本文選題：色胺酮 + Caco-2單細(xì)胞層　； 參考：《復(fù)旦大學(xué)》2011年碩士論文

【摘要】：色胺酮屬吲哚喹唑啉類生物堿,具有抗細(xì)菌、抗真菌、抗腫瘤等多種生物學(xué)活性,目前作為抗結(jié)核病及抗腫瘤候選藥物已經(jīng)進(jìn)入臨床前研究階段。現(xiàn)有研究多集中在其藥效及藥理學(xué)研究,但是其藥代動(dòng)力學(xué)方面的研究幾乎沒有。本課題第一部分的研究?jī)?nèi)容是：使用體外培養(yǎng)的Caco-2單細(xì)胞層作為腸道吸收模型,研究色胺酮的腸道吸收轉(zhuǎn)運(yùn)特性。同時(shí)研究主要的外排蛋白P-糖蛋白(P-glycoprotein, encoded by MDR1, P-gp)和多藥耐藥相關(guān)蛋白2(multidrug resistance-associated protein 2, MRP2)是否參與色胺酮的腸道轉(zhuǎn)運(yùn)過程及色胺酮對(duì)P-gp及MRP2的表達(dá)及功能有何影響。結(jié)果顯示,色胺酮在Caco-2模型上的吸收與外排轉(zhuǎn)運(yùn)都主要是通過被動(dòng)擴(kuò)散的方式進(jìn)行,其從基底側(cè)(BL, Basolateral)到腸腔側(cè)(AP, Apical)以及從AP到BL的表觀滲透系數(shù)Papp分別為4.731±0.101×10-5 cm/sec和6.138±0.291×10-5 cm/sec,外排系數(shù)Papp (BL→AP)/Papp (AP→BL)為0.77,表明其吸收方向(AP→BL)的轉(zhuǎn)運(yùn)占主導(dǎo)地位。P-gp抑制劑維拉帕米和MRP2抑制劑格列苯脲都沒有對(duì)色胺酮的外排轉(zhuǎn)運(yùn)產(chǎn)生影響。而色胺酮在沒有影響這兩種外排蛋白的表達(dá)的情況下,抑制了P-gp底物地高辛及MRP2底物普伐他汀的外排轉(zhuǎn)運(yùn)。以上結(jié)果表明,色胺酮在Caco-2細(xì)胞模型上顯示為由被動(dòng)擴(kuò)散介導(dǎo)的吸收良好的藥物,主要的外排蛋白P-gp和MRP2都沒有參與它的轉(zhuǎn)運(yùn)過程,而色胺酮對(duì)這兩種外排轉(zhuǎn)運(yùn)蛋白的功能則具有一定的抑制作用。 Caco-2細(xì)胞模型是一種藥物離體口服特性篩選模型,已廣泛用于藥物在小腸吸收的評(píng)價(jià)和各種轉(zhuǎn)運(yùn)機(jī)制研究中。該模型的建立一般要經(jīng)過21天的培養(yǎng)周期,消耗大量時(shí)間和材料。而短鏈脂肪酸丁酸可促進(jìn)Caco-2細(xì)胞的分化和細(xì)胞間緊密連接的形成。本課題第二部分的研究?jī)?nèi)容是：利用成分不同的含丁酸培養(yǎng)基建立一種短期培養(yǎng)Caco-2單細(xì)胞層的方法,并通過顯微鏡觀察細(xì)胞形態(tài)學(xué)、測(cè)定跨細(xì)胞膜電阻和熒光黃通透量等指標(biāo)對(duì)Caco-2單細(xì)胞層進(jìn)行完整性評(píng)價(jià),通過堿性磷酸酶活性檢測(cè)對(duì)其進(jìn)行細(xì)胞極化評(píng)價(jià),通過RT-PCR方法評(píng)價(jià)對(duì)主要藥物轉(zhuǎn)運(yùn)蛋白表達(dá)的影響。結(jié)果顯示,使用短期培養(yǎng)法所得的Caco-2單細(xì)胞層形態(tài)完整,TEER500Ω·cm2,漏出標(biāo)志物熒光黃Papp0.5×10-6 cm/s,表明單細(xì)胞層的完整性良好。同時(shí)AP側(cè)堿性磷酸酶的活性顯著升高,顯示單細(xì)胞層出現(xiàn)明顯的極性分化。短期培養(yǎng)法與21天培養(yǎng)法所得的Caco-2單細(xì)胞層中,藥物轉(zhuǎn)運(yùn)蛋白P-gp和MRP2的RNA表達(dá)水平基本一致。表明了使用含丁酸的無血清培養(yǎng)基可促進(jìn)Caco-2單細(xì)胞層的快速形成,該方法所建立的Caco-2細(xì)胞模型符合各項(xiàng)指標(biāo)的要求,可用于研究口服藥物的吸收、轉(zhuǎn)運(yùn)機(jī)制。
[Abstract]:Tryptophan is an indole quinazoline alkaloid with many biological activities, such as antibacterial, antifungal, antitumor and so on. Most of the current studies focus on pharmacodynamics and pharmacology, but there are few studies on pharmacokinetics. The first part of this thesis is to study the intestinal absorption and transport characteristics of tryptophan by using the single cell layer of Caco-2 in vitro as the intestinal absorption model. At the same time, whether the main efflux protein P-glycoprotein (encoded by MDR1, P-gp) and multidrug resistance-associated protein 2(multidrug resistance-associated protein 2 (MRP2) are involved in the intestinal transport of tryptophan and the effect of tryptamine on the expression and function of P-gp and MRP2 were studied. The results showed that the absorption and transport of tryptophan in Caco-2 model were mainly carried out by passive diffusion. The apparent osmotic coefficients (Papp) from basal BLL, Basolateralto AP, Apicaland from AP to BL were 4.731 鹵0.101 脳 10-5 cm/sec and 6.138 鹵0.291 脳 10-5 cm / sec.The efflux coefficient Papp BL / AP)/Papp / AP / L was 0.77, indicating that the transport of AP BLs in the absorption direction was dominant. P-gp inhibitor was dominant. Neither verapamil nor glibenclamide, an inhibitor of MRP2, had any effect on the transport of tryptamine. Tryptamine inhibited the efflux transport of P-gp substrate digoxin and MRP2 substrate pravastatin without affecting the expression of these two efflux proteins. These results suggest that tryptamine is a well absorbed drug mediated by passive diffusion in Caco-2 cell model. Neither P-gp nor MRP2 is involved in its transport process. Tryptophan has a certain inhibitory effect on the function of these two efflux transporters. Caco-2 cell model is a drug screening model in vitro, which has been widely used in the evaluation of drug absorption in the small intestine and in the study of various transport mechanisms. The establishment of the model usually takes 21 days of culture cycle and consumes a lot of time and materials. Short chain fatty acid butyric acid can promote the differentiation of Caco-2 cells and the formation of tight intercellular junctions. In the second part of this paper, the method of short-term culture of Caco-2 single cell layer was established by using different butyric acid medium, and cell morphology was observed by microscope. The integrity of Caco-2 single cell layer was evaluated by measuring the transmembrane resistance and fluorescence yellow permeability. The cell polarization was evaluated by alkaline phosphatase activity and the effect on the expression of major drug transporters was evaluated by RT-PCR method. The results showed that the morphology of Caco-2 single cell layer was intact and the fluorescent yellow Papp0.5 脳 10 ~ (-6) cm 路s ~ (-1), which indicated that the integrity of the single cell layer was good. At the same time, the activity of alkaline phosphatase in AP side was significantly increased, indicating obvious polar differentiation in single cell layer. The level of RNA expression of drug transporter P-gp and MRP2 in the single cell layer of Caco-2 obtained by short culture and 21 days culture was basically the same. The results showed that serum-free medium containing butyric acid could promote the rapid formation of Caco-2 single cell layer, and the Caco-2 cell model established by this method could be used to study the mechanism of absorption and transport of oral drugs.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363

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相關(guān)期刊論文 前2條

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本文編號(hào)：1940849