CAJ | 학술논문

目的：观察咪唑克生（ idazoxan，IDA）对体外血脑屏障（ blood-brain barrier， BBB）炎症模型的通透性、紧密连接蛋白ZO-1表达和分布及基质金属蛋白酶-9（ matrix metalloproteinases-9，MMP-9）和MMP-9抑制物———金属蛋白酶组织抑制物-1（tissue inhibitor of metalloproteinase-1， TIMP-1）表达的影响。方法：采用培养7 d的小鼠脑微血管内皮细胞株b．End3建立体外BBB模型，采用TNF-α（10 nmol／L）处理24 h诱导BBB炎症模型，并对BBB炎症模型分别采用IDA 50、100、200μmol／L预处理6 h以观察IDA对BBB炎症模型的作用。通过检测异硫氰酸荧光标记的葡聚糖通过率来确立模型的通透性，采用Western blot法检测紧密连接蛋白ZO-1的表达，通过免疫荧光法观察ZO-1的分布情况，并通过RT-PCR法检测MMP-9／TIMP-1的表达。结果：培养7 d的b．End3细胞汇合成稳定的单层连接，有较好的屏障功能，而采用TNF-α处理24 h后诱导的BBB炎症模型的通透性明显升高，紧密连接蛋白ZO-1在膜上的表达明显减少、不连续，Western blot法显示ZO-1蛋白表达水平明显下降，MMP-9的表达明显升高；而采用IDA 50、100、200μmol／L预处理6 h能明显降低其通透性，增加ZO-1蛋白的表达和改善ZO-1的分布异常，降低MMP-9的表达，其中200μmol／L IDA作用最明显，差异有统计学意义（P＜0.01）。结论：IDA能够直接作用于脑血管内皮细胞，降低TNF-α诱导的体外BBB炎症模型MMP-9的表达，增加紧密连接蛋白ZO-1的表达、修复紧密连接ZO-1的异常分布，从而改善其异常增高的通透性。
목적：관찰미서극생（ idazoxan，IDA）대체외혈뇌병장（ blood-brain barrier， BBB）염증모형적통투성、긴밀련접단백ZO-1표체화분포급기질금속단백매-9（ matrix metalloproteinases-9，MMP-9）화MMP-9억제물———금속단백매조직억제물-1（tissue inhibitor of metalloproteinase-1， TIMP-1）표체적영향。방법：채용배양7 d적소서뇌미혈관내피세포주b．End3건입체외BBB모형，채용TNF-α（10 nmol／L）처리24 h유도BBB염증모형，병대BBB염증모형분별채용IDA 50、100、200μmol／L예처리6 h이관찰IDA대BBB염증모형적작용。통과검측이류청산형광표기적포취당통과솔래학립모형적통투성，채용Western blot법검측긴밀련접단백ZO-1적표체，통과면역형광법관찰ZO-1적분포정황，병통과RT-PCR법검측MMP-9／TIMP-1적표체。결과：배양7 d적b．End3세포회합성은정적단층련접，유교호적병장공능，이채용TNF-α처리24 h후유도적BBB염증모형적통투성명현승고，긴밀련접단백ZO-1재막상적표체명현감소、불련속，Western blot법현시ZO-1단백표체수평명현하강，MMP-9적표체명현승고；이채용IDA 50、100、200μmol／L예처리6 h능명현강저기통투성，증가ZO-1단백적표체화개선ZO-1적분포이상，강저MMP-9적표체，기중200μmol／L IDA작용최명현，차이유통계학의의（P＜0.01）。결론：IDA능구직접작용우뇌혈관내피세포，강저TNF-α유도적체외BBB염증모형MMP-9적표체，증가긴밀련접단백ZO-1적표체、수복긴밀련접ZO-1적이상분포，종이개선기이상증고적통투성。
[ ABSTRACT ] AIM: To study the effect of idazoxan on the permeability of inflammatory blood-brain barrier ( BBB) model in vitro and the expression of tight junction protein ZO-1.METHODS:In vitro BBB model was established by murine brain endothelial cell line bEnd.3 incubated for 7 d.The cells were treated with TNF-α(10 nmol/L) for addi-tional 24 h to establish the inflammatory BBB model, which was pretreated with IDA at doses of 50, 100 and 200μmol/L, respectively.The permeability was measured using fluorescein isothiocyanate-conjugated dextran (FD-40, MW 40,000), the expression of ZO-1 was detected by Western blot analysis, the distribution of ZO-1 was observed by immunofluores-cence, and the mRNA expression of MMP-9/TIMP-1 was measured by RT-PCR.RESULTS:After incubated for 7 d, b. End3 cells converged to be confluent monolayer with low permeability.The inflammatory BBB model induced by TNF-αtreatment displayed much higher permeability with decreased expression of tight junction protein ZO-1, destroyed distribu-tion of ZO-1 and increased mRNA expression of MMP-9.When pretreated with IDA, the permeability was greatly de-creased, the expression of ZO-1 was greatly increased, the abnormal distribution of ZO-1 was greatly ameliorated and the mRNA expression of MMP-9 was obviously reduced.The effect was most significant in IDA ( 200 μmol/L )-pretreated group (P<0.01).CONCLUSION:IDA directly acts on brain endothelial cells to reduce the expression of MMP-9, in-crease the expression of tight junction protein ZO-1 and ameliorate the destroyed distribution of ZO-1 in the inflammatory BBB, thus reversing the abnormally elevated permeability in a inflammatory BBB model in vitro induced by TNF-α.