enzyme digestion, and dextran centrifugation. Then, the fragments were seeded on dishes and cultured primarily. In vitro BTB models were constructed by co-culture rat brain microvascular endothelial cells （BMEC） with C6 glioma cells. Confluent monolayers of co-cultured BMEC were divided into 3 groups ： control, EMAP- Ⅱ , and H7 ＋ EMAP- Ⅱ groups. Transendo- thelial electric resistance values and horseradish peroxidase flux were measured to evaluate changes in BTB permeability. The expression level of tight junction-related protein clandin-5 of BMEC was measured by Western blot and immunofluorescent assays. Confluent monolayers of co-cultured BMEC were randomly divided into 5 groups ： EMAP-Ⅱ 0 h, 0.5 h, 1 h, 2 h and 4 h groups. The expression levels of PKC isoforms （ α, β, γ, ε, δ, and ζ ） on membrane of BMEC after EMAP- Ⅱ infusion were detected by Western blot. Results Compared with control group, BTB permeability of EMAP- Ⅱ group was increased significantly （P = 0.002）. Meanwhile, the expression level of claudin-5 on BMEC was significantly decreased （P = 0.005 ）. These above-mentioned effects of EMAP-Ⅱwere significantly inhibited by H7 pretreatment, which is an inhibitor of PKC （ P = 0.036）. Fur- thermore, the EMAP- Ⅱ - induced BTB opening was accompanied by upregulated protein expression of PKC -α, - β and - ζ on BMEC membrane. Conclusion Signaling molecule PKC is invovled in EMAP-Ⅱ -induced increase in BTB permeability, which may be associated with the upregula-tion of PKC-α, -βand -ζ on BMEC membrane.