药学学报  2015, Vol. 50 Issue (7): 842-847   PDF    
稳定表达hMATE1及共表达hMATE1与hOCT1或hOCT2细胞模型的构建
雷红梅, 孙思源, 李丽萍, 涂美娟, 周慧, 曾苏, 蒋惠娣     
浙江大学药学院, 浙江 杭州 310058
摘要:为构建稳定表达人多药及毒素外排转运体1 (hMATE1) 的转基因细胞模型, 提取人肾总mRNA, 经逆转录PCR获得hMATE1 cDNA, 借助HindⅢ、KpnⅠ两个酶切位点与pcDNA3.1(+) 重组获得重组质粒。将pcDNA3.1(+)-hMATE1重组质粒转染至MDCK、MDCK-hOCT1和MDCK-hOCT2细胞中, 经潮霉素B抗性筛选后, 以4',6-二脒基-2-苯基吲哚 (DAPI) 和N-甲基-4-苯基吡啶 (MPP+) 的积聚实验筛选获得具有良好hMATE1功能的单克隆。测定筛选获得的细胞中转运体mRNA的表达量, 并表征其对二甲双胍的积聚或对西咪替丁的转运能力。结果表明, 本研究构建的MDCK-hMATE1、MDCK-hOCT1/hMATE1、MDCK-hOCT2/hMATE1细胞模型均高表达hMATE1 mRNA, MDCK-hMATE1细胞对二甲双胍的积聚为转染空载体细胞的17.6倍; MDCK-hOCT1/hMATE1和MDCK-hOCT2/hMATE1细胞对西咪替丁的净外排率分别为17.5和3.65。因此, 本研究成功构建了稳定表达hMATE1及共表达hMATE1与hOCT1或hOCT2的细胞模型, 可用于hMATE1及其与hOCT1或hOCT2共同参与的药物转运或药物-药物相互作用的体外研究。
关键词细胞模型     人多药及毒素外排转运体1     人有机阳离子转运体1     人有机阳离子转运体2     共表达    
Establishment of MDCK cell models expressing human MATE1 or co-expressing with human OCT1 or OCT2
LEI Hong-mei, SUN Si-yuan, LI Li-ping, TU Mei-juan, ZHOU Hui, ZENG Su, JIANG Hui-di     
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
Abstract: To establish single-and double-transfected transgenic cells stably expressing hMATE1, hMATE1 cDNA was cloned by RT-PCR from human cryopreserved kidney tissue, and subcloned into pcDNA3.1(+) plasmid by virtue of both HindⅢ and KpnⅠ restriction enzyme sites. Subsequently, the recombined pcDNA3.1(+)-hMATE1 plasmid was transfected into MDCK, MDCK-hOCT1 or MDCK-hOCT2 cells using Lipofectamine 2000 Reagent. After a 14-day-cultivation with hygromycin B at the concentration of 400 μg·mL-1, all clones were screened with DAPI and MPP+ as substrates to identify the best candidate. The mRNA content of hMATE1, the cellular accumulation of metformin with or without cimetidine as inhibitor, or transportation of cimetidine was further valuated. The results showed that all of the three cell models over expressed hMATE1 mRNA. The cellular accumulation of metformin in MDCK-hMATE1 was 17.6 folds of the control cell, which was significantly inhibited by 100 μmol·L-1 cimetidine. The transcellular transport parameter net efflux ratios of cimetidine across MDCK-hOCT1/hMATE1 and MDCK-hOCT2/hMATE1 monolayer were 17.5 and 3.65, respectively. In conclusion, cell models with good hMATE1 function have been established successfully, which can be applied to study the drug transport or drug-drug interaction involving hMATE1 alone or together with hOCT1/2 in vitro.
Key words: cell model     human multidrug and toxin extrusion 1     human organic cation transporter 1     human organic cation transporter 2     coexpression    

多药及毒素外排蛋白家族 (multidrug and toxin extrusion proteins,MATEs) 是广泛存在于古生菌、细菌和真核生物中、以质子或钠离子的跨膜电势差为驱动力的外排转运体[1],是溶质转运体 (solute carrier family,SLC) 超家族中的成员。MATEs可介导大肠 杆菌、金葡菌、多形拟杆菌以及拟南芥等的耐药。人多药及毒素外排转运体1 (human multidrug and toxin extrusion 1,hMATE1) 是人MATEs家族的一员,含有570个氨基酸残基[2],包含13个跨膜结构域[2,3],由位于17号染色体上的SLC47A1基因编码。hMATE1在肾小管上皮细胞刷状缘侧和肝细胞胆毛细管侧有较丰富的表达[3],以反向质子势能为驱动力[2]介导多种药物、毒物及内源性物质从细胞中排出[2,4]。hMATE1的底物包括N-甲基-4-苯基吡啶 (N-methyl-4- phenylpridinium,MPP+)、二甲双胍、西咪替丁、4',6-二脒基-2-苯基吲哚 (4',6-diamidino-2-phenylindole,DAPI)、奥沙利铂、非索非那定、百草枯、肌酐及两

性化合物头孢氨苄等,已发现的底物中多数也是有机阳离子转运体家族 (organic cation transporters,OCTs) 的底物。人有机阳离子转运体1 (human organic cation transporter 1,hOCT1) 主要分布于肝血窦侧肝细胞的基底侧膜上,hOCT2则分布于肾小管上皮细胞的基底侧[3],已有研究证明hOCT2和hMATE1分别位于同一近端肾小管上皮细胞的基底侧和刷状缘侧[5],推测hMATE1与hOCTs共同参与其底物的处置。

由转运体介导的药物-药物相互作用越来越受到重视,因此在创新药物非临床药代动力学研究中,各国药政部门要求体外考察候选药物与转运体的相互作用。由于人原代细胞来源困难,且原代细胞在体外培养中转运体易丢失,因此,稳定高表达人源转运体的转基因细胞是国际上公认的细胞模型。目前,已有多篇文献[6, 7, 8]报道在细胞中成功转染共表达两个甚至3个外源基因,也有报道[9, 10]将一个摄取转运体和一个外排转运体转染至细胞中共表达以研究两个转运体共同底物的转运。因此,本研究拟构建稳定表达人hMATE1,及共表达hMATE1与hOCT1或hOCT2的MDCK细胞模型,以研究由hMATE1及与hOCT1/2共同参与转运的底物药物、内源物的细胞处置,以及由上述转运体介导的药物-药物相互作用。

材料与方法 材料与试剂

人肾癌组织 (浙江省肿瘤医院医学伦理委员会批准使用); MDCK、MDCK-hOCT1和MDCK-hOCT2,E.coli DH5α及pcDNA3.1(+) 质粒 (本实验室保存); 特异性引物 (上海生物工程有限公司合成); PrimeScript RT试剂盒,HindⅢ、KpnⅠ限制性内切酶,T4 DNA连接酶 (日本Takara公司); 质粒抽提试剂盒和凝胶纯化试剂盒 (德国Qiagen公司)。DMEM培养基、胎牛血清、胰酶 (美国Gibco公司); 潮霉素B、DAPI、MPP+、二甲双胍、西咪替丁 (美国Sigma公司)。

细胞培养

MDCK细胞生长于含10% 胎牛血清、100 u·mL-1青霉素和100 μg·mL-1链霉素的DMEM培养基中,在37 ℃、5% CO2,饱和湿度条件下培养,贴壁生长。MDCK-hOCT1/2的培养基添加350 μg·mL-1 G418,MDCK-hMATE1的培养基添加200 μg·mL-1潮霉素B,两种共表达细胞的培养基添加350 μg·mL-1 G418和200 μg·mL-1潮霉素B,其他培养条件同MDCK细胞。细胞生长至汇合度80% 时用含EDTA的0.25% 胰酶消化传代。

hMATE1基因的克隆

设计扩增hMATE1 cDNA (GenBank accession number: NM_018242.2) 的特异性引物 (表 1),并引入HindⅢ和KpnⅠ两个酶切位点 (下划线标示)。提取人肾总mRNA,经逆转录PCR获得目的基因片段。经1% 琼脂糖凝胶电泳回收纯化目的片段。测序验证以获得正确的hMATE1 cDNA。hMATE1 cDNA与含潮霉素B抗性基因的pcDNA3.1(+) 质粒均进行HindⅢ和KpnⅠ双酶切后,用T4 DNA连接酶连接上述酶切后片段,连接产物转化至E. coli DH 5α细菌中扩增后,提取质粒,经酶切和测序鉴定获得正确pcDNA3.1(+)-hMATE1重组质粒。

Table 1 Primers used in PCR and real-time PCR. The two sequences underlined were restriction enzyme sites in PCR primers
细胞转染和抗性克隆筛选

细胞接种于6孔 细胞培养板,待细胞生长至汇合度80% 时,按照LipofectaminTM2000试剂说明书转染MDCK、MDCK- hOCT1和MDCK-hOCT2细胞。以转染空载体的MDCK (MDCK-3.1,M-3.1)、MDCK-hOCT1 (MDCK-hOCT1/3.1,M-1/3.1) 和MDCK-hOCT2 (MDCK-hOCT2/3.1,M-2/3.1) 细胞作为阴性对照。转染6 h后正常培养48 h,再换成添加400 μg·mL-1潮霉素B的培养基持续筛选14天。采用有限稀释法接种于96孔板中,进行单克隆标记。

底物积聚实验

通过荧光底物DAPI的积聚实验筛选单克隆。 将细胞接种于96孔黑色底透酶标板,培养3~4天后进行DAPI积聚实验。MATE1以H+ 跨膜浓度差为驱动力进行底物转运,可利用此特性人为制造胞内高H+ 浓度环境将MATE1转运方向转变为摄取。具体方法为: 弃去培养基后,细胞用200 μL 37 ℃含30 mmol·L-1 NH4Cl的人工摄取缓冲液 (artificial uptake buffer,AUB,KCl 130 mmol·L-1、K2HPO4 2.0 mmol·L-1、MgSO4 1.2 mmol·L-1、CaCl2 1.0 mmol·L-1、葡萄糖5.6 mmol·L-1、HEPES 25 mmol·L-1) 预孵育20 min后,换成AUB继续孵育 5 min[11, 12],除去预孵育液,加入含1.0 μmol·L-1 DAPI的AUB 150 μL孵育20 min,立即用冰AUB洗3遍。用酶标仪测定荧光强度,并测定样品的蛋白浓度。MPP+和二甲双胍的积聚实验方法与DAPI积聚类似,底物孵育液体积为200 μL,孵育时间为3 min; 用0.1% SDS裂解细胞,经2倍体积乙腈沉淀蛋白质,13 000 r·min-1离心15 min后取上清液,以LC-MS/MS法[6,13]测定上清液中MPP+ 或二甲双胍浓度 (Waters UPLC- TQD三重四级杆质谱系统),并进行总蛋白校正。

测定细胞中转运体mRNA的表达

将细胞接种于24孔细胞培养板培养3~4天,提取细胞总mRNA并逆转录后,使用特异性引物以GAPDH为内参基因进行RT-PCR,测定细胞中相关转运体mRNA的表达量。

西咪替丁的转运实验

将MDCK-hOCT1/ hMATE1、MDCK-hOCT2/hMATE1及各自的阴性对照细胞均匀种于Costar 12孔transwell板中,第2、4天换液培养4~5天后,进行转运实验。采用测定荧光黄表观透过系数 (apparent permeability coefficient,Papp) 的方法验证细胞层致密性[14],按照公式1计算荧光黄的Papp

$\begin{array}{*{20}{l}} {{P_{app}} = ({V_{acceptor}}/A \cdot t)\times ({{\left[ {drug} \right]}_{acceptor}}/{{\left[ {drug} \right]}_{initial,donor}})}\\ \end{array}$ (1)

式1中A为多聚碳酸酯膜的面积 (1.12 cm2),t为转运时长 (s)。

选择符合细胞层致密性要求的细胞孔进行西咪替丁的转运实验。每20 min取接收液100 μL,并加入新的缓冲液补足体积。转运实验持续120 min。接收液100 μL以乙酸乙酯1 mL提取,13 000 r·min-1离心20 min后,有机相离心浓缩至干,复溶后进行LC- MS/MS (Agilent1290/6460)[15]测定西咪替丁浓度。按照公式2计算西咪替丁的表观渗透系数。

${P_{app}} = dQ/\left( {dt\cdot A\cdot{C_0}} \right)$ (2)

式2中C0为西咪替丁在给药室的初始浓度 (20 μmol·L-1),dQ/dt为药物的转运速率。

按照公式3和4计算西咪替丁的外排率 (efflux ratio,ER) 和净外排率 (net efflux ratio,net ER)。

$ER = {P_{app\left( {BL \to AP} \right)}}/{P_{app\left( {AP \to BL} \right)}}$ (3)

式3中BL→AP表示底侧到顶侧方向,AP→BL表示顶侧到底侧方向。

$net ER = ER(transgenic cell) / ER(negative control cell)$ (4)
结果

1 pcDNA3.1-hMATE1重组质粒的制备

人肾总mRNA经逆转录PCR获得长度约1 700 bp的目的条带。胶回收纯化获得的纯化产物测序后,经比对与hMATE1 cDNA (GenBank accession number: NM_018242.2) 100% 匹配。转化入E.coli DH5α扩增后的pcDNA3.1(+)-hMATE1重组质粒,经HindⅢ、KpnⅠ双酶切和SalⅠ单酶切后进行琼脂糖凝胶电泳,结果 (图 1) 显示存在含hMATE1 cDNA片段长度插入片段的重组质粒; 经测序比对,1号菌株中重组质粒所含插入的基因片段为正确的hMATE1 cDNA。

Figure 1 Restriction enzyme analysis of pcDNA3.1(+)-hMATE1. Lane M1: Marker 2000; Lane P: PCR product; Lane 1-3: Double digestion with HindⅢ and KpnⅠ of plasmid extracted from clone 1-3; Lane M2: Marker 15 000; Lane 4-6: Single digestion with SalⅠ of plasmid extracted from clone 1-3
2 以荧光底物DAPI积聚筛选单克隆细胞株

MDCK-hMATE1 (51株)、MDCK-hOCT1/hMATE1 (37株) 和MDCK-hOCT2/hMATE1 (38株) 的单克隆细胞株,经荧光底物DAPI积聚实验初步筛选后,分别得到4、6和4株积聚量明显高于阴性对照细胞的单克隆细胞株。进一步考察经典底物MPP+ 在上述细胞株中的积聚量,结果 (图 2) 显示,单克隆细胞株对MPP+ 的积聚均明显高于各自的阴性对照细胞,且西咪替丁能显著抑制MPP+ 的积聚。挑选对MPP+ 积聚能力最强的MDCK-hMATE1 (M-M A28)、MDCK- hOCT1/hMATE1 (M-1/M 25) 和MDCK-hOCT2/hMATE1 (M-2/M 29) 细胞株,进行mRNA及功能验证。

Figure 2 Selection of MDCK-hMATE1 (a),MDCK-hOCT1/hMATE1 (b) and MDCK-hOCT2/hMATE1 (c) monoclones by MPP+ accumulation. 10 μmol·L-1 MPP+ was incubated with or without cimetidine for 3 min. MPP+ accumulation of each sample was calibrated with protein content,and define that of negative control cell (M-3.1,M-1/3.1 and M-2/3.1,separately) as 1. n = 3,x± s. P < 0.05,**P < 0.01,***P < 0.001 vs negative cell group; P < 0.05,△△P < 0.01,△△△P < 0.001 vs incubatedwithout cimetidine group
3 转运体mRNA含量的测定

提取各模型细胞中的总mRNA,经逆转录后进行相关转运体mRNA的含量测定。结果 (图 3) 显示,筛选出的MDCK-hMATE1 (M-M)、MDCK-hOCT1/ hMATE1 (M-1/M) 和MDCK-hOCT2/hMATE1 (M-2/M) 3种转基因细胞模型中均高表达hMATE1 mRNA,hMATE1 mRNA的表达量分别为阴性对照细胞的 7 300、27 600和2 400倍。

Figure 3 Quantification of hMATE1 mRNA in MDCK-hMATE1 (a),MDCK-hOCT1/hMATE1 (b) and MDCK-hOCT2/hMATE1 (c). Define hMATE1 mRNA content in negative control cell (M-3.1,M-1/3.1 and M-2/3.1,separately) as 1. n = 3,x± s. ***P < 0.001 vs negative control

4 MDCK-hMATE1对二甲双胍的积聚

以每毫克蛋白的积聚量表示二甲双胍在MDCK- hMATE1中的积聚,结果 (图 4) 显示,二甲双胍 (10 μmol·L-1) 在M-M中的积聚量为其阴性对照细胞 (M-3.1) 的17.6倍,西咪替丁 (100 μmol·L-1) 能显著减少其积聚量,表明所选择的MDCK-hMATE1模型具有显著的hMATE1功能。

Figure 4 Function verification of MDCK-hMATE1 by metformin accumulation. 10 μmol·L-1 metformin was incubated with or without 100 μmol·L-1 cimetidine for 3 min. Metformin accumulation of each sample was calibrated with protein content,and define that of negative control (M-3.1) as 1. n = 3,x± s. ***P < 0.001 vs negative control; △△△P < 0.001 vs incubated without cimetidine group
5 MDCK-hOCT1/hMATE1MDCK-hOCT2/ hMATE1对西咪替丁的转运

按照公式1计算荧光黄的Papp,选择Papp ≤ 5 × 10-7 cm·s-1的孔进行西咪替丁转运实验。

根据公式2计算西咪替丁的表观渗透系数,结果 (图 5) 显示,共表达hMATE1与hOCT1或hOCT2的细胞BL→AP方向转运西米替丁的能力显著高于其阴性对照细胞,同时,也显著高于AP→BL方向对西米替丁的转运。根据公式3计算得到M-1/M和M-2/M两个共表达细胞及各自阴性对照细胞对西咪替丁的外排率分别为ER(M-1/M) = 33.97,ER(M-1/3.1) = 1.94; ER(M-2/M) = 10.54,ER(M-2/3.1) = 2.89。

Figure 5 Transportation of cimetidine across MDCK-hOCT1/ hMATE1 and MDCK-hOCT2/hMATE1 cell monolayer. 100 μL buffer in reception tank was sampled every 20 min until transported up to 120 min. n = 3,x± s. **P < 0.01,***P < 0.001 vs negative control (M-1/3.1 and M-2/3.1,respectively) cell; △△P < 0.01,△△△P < 0.001 vs Papp(BL→AP)

根据公式4计算得到MDCK-hOCT1/hMATE1 和MDCK-hOCT2/hMATE1对西咪替丁的净外排率 分别为17.5和3.65,均大于2,表明MDCK-hOCT1/ hMATE1和MDCK-hOCT2/hMATE1可分别用于hMATE1与hOCT1和hMATE1与hOCT2共同底物的转运研究。

讨论

hMATE1是以反向质子跨膜电势差为驱动力的转运体,以肾小管上皮细胞为例,在正常生理状态 下,细胞内代谢产生的质子排泄到肾小管,随着水的重吸收,肾小管中质子的浓度越来越高,因此肾小管腔的质子浓度高于肾小管上皮细胞,因而有利于肾小管细胞排泄药物[1]。体外实验中,化合物须先进 入细胞,再由外排转运体排出,在稳定表达单一外排转运体的转基因细胞模型上,如果化合物通过被动转运进入细胞的量很少,外排转运体的作用很难体现,从而会限制转基因细胞模型的应用。因而可利用hMATE1以反向质子电势差为驱动力的特性,对细胞进行预处理,人为制造细胞内的质子浓度高于细胞外质子浓度的状态,翻转hMATE1的转运方向。因 此,本文采用含30 mmol·L-1 NH4Cl的AUB预孵育20 min,替换成AUB继续预孵育5 min的方法,增加细胞内H+浓度,将hMATE1转运方向变为摄取,简化体外研究hMATE1转运药物的过程。

二甲双胍主要以原形经肾脏排泄,除肾小球的滤过作用外,肾小管的分泌排泄也发挥重要作用。有报道OCT2与MATE1分别分布于同一肾小管上皮细胞的基底侧和刷状缘侧[5]。肾小管上皮细胞基底侧的OCT2可将二甲双胍从血液中摄取至肾小管上皮细胞,再由MATE1外排至肾小管中。有研究证实OCT2[17]和MATE1[18]的基因多态性与二甲双胍疗效个体差异的相关性,且二甲双胍在肾脏积累过多会导致乳酸性酸中毒,影响临床安全用药。二甲双胍口服吸收后,可由OCT1转运进入肝脏细胞发挥降血糖作用,有研究表明,OCT1的功能降低或缺失可显著降低二甲双胍的疗效[19]。因此,本文构建的共表达细胞模型可用于研究药物是否影响二甲双胍等OCTs和MATE1共同底物的跨细胞转运和细胞内积聚情况,为研究体内由OCTs和MATE1介导药物-药物相互作用提供依据。

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