人源有機(jī)陰離子轉(zhuǎn)運(yùn)多肽1B1和多藥耐藥相關(guān)蛋白2雙轉(zhuǎn)MDCKⅡ細(xì)胞株的構(gòu)建及其功能驗(yàn)證

胡 楠 馬 國 楊 青,3△

(1復(fù)旦大學(xué)生命科學(xué)學(xué)院生物化學(xué)系 上海 200438;2復(fù)旦大學(xué)藥學(xué)院臨床藥學(xué)教研室 上海 201203;3云南天然產(chǎn)物與生物制藥協(xié)同創(chuàng)新中心 昆明 650091)

人源有機(jī)陰離子轉(zhuǎn)運(yùn)多肽1B1和多藥耐藥相關(guān)蛋白2雙轉(zhuǎn)MDCKⅡ細(xì)胞株的構(gòu)建及其功能驗(yàn)證

胡 楠1馬 國2楊 青1,3△

(1復(fù)旦大學(xué)生命科學(xué)學(xué)院生物化學(xué)系 上海 200438;2復(fù)旦大學(xué)藥學(xué)院臨床藥學(xué)教研室 上海 201203;3云南天然產(chǎn)物與生物制藥協(xié)同創(chuàng)新中心 昆明 650091)

目的 構(gòu)建人源有機(jī)陰離子轉(zhuǎn)運(yùn)多肽1B1 (human organic anion transporting polypeptide 1B1,hOATP1B1)和多藥耐藥相關(guān)蛋白2 (multidrug resistance-associated protein 2,hMRP2)雙轉(zhuǎn)MDCKⅡ細(xì)胞株,驗(yàn)證其功能,并應(yīng)用其考察創(chuàng)新藥物2,3-雙加氧化酶 (indoleamine 2,3-dioxygenase,IDO)抑制劑1-甲基色氨酸(1-methyl-tryptophan,1-MT)的轉(zhuǎn)運(yùn)特性。方法 采用基因工程手段獲得hOATP1B1和hMRP2表達(dá)真核載體pVITRO2-SLCO1B1-ABCC2,轉(zhuǎn)染MDCKⅡ細(xì)胞,通過遺傳霉素G418篩選得到穩(wěn)定表達(dá)的細(xì)胞株;通過Real-time PCR、Western blot、免疫熒光共聚焦顯微鏡確認(rèn)目的蛋白特異性;利用該雙轉(zhuǎn)模型考察普伐他汀(不同pH環(huán)境和不同底物濃度)和1-MT的轉(zhuǎn)運(yùn)。結(jié)果 經(jīng)電泳分析、雙酶切、DNA測序鑒定表明重組質(zhì)粒構(gòu)建成功;通過Real-time PCR、Western blot、免疫熒光共聚焦顯微鏡證明經(jīng)遺傳霉素篩選的MDCK-OATP1B1/MRP2細(xì)胞構(gòu)建成功;pH=6.5時(shí),普伐他汀在所構(gòu)建雙轉(zhuǎn)細(xì)胞模型上轉(zhuǎn)運(yùn)最佳;在0～500 μmol/L的濃度范圍內(nèi),普伐他汀在該模型上的轉(zhuǎn)運(yùn)呈現(xiàn)濃度依賴性。1-MT在該細(xì)胞模型上無明顯轉(zhuǎn)運(yùn)。結(jié)論 成功構(gòu)建人源MDCK-OATP1B1/MRP2雙轉(zhuǎn)細(xì)胞株,發(fā)現(xiàn)1-MT既不是OATP1B1蛋白也不是MRP2蛋白的底物,該細(xì)胞株可用于OATP1B1/MRP2介導(dǎo)的外源性物質(zhì)(如藥物)和內(nèi)源性物質(zhì)(如膽紅素)的轉(zhuǎn)運(yùn)研究。

有機(jī)陰離子轉(zhuǎn)運(yùn)多肽1B1; 多藥耐藥相關(guān)蛋白2; MDCKⅡ; 普伐他汀; 1-甲基色氨酸; 轉(zhuǎn)運(yùn)

肝臟是許多內(nèi)源性和外源性物質(zhì)(如藥物)代謝的重要場所。藥物轉(zhuǎn)運(yùn)蛋白(drug transport)是完整的膜蛋白,廣泛分布于腸道、肝臟、腎臟、腦等組織器官,可以介導(dǎo)藥物在體內(nèi)的吸收、分布和排泄[1]。根據(jù)功能不同,藥物轉(zhuǎn)運(yùn)體可分為攝取轉(zhuǎn)運(yùn)體和外排轉(zhuǎn)運(yùn)體。攝取轉(zhuǎn)運(yùn)體如有機(jī)陰離子多肽1B1 (organic anion transporting polypeptide 1B1,OATP1B1),外排轉(zhuǎn)運(yùn)體如P-gp及多藥耐藥相關(guān)蛋白2 (multidrug resistance-associated protein 2,MRP2)等ABC轉(zhuǎn)運(yùn)體。

OATP1B1蛋白由SLCO1B1基因編碼,主要位于肝細(xì)胞基底外側(cè)膜。肝臟中的OATP1B1在mRNA和蛋白水平上均有表達(dá)[2-7],在小腸等組織中僅有少量在mRNA水平上的表達(dá)。在肝細(xì)胞中,OATP1B1主要介導(dǎo)外源性物質(zhì)(他汀類藥物、血管緊張素酶抑制劑、血管緊張素Ⅱ受體拮抗劑)和內(nèi)源性物質(zhì)(如非結(jié)合膽紅素)的攝取[8]。

MRP2蛋白由ABCC2基因編碼,主要位于肝細(xì)胞毛細(xì)膽管膜側(cè),主要介導(dǎo)與葡萄糖醛酸和谷胱甘肽結(jié)合的親脂性物質(zhì)(如他汀類藥物)的外排(分泌),在藥物的體內(nèi)清除過程中發(fā)揮重要作用。臨床上許多藥物是OATP1B1和MRP2的底物和/或調(diào)控劑(抑制劑、誘導(dǎo)劑),其中一些是二者的共同底物和/或調(diào)控劑。因此,構(gòu)建一種合適的細(xì)胞模型,用于研究這兩種轉(zhuǎn)運(yùn)蛋白介導(dǎo)的藥物轉(zhuǎn)運(yùn)、藥物相互作用等具有重要的意義。為此,本研究構(gòu)建了人源有機(jī)離子轉(zhuǎn)運(yùn)蛋白OATP1B1和MRP2共同轉(zhuǎn)染的MDCK細(xì)胞株(MDCK-OATP1B1/MRP2),并以二者的特異性底物普伐他汀、專屬性抑制劑MK-571和吉非貝齊對(duì)其進(jìn)行功能驗(yàn)證,并考察了創(chuàng)新藥物吲哚胺2,3-雙加氧化酶(indoleamine 2,3-dioxygenase,IDO)抑制劑1-甲基色氨酸(1-methyltryprophan,1-MT)在該細(xì)胞上的轉(zhuǎn)運(yùn)。所構(gòu)建細(xì)胞模型可為藥物開發(fā)和藥物相互作用研究提供有用的工具。

材 料 和 方 法

藥品與試劑 人源SLCO1B1 cDNA、ABCC2 cDNA(長沙贏潤生物技術(shù)有限公司),限制性內(nèi)切酶(日本TaKaRa公司)、PrimeSTAR Max DNA聚合酶(日本TaKaRa公司),NovoRec重組酶(上海近岸科技有限公司),DH5α菌株(Biovector質(zhì)粒載體菌種細(xì)胞基因保藏中心),Lip2000(美國Invitrogen公司);人源OATP1B1一抗(美國Abcam公司,ab15441),人源MRP2一抗(美國CST公司,4446S),Trizol試劑(日本TaKaRa公司);HRP標(biāo)記二抗、ECL顯色液、BCA法蛋白濃度測定試劑盒(上海碧云天生物技術(shù)有限公司),普伐他汀鈉(上海晶純生化科技股份有限公司),MK571抑制劑(美國Sigma-Aldrich公司),吉非貝齊(上海晶純生化科技股份有限公司),1-甲基色氨酸(美國Sigma-Aldrich公司)。

儀器與設(shè)備 Lengend Micro 17R高速離心機(jī)(美國Thermo Fisher公司);轉(zhuǎn)膜儀、S1000TMPCR儀(美國BioRad公司);SW-CJ-1FD潔凈工作臺(tái)(蘇州安泰空氣技術(shù)有限公司);SCS-24恒溫?fù)u床(江蘇太倉實(shí)驗(yàn)設(shè)備廠);垂直平板電泳儀(北京百晶生物技術(shù)有限公司);DNP型電熱恒溫培養(yǎng)箱(上海精宏實(shí)驗(yàn)設(shè)備有限公司);Clinx Chem Scope(上海勤翔科學(xué)儀器有限公司);超低溫冰箱(美國Thermo Fisher公司);Zeiss Cell Observer SD (德國Zeiss公司);Agilent 1260高效液相色譜儀(美國安捷倫公司)。

重組質(zhì)粒pVITRO2-SLCO1B1-ABCC2的構(gòu)建

利用NovoRec重組酶及目的基因SLCO1B1(2076 bp)和ABCC2(4638 bp)構(gòu)建質(zhì)粒pVITRO2-SLCO1B1-ABCC2,引物序列及酶切位點(diǎn)信息見表1。

表1 pVITRO2-SLCO1B1及pVITRO2-SLCO1B1-ABCC2引物序列及酶切位點(diǎn)Tab 1 Primer sequence and endonuclease sites of pVITRO2-SLCO1B1 and pVITRO2-SLCO1B1-ABCC2

Endonuclease sites are emphasized with linear underline ;pVITRO2-SLCO1B1 was digested withSgrAI andNheI ; pVITRO2-SLCO1B1-ABCC2 was digested withBamHI andAgeI .

以上述SLCO1B1和ABCC2的cDNA (Genepharma)為PCR模板,利用PrimeSTAR Max DNA聚合酶進(jìn)行PCR反應(yīng),獲得目的片段。反應(yīng)條件為:98 ℃預(yù)變性3 min;98 ℃、10 s,56 ℃、15 s,72 ℃、1.5 min,30個(gè)循環(huán);72 ℃延伸10 min。PCR擴(kuò)增產(chǎn)物經(jīng)1%瓊脂糖凝膠電泳鑒定并進(jìn)行膠回收。利用酶切載體質(zhì)粒pVITRO2,回收線性化片段將目的片段和線性化載體以8∶1的摩爾比例加到EP管中,利用NovoRec PCR一步定向克隆試劑盒進(jìn)行重組反應(yīng);混勻后在37 ℃放置20 min;反應(yīng)結(jié)束立即進(jìn)行轉(zhuǎn)化;收集菌體、抽提質(zhì)粒,進(jìn)行菌落PCR及質(zhì)粒雙酶切驗(yàn)證,并將鑒定正確的陽性克隆進(jìn)行測序。

MDCK-OATP1B1/MRP2穩(wěn)轉(zhuǎn)細(xì)胞株篩選 取MDCK細(xì)胞置于37 ℃、5%CO2培養(yǎng)箱中培養(yǎng),待細(xì)胞達(dá)到90%融合度時(shí),使用Lip 2000進(jìn)行轉(zhuǎn)染。取適量轉(zhuǎn)染細(xì)胞鋪于10 cm 細(xì)胞培養(yǎng)板上,用1 mg/mL G418遺傳霉素篩選至出現(xiàn)“細(xì)胞島”;將“細(xì)胞島”轉(zhuǎn)移至6 孔板,繼續(xù)用500 μg/mL G418遺傳霉素篩選;待細(xì)胞長滿后,取適量進(jìn)行鑒定。

MDCK-OATP1B1/MRP2穩(wěn)轉(zhuǎn)細(xì)胞株鑒定

Real-time PCR鑒定 待六孔板中細(xì)胞長滿后,使用Trizol(RNA iso plus)提取得到RNA,按照37 ℃連接15 min ,85 ℃ 15 s進(jìn)行RT-PCR(逆轉(zhuǎn)錄PCR),得到cDNA。然后,按照95 ℃預(yù)變性5 min;95 ℃變性30 s;55 ℃復(fù)性30 s;72 ℃退火30 s;72 ℃延伸5 min,進(jìn)行Real-time PCR。

Western blot鑒定 取MDCK穩(wěn)轉(zhuǎn)細(xì)胞裂解后的總蛋白30 μg,加入5×加樣緩沖液中混勻,100 ℃干浴,得到變性蛋白,然后與蛋白Marker分別上樣,進(jìn)行SDS-PAGE電泳,電泳結(jié)束后利用轉(zhuǎn)膜儀將凝膠中的蛋白轉(zhuǎn)印到PVDF膜上,5% (質(zhì)量濃度,PBS配制)的脫脂牛奶常溫封閉1.5 h;加入適量人OATP1B1(鼠源,1∶2 000稀釋)和MRP2 (兔源,1∶2 000稀釋)抗體,4 ℃孵育過夜,PBST (含3‰ Tween20 的PBS溶液)溶液洗膜(洗3次,一次洗10 min),然后加入相對(duì)應(yīng)的二抗(山羊抗小鼠,1∶2 000;山羊抗兔,1∶2 000),室溫震搖孵育1.5 h,PBST再次洗膜后,使用ECL化學(xué)發(fā)光試劑盒在Clinx Chemi Scope儀器中顯色。

共聚焦顯微鏡檢測 將待測細(xì)胞鋪于共聚焦皿(密度5×104個(gè)/孔),同時(shí)加入10 mmol/L丁酸鈉刺激細(xì)胞24 h[9];兩種一抗共同孵育[1%BSA 90 μL+OATP1B1 一抗 10 μL (1∶10,使用PBS稀釋)+MRP2一抗 0.25 μL (1∶400)],4 ℃過夜;吸去一抗,使用PBS洗3次,每次10 min;室溫避光孵育熒光二抗1 h[PBS 98 μL+OATP1B1熒光二抗1 μL (1∶100)+MRP2熒光二抗1μL(1∶100)];吸去二抗,使用PBS洗3次,每次10 min;DAPI (1 μg/mL)染色10 min,使用PBS洗3次,每次10 min,2 h內(nèi)使用Zeiss Cell Observer SD進(jìn)行檢測。

MDCK-OATP1B1/MRP2穩(wěn)轉(zhuǎn)細(xì)胞株功能驗(yàn)證

不同pH值條件下普伐他汀的MDCK-OATP1B1/MRP2細(xì)胞轉(zhuǎn)運(yùn) 將MDCK-OATP1B1-MRP2細(xì)胞鋪于12孔Transwell板,培養(yǎng)5天;實(shí)驗(yàn)前一天,在Apical室內(nèi)加入終濃度為10 mmol/L的丁酸鈉刺激24 h。Apical側(cè)到Basolateral側(cè)轉(zhuǎn)運(yùn):將普伐他汀溶液加入到細(xì)胞Apical側(cè)(供給室),調(diào)節(jié)細(xì)胞轉(zhuǎn)運(yùn)液pH值分別為6.0、6.5、7.0和7.5;在Basolateral側(cè)加入pH值為7.5的Krebs-Henseleit緩沖液。Basolateral側(cè)到Apical側(cè)轉(zhuǎn)運(yùn):普伐他汀溶液加入到Basolateral側(cè),按同法操作;分別在30 min、60 min、90 min吸取接收室溶液100 μL,13 000×g離心1 min,取上清液進(jìn)行HPLC檢測。

不同濃度普伐他汀的MDCK-OATP1B1/MRP2細(xì)胞轉(zhuǎn)運(yùn) 將終濃度分別為10、50、100、500、1 000、1 500、2 000和2 500 μmol/L的普伐他汀溶液加入到細(xì)胞Basolateral側(cè)(供給室,細(xì)胞轉(zhuǎn)運(yùn)液pH值為6.5),按上法進(jìn)行轉(zhuǎn)運(yùn)實(shí)驗(yàn)。

OATP1B1和MRP2抑制劑對(duì)普伐他汀轉(zhuǎn)運(yùn)的影響 分別將OATP1B1抑制劑吉非貝齊(終濃度8 μmol/L)和MRP2抑制劑MK-571 (終濃度40 nmol/L)加入到MDCK-OATP1B1/MRP2細(xì)胞的Basolateral側(cè)和Apical側(cè),同時(shí)加入普伐他汀,同法研究普伐他汀在細(xì)胞上的轉(zhuǎn)運(yùn)。

IDO抑制劑1-MT在MDCK-OATP1B1/MRP2細(xì)胞上的轉(zhuǎn)運(yùn) 分別將OATP1B1的抑制劑吉非貝齊(終濃度8 μmol/L)和MRP2的抑制劑MK-571(終濃度40 nmol/L)加入到MDCK-OATP1B1/MRP2細(xì)胞的Basolateral側(cè)和Apical側(cè),同時(shí)加入1-MT,同法研究1-MT在細(xì)胞上的轉(zhuǎn)運(yùn)。

結(jié) 果

重組質(zhì)粒pVITRO2-SLCO1B1-ABCC2的構(gòu)建 pVITRO2-SLCO1B1和pVITRO2-SLCO1B1-ABCC2的凝膠電泳圖分別見圖1和2?？梢远ㄐ缘弥猵VITRO2-SLCO1B1-ABCC2質(zhì)粒構(gòu)建成功,后經(jīng)測序公司驗(yàn)證,序列完全正確無突變,確認(rèn)雙轉(zhuǎn)質(zhì)粒構(gòu)建成成功。

MDCK-OATP1B1/MRP2穩(wěn)轉(zhuǎn)細(xì)胞株鑒定

Real-time PCR鑒定 MDCK雙轉(zhuǎn)細(xì)胞中SLCO1B1(編碼OATP1B1)和ABCC2 (編碼 MRP2) mRNA表達(dá)分析結(jié)果見圖3。在MDCK-OATP1B1/MRP2細(xì)胞的mRNA水平上,SLCO1B1基因表達(dá)量比對(duì)照組高72倍,ABCC2基因表達(dá)量比對(duì)照組高220倍。這說明MDCK-OATP1B1/MRP2細(xì)胞在RNA水平上顯著表達(dá)SLCO1B1和ABCC2兩個(gè)基因。

A:Lane 1:pVITRO2 plasmids;Lane 2:Double digested pVITRO2-SLCO1B1.B:Lane 1:The linear pVITRO2;Lane 2:Full-length SLCO1B1 fragments; Lane 3:pVITRO2-SLCO1B1 plasmid;Lane 4:Double digested pVITRO2-SLCO1B1.

Lane 1:The linear pVITRO2-SLCO1B1;Lane 2:Full-length ABCC2 fragments; Lane 3:pVITRO2-SLCO1B1-ABCC2 plasmid;Lane 4:Double digested pVITRO2-SLCO1B1-ABCC2.

Control group vs.Wild type MDCK Ⅱ cells,(1)P<0.05,(2)P<0.01.

Western blot鑒定 MDCK-OATP1B1/MRP2的Western blot鑒定結(jié)果見圖4。所構(gòu)建的MDCK-OATP1B1/MRP2細(xì)胞在蛋白水平上都表達(dá)OATP1B1和MRP2蛋白,同時(shí)發(fā)現(xiàn)丁酸鈉濃度為10 mmol/L時(shí)蛋白表達(dá)量顯著,效果最佳。

共聚焦顯微鏡檢測 MDCK-OATP1B1/MRP2細(xì)胞中OATP1B1和MRP2的免疫熒光分析結(jié)果見圖5。較之野生型MDCK細(xì)胞,MDCK-OATP1B1/MRP2雙轉(zhuǎn)細(xì)胞的細(xì)胞膜上穩(wěn)定表達(dá)OATP1B1 (綠色熒光)和MRP2 (紅色熒光)兩個(gè)蛋白。

上述Real-Time PCR、Western blot和共聚焦顯微實(shí)驗(yàn)分別從RNA水平、蛋白水平、免疫熒光水平3個(gè)方面證明兩種目的蛋白在MDCK-OATP1B1/MRP2細(xì)胞上均有表達(dá)。

MDCK-OATP1B1-MRP2穩(wěn)轉(zhuǎn)細(xì)胞株功能驗(yàn)證

不同pH值條件下普伐他汀的MDCK-OATP1B1/MRP2細(xì)胞轉(zhuǎn)運(yùn) 由圖6可知,pH在6.0～7.5范圍內(nèi),普伐他汀從Basal側(cè)至Apical側(cè)或者Apical至Basal側(cè)轉(zhuǎn)運(yùn)量隨時(shí)間的增加而增加;在pH為7.0 (中性)時(shí),普伐他汀雙向轉(zhuǎn)運(yùn)均高于pH為6.0和6.5 (弱酸性)時(shí)的轉(zhuǎn)運(yùn)。中性環(huán)境比弱酸性環(huán)境更有利于普伐他汀的轉(zhuǎn)運(yùn)(P<0.05)。pH為7.5 (弱堿性)時(shí),普伐他汀Apical側(cè)至Basal側(cè)轉(zhuǎn)運(yùn)高于Basal側(cè)至Apical側(cè)轉(zhuǎn)運(yùn),說明弱堿性條件利于普伐他汀從Apical側(cè)至Basal側(cè)轉(zhuǎn)運(yùn),因而不利于其消除;pH為6.5時(shí),普伐他汀轉(zhuǎn)運(yùn)的R值(R=b-a/a-b)為2.0,遠(yuǎn)高于其他組,說明在此條件下,該細(xì)胞模型符合體內(nèi)實(shí)際情況。

不同濃度普伐他汀的MDCK-OATP1B1/MRP2細(xì)胞轉(zhuǎn)運(yùn) 由圖7和圖8可知,普伐他汀在MDCK-OATP1B1/MRP2細(xì)胞上的轉(zhuǎn)運(yùn)呈濃度依賴性。當(dāng)?shù)孜餄舛雀哂?00 μmol/L,普伐他汀在細(xì)胞上的轉(zhuǎn)運(yùn)趨于飽和。普伐他汀在野生型細(xì)胞上幾乎沒有轉(zhuǎn)運(yùn);當(dāng)OATP1B1或MRP2任一蛋白被抑制,普伐他汀轉(zhuǎn)運(yùn)都大大降低。普伐他汀在OATP1B1/MRP2雙轉(zhuǎn)染細(xì)胞上的轉(zhuǎn)運(yùn)量顯著高于OATP1B1抑制組(含gemfibrozil組)、MRP2抑制組(含MK571組)及野生型組(P<0.01)。

A:The expression analysis of OATP1B1 and MRP2 in MDCK and MDCK-OATP1B1/MRP2 cells by Western blot.B:The expression analysis of OATP1B1 and MRP2 in MDCK-OATP1B1/MRP2 cells by Western blot,after the stimulation of Na-butyrate ranging from 1 mmol/L to 15 mmol/L at 24 hours.A:Lane 1:MDCK cells; Lane 2:MDCK-OATP1B1/MRP2 cells.B:Lane1:MDCK-OATP1B1/MRP2 control;Lane 2:MDCK-OATP1B1/MRP2-1 mmol/L Na-butyrate;Lane 3:MDCK-OATP1B1/MRP2-5 mmol/L Na-butyrate;Lane 4:MDCK-OATP1B1/MRP2-10 mmol/L Na-butyrate;Lane 5:MDCK-OATP1B1/MRP2-15 mmol/L Na-butyrate.

MDCK-OATP1B1/MRP2 cells were stained with the polyclonal antibody against humanOATP1B1 (green fluorescence) and the monoclonal antibody against human MRP2 (red fluorescence).Nuclei were stained with DAPI (blue fluorescence).

IDO抑制劑1-MT在MDCK-OATP1B1/MRP2細(xì)胞上的轉(zhuǎn)運(yùn) 由圖9可知,1-MT (100 μmol/L)在MDCK-OATP1B1/MRP2細(xì)胞上的轉(zhuǎn)運(yùn)量與OATP1B1抑制組、MRP2抑制組及野生型組比較,差異無統(tǒng)計(jì)學(xué)意義(P> 0.05)。這說明1-MT既不是OATP1B1的底物,也不是MRP2的底物。

pH dependence of pravastatin (500 μmol/L) basal to apical (b-a) and apical to basal (a-b) transcellulartransportin monolayers of MDCK-OATP1B1-MRP2 cells after administration of pravastatin either the basal or the apical side of the cell monolayers.pH was 6.0(A),6.5(B),7.0(C),7.5(D).Data are shown as mean value± standard deviation.The transport ratio (R) was calculated as the quotient of the mean of the apically directed transport and the mean of the basally directed transport at 1.5 h.

Concentration-dependent transcellular transport of pravastatin in monolayers of MDCK-wild type,MDCK-OATP1B1/MRP2 pretreated with MK571 (40 nmol/L),MDCK-OATP1B1/MRP2 pretreated with gemfibrozil (8 μmol/L),and MDCK-OATP1B1-MRP2 cells.Gemfibrozil is the specific inhibitor of OATP1B1 protein,and MK571 is the specific inhibitor of MRP2 protein.

A:Transcellular transport of pravastatin (500 μmol/L) in monolayers of MDCK-Wild type;B:MDCK-OATP1B1/MRP2 pretreated with MK571 (40 nmol/L);C:MDCK-OATP1B1/MRP2 pretreated with gemfibrozil (8 μmol/L);D:MDCK-OATP1B1-MRP2 cells.Gemfibrozil is the specific inhibitor of OATP1B1 protein,and MK571 is the specific inhibitor of MRP2 protein.(1)P<0.01.

A:1-MT (100 μmol/L)was administered to the basal side of monolayers of MDCK wild type;B:MDCK-OATP1B1/MRP2 pretreated with MK-571 (40 nmol/L);C:MDCK-OATP1B1/MRP2 pretreated with gemfibrozil (8 μmol/L);D:MDCK-OATP1B1/MRP2 cells.

討 論

本研究將SLCO1B1和ABCC2基因分別連接至pVITRO2質(zhì)粒的2個(gè)多克隆位點(diǎn)上,構(gòu)建得到pVITRO2-SLCO1B1-ABCC2重組質(zhì)粒,然后通過Lip2000轉(zhuǎn)染進(jìn)MDCKⅡ細(xì)胞,使用遺傳霉素篩選得到MDCK-OATP1B1/MRP2細(xì)胞株,相關(guān)方法未見文獻(xiàn)報(bào)道。

本實(shí)驗(yàn)除了研究OATP1B1和MRP2轉(zhuǎn)運(yùn)蛋白共同介導(dǎo)的普伐他汀和1-MT轉(zhuǎn)運(yùn)外,同時(shí)也考察了單一轉(zhuǎn)運(yùn)蛋白OATP1B1或者M(jìn)RP2對(duì)普伐他汀和1-MT轉(zhuǎn)運(yùn)的影響。對(duì)于后者的研究,常采用的方法有兩種,一是分別構(gòu)建單轉(zhuǎn)的MDCK-OATP1B1和MDCK-MRP2細(xì)胞;二是分別使用2個(gè)轉(zhuǎn)運(yùn)蛋白的專屬性抑制劑,即OATP1B1專屬性抑制劑吉非貝齊[10]和MRP2專屬性抑制劑MK571[11],本實(shí)驗(yàn)采用后一種方法。MDCK-OATP1B1/MRP2經(jīng)相關(guān)特異性蛋白抑制劑預(yù)處理后,可以達(dá)到單轉(zhuǎn)MDCK-OATP1B1和MDCK-MRP2細(xì)胞相同的研究效果(圖8、9)。

普伐他汀在MDCK-OATP1B1/MRP2細(xì)胞上的轉(zhuǎn)運(yùn)量顯著高于野生型組及加入OATP1B1抑制劑gemfibrozil組和MRP2抑制劑MK571組(P<0.01,圖8)。這說明OATP1B1和MRP2均參與了普伐他汀的跨細(xì)胞轉(zhuǎn)運(yùn),二者在普伐他汀的主動(dòng)轉(zhuǎn)運(yùn)過程中均發(fā)揮重要作用。二者中任意一個(gè)蛋白被抑制,均可使普伐他汀的轉(zhuǎn)運(yùn)顯著減少。IDO作為色氨酸經(jīng)犬尿氨酸代謝途徑代謝的限速酶[12-13],與腫瘤及阿爾茨海默癥等疾病密切相關(guān),是備受關(guān)注的藥物作用靶點(diǎn)。1-MT是發(fā)現(xiàn)較早、與底物色氨酸結(jié)構(gòu)最接近的競爭性IDO抑制劑,也是公認(rèn)的IDO抑制劑[14-15],目前廣泛應(yīng)用于IDO抑制劑的抗腫瘤研究中。1-MT在MDCK-OATP1B1/MRP2細(xì)胞上的轉(zhuǎn)運(yùn)與野生型組比較差異無統(tǒng)計(jì)學(xué)意義,說明OATP1B1和MRP2未參與1-MT的跨細(xì)胞轉(zhuǎn)運(yùn),據(jù)此推測1-MT的轉(zhuǎn)運(yùn)可能是被動(dòng)轉(zhuǎn)運(yùn)和/或其他轉(zhuǎn)運(yùn)蛋白介導(dǎo)的轉(zhuǎn)運(yùn)。

綜上所述,本研究成功構(gòu)建了MDCK-OATP1B1/MRP2雙轉(zhuǎn)細(xì)胞模型,其可為OATP1B1/MRP2介導(dǎo)的外源性物質(zhì)(如他汀類藥物)和內(nèi)源性物質(zhì)(如結(jié)合膽紅素)的轉(zhuǎn)運(yùn)研究提供一個(gè)良好的研究工具,并將在新藥開發(fā)及藥物相互作用研究中發(fā)揮重要作用。

[1] KLAASSEN CD,ALEKSUNES LM.Xenobiotic,bile acid,and cholesterol transporters:function and regulation [J].PharmacolRev,2010,62(1):1-96.

[2] KONIG J,CUI Y,NIES AT,etal.Localization and genomic organization of a new hepatocellular organic anion transporting polypeptide [J].JBiolChem,2000,275(30):23161-23168.

[3] KONIG J,CUI Y,NIES AT,etal.A novel human organic anion transporting polypeptide localized to the basolateral hepatocyte membrane [J].AmJPhysiolGastrointestLiverPhysiol,2000,278(1):G156-164.

[4] ABE T,KAKYO M,TOKUI T,etal.Identification of a novel gene family encoding human liver-specific organic anion transporter LST-1 [J].JBiolChem,1999,274(24):17159-17163.

[5] HSIANG B,ZHU Y,WANG Z,etal.A novel human hepatic organic anion transporting polypeptide (OATP2).Identification of a liver-specific human organic anion transporting polypeptide and identification of rat and human hydroxymethylglutaryl-CoA reductase inhibitor transporters [J].JBiolChem,1999,274(52):37161-37168.

[6] HO RH,TIRONA RG,LEAKE BF,etal.Drug and bile acid transporters in rosuvastatin hepatic uptake:function,expression,and pharmacogenetics[J].Gastroenterology,2006,130(6):1793-1806.

[7] KARTENBECK J,LEUSCHNER U,MAYER R,etal.Absence of the canalicular isoform of the MRP gene-encoded conjugate export pump from the hepatocytes in Dubin-Johnson syndrome [J].Hepatology,1996,23(5):1061-1066.

[8] 林佳媛,馬國.膽紅素的代謝及其調(diào)節(jié)研究進(jìn)展[J].復(fù)旦學(xué)報(bào)(醫(yī)學(xué)版),2014,41 (3):405-411.

[9] KONIG J,ZOLK O,SINGER K,etal.Double-transfected MDCK cells expressing human OCT1/MATE1 or OCT2/MATE1:determinants of uptake and transcellular translocation of organic cations[J].BrJPharmacol,2011,163(3):546-555.

[10] NAKAGOMI HR,NAKAI D,TOKUI T,etal.Gemfibrozil and its glucuronide inhibit the hepatic uptake of pravastatin mediated by OATP1B1[J].Xenobiotica,2007,37(5):474-486.

[11] YAMAZAKI M,LI B,LOUIE SW,etal.Effects of fibrates on human organic anion-transporting polypeptide 1B1-,multidrug resistance protein 2- and P-glycoprotein-mediated transport[J].Xenobiotica,2005,35(7):737-753.

[12] LOB S,KONIGSRAINER A,RAMMENSEE HG,etal.Inhibitors of indoleamine 2,3-dioxygenase for cancer therapy:can we see the wood for the trees?[J].NatRevCancer,2009,9(6):445-452.

[13] MOFFETT JR,NAMBOODIRI MA.Tryptophan and the immune response[J].ImmunolCellBiol,2003 ,81(4):247-265.

[14] CADY SG,SONO M.1-Methyl-DL-tryptophan,beta-(3-benzofuranyl)-DL-alanine (the oxygen analog of tryptophan),and beta-[3-benzo(b)thienyl]-DL-alanine (the sulfur analog of tryptophan) are competitive inhibitors for indoleamine 2,3-dioxygenase[J].ArchBiochemBiophys,1991,291(2):326-333.

[15] KATZ JB,MULLER AJ,PRENDERGAST GC.Indoleamine 2,3-dioxygenase in T-cell tolerance and tumoral immune escape[J].ImmunolRev,2008,222:206-221.

Establishment of double-transfected MDCK Ⅱ cells expressing human organic anion transporting polypeptide 1B1 and multidrug resistance-associated protein 2 and identification of its functions

HU Nan1, MA Guo2, YANG Qing1,3△

(1DepartmentofBiochemistry,SchoolofLifeSciences,FudanUniversity,Shanghai200438,China;2DepartmentofClinicalPharmacy,SchoolofPharmacy,FudanUniversity,Shanghai201203,China;3theCollaborativeInnovationCenterofYunnanNaturalProductsandBiologicalPharmacy,Kunming650091,YunnanProvince,China)

Objective To establish double-transfected Madin-Darby canine kidney (MDCK ) Ⅱ cells expressing human organic anion transporting polypeptide 1B1(hOATP1B1) and multidrug resistance-associated protein 2 (hMRP2)and to testify their functions,moreover,to study the transcellur transport of indoleamine 2,3-dioxygenase (IDO) inhibitor 1-methyltryptophan (1-MT) in the transfectants.Methods hOATP1B1/hMRP2 eukaryotic vectorspVITRO2-SLCO1B1-ABCC2 was obtained by genetic engineering method and then transfected into MDCK cells.Stably expressed MDCK cells were screened by using the geneticin G418.Real-time PCR,Western blot analysis and immuno fluorescent confocal microscopy were used to verify the proteins expression.Transport of the representative substrate pravastatin in different pH values and substrate concentrations and 1-MT were evaluated using the double transfectants. Results MDCK-OATP1B1/MRP2 was successfully established.Pravastatin displayed the optimal transcellular transport when pH value was 6.5.Transport of pravastatin demonstrated the concentration-dependent in the concertation range of 0 to 500 μmol/L.Transport of 1-MT showed no significant difference in MDCK cells and transfectants. Conclusions MDCK-OATP1B1/MRP2 was successful established;1-MT was not the substrate of OATP1B1 or MRP2 protein;and the eatablished double transfectant cell lines can be used to evaluate OATP1B1/MRP2-medicated transport of xenobiotics (e.g.new drug candidates) and endogenous compounds (e.g.bilirubin).

organic anion transporting polypeptide 1B1; multidrug resistance-associated protein 2;Madin-Darby canine kidney Ⅱ; pravastatin; 1-methyltryptophan; transport

國家自然科學(xué)基金面上項(xiàng)目(81573310,81374051);高等學(xué)校博士學(xué)科點(diǎn)專項(xiàng)科研基金(20130071110037)

R965.1,Q233

A

10.3969/j.issn.1672-8467.2017.02.002

2016-09-26;編輯:王蔚)

△Corresponding author E-mail:yangqing68@fudan.edu.cn

*This work was supported by the General Program of National Natural Science Foundation of China (81573310,81374051) and the Research Fund for the Doctoral Program of Higher Education of China (20130071110037).