摘要
目的探索内皮一单核细胞激活多肽-Ⅱ（endothelial monocyte activating polypeptide-II，EMAP-II）增强血肿瘤屏障（blood—tumor barrier，BTB）通透性的相关信号机制。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BMEC被随机分成5组（每组6例）：对照组、EMAP-Ⅱ组、H7＋EMAP—11组、C3 exoenzyme＋EMAP-II组和C3exoenzyme＋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通透性变化情况；Western blot法检测BMECs上紧密连接相关蛋白occludin和ZO-1的表达水平变化；免疫荧光法检测OC-cludin和ZO-1在BMECs上的分布与表达。Western blot法检测BMECs上肌球蛋白轻链（myosin light chain，MLC）和磷酸化肌球蛋白轻链（phosphomyosin light chain，pMLC）的表达水平变化。结果与对照组相比较，EMAP-II组BTB的通透性明显增高，BMECs上occludin和ZO-1的表达水平明显降低，pMLC的表达水平明显增高；EMAP—II的上述作用受到蛋白激酶c（protein kinaseC，PKC）抑制剂H7或（和）RhoA抑制剂C3exoenzyme预处理的明显抑制。结论信号分子PKC和RhoA在EMAP—II增强BTB通透性过程中发挥着重要的作用，信号通路PKC—pMLC和RhoA—pMLC参与调控该过程。
Aim To investigate the signaling mechanisms in endothelial monocyte-activating polypeptide- II （ EMAP- II ） -induced increase in 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. In vitro BTB models were constructed by co-cultivation of rat brain microvascular endothelial cells （BMECs） with C6 glioma cells. Confluent monolayers of co-cultured BMECs were divided randomly into 5 groups （ each n = 6） ： control, EMAP- II , H7 ＋ EMAP-II, C3 exoenzyme ＋ EMAP-II, and C3 exoenzyme ＋ H7 ＋ EMAP-II groups. Transendothelial electric resistance values and horseradish peroxidase flux were measured to evaluate changes in the BTB permeability. The expression levels of tight junction-re- lated protein occludin and ZO-1 in BMECs were measured by Western blot. Immunofluorescence was used to identify the expression and distribution of occludin and ZO-1 in BMECs. Also, Western blot were used to detect the expression levels of myosin light chain （MLC） and phosphomyosin light chain （pMLC） in BMECs. Results Compared with control group, the BTB permeability of EMAP- II group was increased significantly. The expression levels of occludin and ZO-1 in BMECs were significantly decreased, accompanied with marked increase in the expression level of pMLC. These above-mentioned effects of EMAP-II were significantly inhibited by pretreatment with H7 （an inhibitor of PKC ） or/and C3 exoenzyme （an inhibitor of RhoA）. Conclusion Signaling molecules PKC and RhoA play important roles in EMAP-II-induced increase in BTB permeability; signaling pathways PKC- pMLC and RhoA-pMLC are involved in this process.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2014年第5期632-637,共6页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 81272795,81171131,81172197,81272564,81372484,81072056)
高等学校博士学科点专项科研基金资助项目(No 20092104110015,20102104110009)
沈阳市科学技术计划项目计划(No F13-220-9-15,F13-316-1-16,F13-316-1-19)
