摘要
本实验建立了Caco-2以及MDCK-MDR1和MDCK等细胞模型,并用跨膜电阻值、荧光黄透过量等评价了模型,证明模型得到的数值是真实可用的。而后利用这3种模型研究了川丁特罗的转运以及吸收上的一些性质。川丁特罗(2-(4-氨基-3-氯-5-三氟甲基苯基)-2-叔丁氨基乙醇,trantinterol),是沈阳药科大学程卯生教授在马布特罗的结构基础上,通过结构修饰,自主研制的治疗哮喘疾病的新型β2受体激动剂。
一、利用Caco-2细胞模型对川丁特罗的转运性质进行研究
建立Caco-2单层细胞模型,并通过生长曲线、跨膜电阻值(TEER)以及荧光黄的透过率的测定证明,本实验室所建立的Caco-2细胞模型可应用于药物转运的研究。
利用成功建立的Caco-2细胞模型研究川丁特罗转运性质,将维拉帕米抑制组作为阴性对照组。结果表明,川丁特罗在Caco-2细胞模型上的外排率为1.08;用维拉帕米抑制P-gp后,川丁特罗外排率为1.11。两者无显著性差异(P>0.05),可见川丁特罗不是P-gp的底物,抑制P-gp后,不能显著改变川丁特罗的外排率。
二、利用MDCK和MDCK-MDR1细胞模型对川丁特罗的转运性质进行研究
建立MDCK和MDCK-MDR1体外药物转运细胞模型,通过跨膜电阻值(TEER)、荧光黄的表观渗透率(Papp)以及罗丹明123(Rho123)在细胞上的转运对模型进行评价。MDCK和MDCK-MDR1细胞的跨膜电阻均达到标准,荧光黄的表观渗透率均小于5%。用Rho123进行转运试验验证P-gp的表达量,MDCK-MDR1的外排率为5.16,说明P-gp的表达量很高。结果表明,MDCK和MDCK-MDR1细胞连接紧密,并且P-gp表达量较高,因此MDCK和MDCK-MDR1细胞模型成功建立了,各项指标符合规定,可以用于药物与P-gp相互作用的研究。
用MDCK和MDCK-MDR1细胞模型研究川丁特罗的双向转运。结果显示:川丁特罗在MDCK-MDR1和MDCK细胞上外排率极为相似,没有显著性差异,分别为1.2和1.05,说明川丁特罗不是P-gp的底物。
川丁特罗对Rho123在MDCK-MDR1细胞模型中转运的影响实验中,川丁特罗显著降低了Rho123的外排比率(P<0.05)。这表明川丁特罗抑制了P-gp介导的Rho123外排,可能为P-gp的抑制剂。
在川丁特罗与P糖蛋白(P-gp)的ATP酶(ATPase)相互作用的研究中发现,川丁特罗对P-gp的抑制作用是通过抑制P-gp的ATPase活性实现的。
In this study, I established Caco-2, MDCK-MDR1and MDCK cellmodel, evaluated these cell model for transmembrane resistance value, fluorescentyellow Permeance to prove that the cell model is available. Then use these three kindsof models studies transportion and absorption of trantinterol. Used for trantinterol(2-(4-amino-3-chloro-5-(trifluoromethyl) phenyl)-2-tert aminoethanols, trantinterol), Shenyang PharmaceuticalUniversity, Professor Cheng Maosheng based on the Mabuterol structuredeveloped a new type of β2agonists.
1. Study the transport of trantinterol with Caco-2monolayer cell model
We established Caco-2monolayer cell model, and verificated it through thecell growth curve determination, the Lucifer yellow transmembrane resistancevalue, proofed the Caco-2cell model in this laboratory can be applied to thetransport study of drugs.
Caco-2cell model successfully established used for transportion study, andverapamil inhibition of the group as a negative control. The results show that theefflux ratio of trantintrol in the Caco-2cell model was1.08; negative control was1.11. There was no significant difference (P>0.05). After all trantintrol is not P-gpsubstrate.
2. Transport of trantinterol in MDCK-MDR1cell monolayer model
We established MDCK and MDCK-MDR1monolayer cell model, andverificated it through the cell growth curve determination, the Lucifer yellowtransmembrane resistance value, the TEER and the Rho123transportion. MDCKand MDCK-MDR1cell model were successfully established. Lucifer yellow apparentpermeability was less than5%. The efflux rate of Rho123was5.16, indicatingthat P-gp expression is high. The results show that MDCK and MDCK-MDR1cellconnected tightly, and highly expressed P-gp. After all MDCK and MDCK-MDR1cell model was successfully established, all the indexes required, can be used for thestudy of interaction of drugs with P-gp.
Study the transportion of trantintrol in MDCK and MDCK-MDR1cell model. The results showed that: the efflux rate of trantinterol in MDCK-MDR1andMDCK cells are1.2and1.05, respectively. There is no significant difference, sotrantinterol is not a P-gp substrate.
The effect of trantinerol on the transport of Rho123in MDCK-MDR1cell model,trantinterol significantly reduce the efflux ratio of Rho123(P <0.05). In thistable, trantinterol inhibits P-gp-mediated Rho123efflux, may be P-gp inhibitor.
Interaction studies for trantinerol and ATPase (ATPase) on P-glycoprotein (P-gp),show that trantinterol inhibit P-gp achieved by inhibiting the ATPase activity of P-gp.
引文
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