体外血脑屏障的构建及川芎嗪与葛根素合用对P-gp基因表达的影响

Establishment of artificial blood-brain barrier and its permeability affected by tetramethylpyrazine combined with puerarin

目的:探讨川芎嗪葛根素合用对糖氧剥夺诱导的体外血脑屏障(BBB)模型通透性增高的保护作用机制。方法:利用大鼠脑微血管内皮细胞与星型胶质细胞共培养建立体外血脑屏障模型,随机分为正常对照组、模型组、川芎嗪组、葛根素组、葛根素川芎嗪合用组和尼莫地平组,采用测定TEER、γ-GT、AKP活性及LDH含量等方法比较和鉴定BBB的特征,RT-PCR检测各组P-gp的基因表达。结果:与单层细胞模型相比,BMECs与星形胶质细胞共培养电阻抗可达到(252.61±13.14)Ω·cm2,γ-GT、AKP均显著增加(P<0.01),各用药组较模型组均能下调P-gp基因表达(P<0.01),川芎嗪与葛根素合用组较川芎嗪、葛根素单用显著下调P-gp基因表达(P<0.05)。结论:体外BBB构建成功。川芎嗪、葛根素合用下调P-gp的表达,减少对药物泵出外排,其机制可能与其协同增效治疗BBB损伤有关。

Objective：The purpose of study was to establish blood-brain barrier（BBB）model in vitro neonatal rats in order to simulate physiological and pathological state,puerarin and tetramethylpyrazine（TMP）were used to study the structure and morphology changes of BBB,which induced by normal blood-brain barrier and damaged the brain exposed to oxygen deprivation of sugar（OGD）.To study the protective mechanism and the mechanism of drug transport into brain,illustrate the principles and mechanism the Chinese herbs Gegen,Chuanxiong and its compatibility for providing experimental basis and clinical use.Methods：Use the rat brain microvascular endothelial cells and astrocytes to culture BBB model in vitro successfully to construct the BBB model injury by OGD-induced.There were normal control group,model group,TMP group,puerarin group,nimodipine group and the TMP-puerarin group.Use TEER,γ-glutamyl transpeptidase,alkaline phosphatase activity in enzyme methods to contrast and identify the features of BBB.Analysis gene expression P-glycoprotein changes by Semi-quantitative RT- PCR.Results：Compared with the single-cell model,the TEER of BMECs with astrocytes up to（252.61±13.14）Ω？cm2,γ-GT,AKP were significantly increased（P0.01）.The P-gp gene were significantly down-regulate in treatment group compared with model group（P0.01）.The P-gp gene were significantly down-regulate in TMP group,puerarin group compared with TMP-puerarin group（P0.05）.Conclusion：The artificial BBB model of building successfully.TMP-puerarin can reduce P-glycoprotein expression coordinatedly,can synergies reduce drug pump out,it may be one of the mechanisms.