【摘要】：吡咯里西啶生物堿(Pyrrolizidine alkaloids, PAs)廣泛分布于紫草科、菊科、豆科植物中。PAs可能是目前對(duì)人和動(dòng)物最重要的天然毒性成分,近50%的PAs為毒性化合物,其毒性表現(xiàn)為肝毒性、遺傳毒性和致癌性等,有的還有肺毒性。毒性PAs主要分為retronecine型和otonecine型,其中野百合堿和倒千里光堿為retronecine型肝毒PAs。大多數(shù)PAs本身無毒或低毒,經(jīng)CYP酶代謝激活后產(chǎn)生毒性。CYP主要分布在肝臟,因此,肝臟是PAs首要的毒性靶器官。但CYPs位于肝細(xì)胞的內(nèi)質(zhì)網(wǎng)膜上,PAs首先需通過一定途徑進(jìn)入肝細(xì)胞,才能被CYPs代謝。肝細(xì)胞膜表達(dá)多種轉(zhuǎn)運(yùn)體,可以介導(dǎo)藥物/化合物的細(xì)胞攝取及轉(zhuǎn)運(yùn),因此肝細(xì)胞膜上的轉(zhuǎn)運(yùn)體也可能參與PAs的肝臟攝取和轉(zhuǎn)運(yùn)。 SLC (solute carrier)22家族的有機(jī)陽離子轉(zhuǎn)運(yùn)體(organic cation transporters, OCTs),包括三個(gè)成員(OCT1-OCT3),它們對(duì)有機(jī)陽離子化合物、弱堿性化合物及少量中性化合物的細(xì)胞攝取起著關(guān)鍵作用。其中OCT1主要表達(dá)在肝臟,定位于肝細(xì)胞的基底側(cè)膜上,介導(dǎo)其底物化合物的肝臟攝取。由于部分PAs屬于弱堿性化合物,在生理?xiàng)lpH條件下可部分電離,推測(cè)它們可能與以O(shè)CT1為代表的肝臟陽離子轉(zhuǎn)運(yùn)體發(fā)生相互作用。本論文構(gòu)建并應(yīng)用穩(wěn)定表達(dá)hOCT1、CYP3A4的轉(zhuǎn)基因細(xì)胞模型,研究四種PAs與肝臟主要陽離子轉(zhuǎn)運(yùn)體的相互作用,并通過原代肝細(xì)胞及共表達(dá)CYP3A4與hOCTl的細(xì)胞,研究OCT1在MCT及RTS肝臟轉(zhuǎn)運(yùn)及毒性中的作用。 1.穩(wěn)定表達(dá)hOCT1的MDCK細(xì)胞模型的構(gòu)建 本章旨在建立穩(wěn)定表達(dá)人OCT1(hOCT1)野生型及兩個(gè)突變體的馬丁達(dá)比狗腎上皮(Madin-Darby canine kidney, MDCK)細(xì)胞模型。首先從人肝組織中提取hOCT1野生型基因,經(jīng)定點(diǎn)突變獲得]hOCT1P341L, hOCT1M420del兩個(gè)突變型基因,構(gòu)建表達(dá)質(zhì)粒pcDNA3.1(+)-hOCT1, pcDNA3.1(+)-hOCT1P341L, pcDNA3.1(+)-hOCT1M420del。將質(zhì)粒轉(zhuǎn)染MDCK細(xì)胞,通過G418篩選獲得抗性克隆,并通過hOCT1的熒光底物4-(4-(dimethylamino)-styryl)-N-methylpyridinium(ASP+)及抑制劑四乙胺(TEA),篩選得到具有較高活性的MDCK-hOCTl,-hOCT1p341L,-hOCT1M420del單克隆細(xì)胞。經(jīng)反轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)及經(jīng)典底物1-methyl-4-phenylpyridinium (MPP+)和二甲雙胍的攝取實(shí)驗(yàn),鑒定篩選得到的單克隆細(xì)胞株hOCT1mRNA的表達(dá)及功能。結(jié)果顯示,本研究獲得的hOCT1野生型及兩個(gè)突變體細(xì)胞模型與mock細(xì)胞相比,其hOCT1mRNA表達(dá)量顯著增高,其對(duì)經(jīng)典底物MPP+及二甲雙胍的攝取也顯著升高,MDCK-hOCT1,-hOCT1P341L,-hOCT1M420del三種細(xì)胞對(duì)二甲雙胍的積聚動(dòng)力學(xué)參數(shù)分別為791.5±24.1,779.1±165.3,537.5±62.8(Vmax, pmol/mg protein/min);409.0±55.1,523.2±36.3,913.4±99.1(Km,μmol/L);1.94,1.49,0.59(Clint, Vmax/Km)。上述結(jié)果表明,穩(wěn)定表達(dá)hOCT1的細(xì)胞模型構(gòu)建成功,可以作為研究hOCT1與藥物相互作用的細(xì)胞模型。 2.PAs與肝臟陽離子轉(zhuǎn)運(yùn)體的相互作用研究 本章利用MDCK-hOCT1細(xì)胞模型,首先考察了野百合堿(MCT)、Isoline、倒千里光堿(RTS)、千里光寧四種PAs,對(duì)hOCT1介導(dǎo)的MPP+攝取的抑制作用。結(jié)果表明,四種PAs均為hOCT1的抑制劑,IC50值分別為：MCT為5.52μmol/L,Isoline為5.35μmol/L, RTS為2.25μmol/L,千里光寧為3.50μmol/L。進(jìn)一步對(duì)上述PAs在MDCK-hOCT1及mock細(xì)胞中的積聚進(jìn)行研究,發(fā)現(xiàn)Isoline及千里光寧在兩種細(xì)胞的積聚無顯著差異,而MCT及RTS在MDCK-hOCTl細(xì)胞中的積聚顯著高于mock細(xì)胞,且兩者在MDCK-hOCT1細(xì)胞內(nèi)的積聚可以被OCT1的抑制劑,TEA、ASP+、奎尼丁、右旋延胡索乙素((+)-THP)抑制,提示MCT及RTS均為hOCTl的底物；進(jìn)一步研究獲得MCT和RTS在MDCK-hOCTl中積聚的動(dòng)力學(xué)參數(shù),Km值分別為25.0±6.7μmol/L,23.6±3.0μmol/L; Vmax值分別為266.0±63.9pmol/mg protein/min,209.9±69.3pmol/mg protein/min。 此外,我們還應(yīng)用穩(wěn)定表達(dá)hOCT2、hOCT3.人多藥及毒素外排蛋白轉(zhuǎn)運(yùn)體1(hMATEl)、或P-糖蛋白(P-gp, MDR1)的MDCK細(xì)胞,及穩(wěn)定表達(dá)人乳腺癌耐藥蛋白(BCRP)的LLC-PK1細(xì)胞模型,考察MCT及RTS是否為上述轉(zhuǎn)運(yùn)體的底物,以闡明除OCT1外,是否還有其它轉(zhuǎn)運(yùn)體參與MCT及RTS的肝臟轉(zhuǎn)運(yùn)。結(jié)果顯示,MCT及RTS不是或僅是上述轉(zhuǎn)運(yùn)體的弱底物。 最后,我們應(yīng)用原代培養(yǎng)的大鼠肝細(xì)胞(PRCH),進(jìn)一步驗(yàn)證OCT1在MCT和RTS肝細(xì)胞處置中的作用。結(jié)果顯示,MCT及RTS在肝細(xì)胞內(nèi)的積聚均可被OCT1的抑制劑,(+)-THP或奎尼丁抑制；MCT、RTS均明顯降低肝細(xì)胞存活率,升高培養(yǎng)基中乳酸脫氫酶(LDH)活力,(+)-THP、奎尼丁可顯著削弱MCT和RTS引起細(xì)胞存活降低及LDH活力升高。上述結(jié)果說明,MCT及RTS具有肝細(xì)胞毒性,OCT1介導(dǎo)MCT及RTS的肝細(xì)胞攝取,OCT1抑制劑對(duì)抗MCT及RTS的肝細(xì)胞毒性。 3.共表達(dá)hOCTl及CYP3A4細(xì)胞模型的構(gòu)建 雙轉(zhuǎn)染、三轉(zhuǎn)染甚至四重轉(zhuǎn)染轉(zhuǎn)運(yùn)體或/和代謝酶的細(xì)胞模型被認(rèn)為是研究幾個(gè)蛋白在肝臟藥物處置中共同作用的有效工具。OCT1和CYP3A4均在肝細(xì)胞內(nèi)高表達(dá),且兩者的底物和抑制劑譜有一定的重合性,我們推測(cè)兩者在MCT和RTS致肝毒中共同發(fā)揮作用。為了給上述假設(shè)的驗(yàn)證提供理想的研究模型,本章建立了穩(wěn)定單表達(dá)CYP3A4及共表達(dá)hOCT1、CYP3A4的細(xì)胞模型。在已有單轉(zhuǎn)染細(xì)胞MDCK-hOCT1, MDCK-pcDNA3.1(+)的基礎(chǔ)上,繼續(xù)向細(xì)胞轉(zhuǎn)入CYP3A4的表達(dá)質(zhì)粒(pcDNA3.1(+)-Hygro-CYP3A4)或其相應(yīng)的空載體(pcDNA3.1(+)-Hygro empty vector)。經(jīng)G418及潮霉素B雙重抗性篩選,獲得單克隆細(xì)胞,通過Western blot和定量PCR (quantitative Real-time PCR)挑選并鑒定陽性克隆。陽性克隆中hOCTl的功能通過熒光底物ASP+的積聚和抑制劑的抑制實(shí)驗(yàn)鑒定,CYP3A4的功能則通過P450-Glo CYP3A4Assay (Luciferin-IPA, CYP3A4的靈敏底物)鑒定。經(jīng)過篩選,共獲得四種細(xì)胞MDCK-mock, MDCK-hOCT1, MDCK-CYP3A4, MDCK-hOCT1-CYP3A4。功能鑒定結(jié)果顯示,MDCK-hOCT1及MDCK-hOCT1-CYP3A4的ASP+積聚量均約為mock細(xì)胞的16倍；而MDCK-CYP3A4和MDCK-hOCT1-CYP3A4細(xì)胞中CYP3A4的活力均約為mock細(xì)胞的66倍。上述結(jié)果表明,挑選出的陽性克隆均表達(dá)了相應(yīng)的目的蛋白,細(xì)胞模型可用于研究hOCTl及CYP3A4各自及共同作用。 4. hOCTl及CYP3A4在RTS致細(xì)胞毒中的作用 由于OCT1對(duì)RTS的肝臟攝取起重要作用,同時(shí)有報(bào)道指出CYP3A4可能介導(dǎo)RTS的代謝激活,鑒于人肝臟同時(shí)表達(dá)OCT1及CYP3A4,本章應(yīng)用單表達(dá)及共表達(dá)hOCT1、CYP3A4的細(xì)胞模型,考察RTS的細(xì)胞毒性,以闡明hOCT1及CYP3A4在RTS致肝細(xì)胞毒性中的作用。將不同濃度的RTS分別與mock, MDCK-hOCT1, MDCK-CYP3A4, MDCK-hOCTl-CYP3A4細(xì)胞共孵育,通過細(xì)胞形態(tài)觀察、MTT實(shí)驗(yàn)及流式細(xì)胞術(shù)測(cè)定細(xì)胞周期,比較RTS對(duì)不同細(xì)胞的毒性差異。結(jié)果顯示,RTS對(duì)mock及MDCK-hOCT1細(xì)胞未顯示明顯的毒性,而對(duì)MDCK-CYP3A4細(xì)胞,顯示出時(shí)間、濃度依賴性的細(xì)胞損傷,提示CYP3A4介導(dǎo)的代謝激活是RTS發(fā)揮毒性的關(guān)鍵步驟；此外,RTS對(duì)MDCK-hOCT1-CYP3A4細(xì)胞的毒性明顯大于對(duì)其它細(xì)胞的毒性,提示OCT1介導(dǎo)的攝取及CYP3A4介導(dǎo)的代謝激活,對(duì)RTS的細(xì)胞毒性均起了重要作用。RTS的毒性表現(xiàn)出以下特點(diǎn)：細(xì)胞、細(xì)胞核顯著增大,細(xì)胞生長(zhǎng)被顯著抑制；細(xì)胞周期分析實(shí)驗(yàn)則顯示RTS誘導(dǎo)細(xì)胞G2/M期的阻滯從而抑制有絲分裂。對(duì)于RTS毒性機(jī)制的了解可以為我們尋找RTS的解毒方法提供新的策略。
[Abstract]:Pyrrolizidine alkaloids (PAs) are widely distributed in Arnebiaceae, Compositae and Legumes. PAs may be the most important natural toxic components in humans and animals. Nearly 50% of PAs are toxic compounds. Their toxicity manifests as hepatotoxicity, genetic toxicity and carcinogenicity, and some are also pulmonary toxicity. Roecine and otonecine, of which monocrotaline and castrarizine are retronecine-type hepatotoxic PAs. Most PAs are non-toxic or low toxic and produce toxicity after metabolic activation by CYP enzymes. CYP mainly distributes in the liver, therefore, the liver is the primary toxic target organ of PAs. However, CYPs are located on the endoplasmic reticulum of hepatocytes, and PAs must first pass a certain route. The hepatocyte membrane expresses a variety of transporters, which can mediate the uptake and transport of drugs and compounds. Therefore, the transporters on the hepatocyte membrane may also participate in the uptake and transport of PAs in the liver.
Organic cation transporters (OCTs) of the SLC (solute carrier) 22 family, including three members (OCT1-OCT3), play a key role in cellular uptake of organic cationic compounds, weak alkaline compounds and a small number of neutral compounds. OCT1 is mainly expressed in the liver and localized on the basolateral membrane of hepatocytes. Because some PAs belong to weak alkaline compounds, they can be partially ionized at pH in physiological strips. It is speculated that they may interact with OCT1 as a representative of liver cationic transporters. The interaction of major cation transporters in the viscera and the role of OCT1 in the transport and toxicity of MCT and RTS in the liver were studied by primary hepatocytes and co-expression of CYP3A4 and hOCTl.
1. construction of MDCK cell model with stable expression of hOCT1
The aim of this chapter is to establish a stable expression model of human OCT1 wild-type and two mutants in Madin-Darby canine kidney (MDCK) cells. Firstly, the wild-type genes of hOCT1 were extracted from human liver tissues, and two mutant genes, hOCT1P341L and hOCT1M420del, were obtained by site-directed mutagenesis. MDCK cells were transfected with pcDNA3.1 (+) - hOCT1P341L and pcDNA3.1 (+) - hOCT1M420del. Resistant clones were obtained by G418 screening. Monoclonal MDCK cells with high activity were screened by hOCT1 fluorescent substrate 4 - (dimethylamino) - styryl - N - methylpyridinium (ASP +) and inhibitor tetraethylamine (TEA). Cells. The expression and function of hOCT1 mRNA were identified by reverse transcription polymerase chain reaction (RT-PCR) and the uptake of 1-methyl-4-phenylpyridinium (MPP+) and metformin. The results showed that the wild type and two mutant cell models of hOCT1 obtained in this study were compared with mock cells. The accumulation kinetic parameters of metformin in MDCK-hOCT1, -hOCT1P341L, -hOCT1M420del cells were 791.5 [24.1], 779.1 [165.3], 537.5 [62.8] (Vmax, pmol / mg protein / min), 409.0 [55.1], 523.2 [36.3] and 913.4 [99.1] respectively. These results indicate that the cell model stably expressing hOCT1 has been successfully constructed and can be used as a cell model to study the interaction between hOCT1 and drugs.
Interaction between 2.PAs and hepatic cationic transporters
In this chapter, MDCK-hOCT1 cell model was used to investigate the inhibitory effects of four kinds of PAs, monocrotaline (MCT), Isoline, Rotundine (RTS) and seneclinine, on the uptake of MPP+ mediated by hOCT1. The results showed that all of the four PAs were inhibitors of hOCT1. IC50 values were MCT 5.52 micromol/L, Isoline 5.35 micromol/L, RTS 2.25 micromol/L, seneclinine 5.25 micromol/L, respectively. The accumulation of PAs in MDK-hOCT1 and mock cells was further studied. It was found that there was no significant difference in the accumulation of Isoline and Seneclonin between the two kinds of cells, while the accumulation of MCT and RTS in MDK-hOCTl cells was significantly higher than that in mock cells. The accumulation of PAs in MDK-hOCT1 cells could be inhibited by OCT1 inhibitors, TEA, ASP+, quinine. Nicotine and dextran tetrahydropalmatine (+) - THP inhibition suggested that both MCT and RTS were substrates of hOCTl. The kinetic parameters of accumulation of MCT and RTS in MDCK-hOCTl were obtained by further study. The Km values were 25.0 (6.7) micromol/L, 23.6 (3.0) micromol/L, Vmax values were 266.0 (63.9) pmol/mg protein/min, 209.9 (69.3) pmol/mg protein/min, respectively.
In addition, we investigated whether MCT and RTS were the substrates of these transporters by stably expressing hOCT2, hOCT3, human multidrug and toxin efflux protein transporter 1 (hMATEl), or P-glycoprotein (P-gp, MDR1) MDK cells, and LC-PK1 cells stably expressing human breast cancer resistance protein (BCRP). The liver transport of MCT and RTS showed that MCT and RTS were not or only the weak substrates of the transporters.
The results showed that the accumulation of MCT and RTS in hepatocytes could be inhibited by OCT1 inhibitors, (+) - THP or quinidine. Both MCT and RTS significantly decreased the survival rate of hepatocytes and increased the activity of lactate dehydrogenase (LDH) in the medium. These results suggest that MCT and RTS have hepatotoxicity, OCT1 mediates hepatocyte uptake of MCT and RTS, and OCT1 inhibitors antagonize hepatotoxicity of MCT and RTS.
3. co expression of hOCTl and CYP3A4 cell models
Cell models of double, triple or even quadruple transfection transporters or/and metabolic enzymes are considered to be effective tools for studying the interaction of several proteins in liver drug disposal. OCT1 and CYP3A4 are highly expressed in hepatocytes, and their substrates and inhibitor profiles have some coincidence. We speculate that OCT1 and CYP3A4 are involved in hepatotoxicity induced by MCT and RTS. In order to provide an ideal research model for validating the above hypothesis, a stable single-expression CYP3A4 and co-expression of hOCT1, CYP3A4 cell model were established. On the basis of existing single-transfected cells MDCK-hOCT1, MDCK-pcDNA3.1 (+), CYP3A4 expression plasmids (pcDNA3.1 (+) - Hygro-CYP3A4) or their corresponding plasmids were transfected into the cells. Vacuum vector pcDNA3.1 (+) - Hygro empty vector. Monoclonal cells were screened by G418 and hygromycin B. Positive clones were selected and identified by Western blot and quantitative Real-time PCR. The function of hOCTl in the positive clones was identified by fluorescent substrate ASP + accumulation and inhibitor inhibition test, and the function of CYP3A4 was identified. P450-Glo CYP3A4Assay (sensitive substrate of Luciferin-IPA, CYP3A4) was used to identify the four cell lines. After screening, MDCK-mock, MDCK-hOCT1, MDCK-CYP3A4, MDCK-hOCT1-CYP3A4, MDCK-hOCT1-CYP3A4, MDCK-hOCT1-CYP3A4, MDCK-hOCT1 and MDCK-hOCT1-CYP3A4 were obtained. The results of functional identification showed that the accumulation of ASP + in MDCK-hOCT1 and MDCK-hOCT1-CYP3A4 was 16 times as much as that in moCK-CYP3A4. CYP3A4 activity in A4 cells was 66 times higher than that in mock cells. These results showed that the selected positive clones expressed the corresponding target proteins. Cell models could be used to study the interaction of hOCTl and CYP3A4.
4. the role of hOCTl and CYP3A4 in cytotoxicity induced by RTS
Because OCT1 plays an important role in the uptake of RTS by liver, it has been reported that CYP3A4 may mediate the activation of RTS metabolism. In view of the simultaneous expression of OCT1 and CYP3A4 in human liver, this chapter investigated the cytotoxicity of RTS by using a single expression and co-expression cell model of hOCT1 and CYP3A4 to clarify the role of hOCT1 and CYP3A4 in RTS-induced hepatotoxicity. RTS at the same concentration were co-incubated with mock, MDCK-hOCT1, MDCK-CYP3A4, MDCK-hOCTl-CYP3A4 cells respectively. Cell cycle was determined by cell morphology, MTT assay and flow cytometry. The results showed that RTS had no significant toxicity to mock and MDCK-hOCT1 cells, but to MDCK-CYP3A4 cells. Time-and concentration-dependent cell damage suggests that CYP3A4-mediated metabolic activation is a key step in the toxicity of RTS; moreover, the toxicity of RTS to MDCK-hOCT1-CYP3A4 cells is significantly greater than that to other cells, suggesting that OCT1-mediated uptake and CYP3A4-mediated metabolic activation play an important role in the cytotoxicity of RTS. Cell cycle analysis showed that RTS could induce G2/M phase arrest and inhibit mitosis. The understanding of the toxic mechanism of RTS could provide a new strategy for the detoxification of RTS.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R917

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相關(guān)期刊論文 前1條

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