三磷酸腺苷结合盒转运体A1在转基因小鼠中的作用

1999年人们发现Tangier病病人缺乏三磷酸腺苷结合盒转运体A1(ATP binding cassette transporter A1，ABCAl)基因，Tangier病以血浆高密度脂蛋白胆固醇(HDL-C)降低、组织巨噬细胞内胆固醇聚积和心血管疾病事件增加为特征。此后，许多实验室为进一步了解ABCA1的功能作了大量的工作。研究发现ABCAl促进胆固醇逆转运的第一步，即细胞内胆固醇和磷脂流出到载脂蛋白接受体。     

膜转运蛋白三磷酸腺苷结合盒转运体A1的表达调控与信号传导

<正>动脉硬化性心血管疾病(atherosclerotic cardiovascular disease,CVD)是最常见的死亡原因。高密度脂蛋白(High density lipoprotein,HDL)主要通过转运动脉巨噬细胞多余的胆固醇到肝脏代谢来预防CVD。这个途径的第1步称为胆固醇逆转运,由整合膜蛋白——三磷酸腺苷结合盒转运体A1(ATP-binding cassette transporter A1,ABCA1)介导。ABCA1转运细胞的胆固醇和磷脂到乏脂载脂蛋白     

三磷酸腺苷结合盒转运蛋白G2与痛风/高尿酸血症

三磷酸腺苷结合盒转运蛋白G2 (ABCG2)是ABC转运蛋白超家族成员之一,编码基因位于4号染色体上,为定位于细胞膜的转运蛋白,在肝、肾、肠等组织广泛表达,具有转运尿酸的功能,其功能异常会导致尿酸排泄减少.目前已经发现,多种单基因多态性影响ABCG2的尿酸转运功能,从而引起高尿酸血症和痛风.所以研究ABCG2靶点药物意义重大,但仍需进一步研究.     

三磷酸腺苷结合盒转运体A1在动脉粥样硬化发病学中的作用

三磷酸腺苷结合盒转运体 (ATP binding cassette trans-porter,ABC)超家族目前已发现有 6个家族分别为 A、B、C、D、E、F,共 4 8个成员 ,其中 A家族 12个成员〔1〕。尽管人 ABCA1基因在 1994年就已经克隆获得 ,但对其功能的认识直到 1999年才得到突破。ABCA1是一种整合膜蛋白 ,它以 ATP为能源 ,促进细胞内游离胆固醇和磷脂的流出。结合到细胞表面的载脂蛋白 A- I与经 ABCA1转运出的游离胆固醇和磷脂结合 ,形成新生的 HDL。由于 ABCA1在胆固醇逆转运 (RCT)和 HDL生成的起始步骤中起重要作用 ,被称作 RCT的守门者 (gatekee…     

三磷酸腺苷结合盒转运体A1的组织特异性功能与动脉粥样硬化

动脉粥样硬化是一种脂质沉积引发的慢性血管炎症。动脉粥样硬化的发生发展与细胞胆固醇稳态的破坏密切相关。三磷酸腺苷结合盒转运体A1(ABCA1)介导了细胞胆固醇流出至载脂蛋白AⅠ、preβ高密度脂蛋白(HDL)和HDL₃。当该通路胆固醇流出障碍可致细胞内胆固醇沉积。更重要的是,ABCA1的功能障碍还影响着动脉粥样硬化斑块的生长及临床冠心病的发生。最近研究显示ABCA1对动脉粥样硬化的调控作用具有组织特异性。本文介绍了胆固醇逆向转运与动脉粥样硬化的相关性,并着重讨论了ABCA1的组织特异性功能及其调控动脉粥样硬化的作用和机制。     

三磷酸腺苷结合盒转运体A7的结构、功能和调控的研究进展

三磷酸腺苷结合盒转运体A7是三磷酸腺苷结合盒转运体家族中新发现的一个和三磷酸腺苷结合盒转运体A1最高同源性的蛋白,体内生理作用尚不清楚。许多研究表明,同三磷酸腺苷结合盒转运体A1对载脂蛋白介导生成高密度脂蛋白作用相似,三磷酸腺苷结合盒转运体A7主要通过影响磷脂或胆固醇的流出参与胆固醇的逆转运过程。但在组织分布和调控等方面又异于三磷酸腺苷结合盒转运体A1,可能具有其他特殊作用。深入研究三磷酸腺苷结合盒转运体A7的功能及其表达的调控机制,对于脂代谢、动脉粥样硬化、冠心病的治疗具有十分重要的意义。     

三磷酸腺苷结合盒转运子A7在脂质转运过程中作用的研究

目的:探讨三磷酸腺苷结合盒转运子A7(ABCA7)在细胞内脂质流出过程中的作用。方法:以apoAI刺激转染ABCA7-或ABCA1-基因的HEK293细胞24h,利用蛋白印迹法以及酶分析法分别测定ABCA7的变化与细胞内胆固醇和磷脂的流出。结果:apoAI分别上调ABCA7或ABCA1蛋白量,ABCA7与ABCA1同样促进细胞内脂质的转运,其中磷脂的流出较胆固醇流出更为明显。结论:ABCA7与ABCA1同样具有促进细胞内脂质转运的功能。     

三氧化二砷逆转肝癌细胞株HepG2/ADM多药耐药的作用

目的:探讨三氧化二砷(As2O3)体外逆转人肝癌细胞多药耐药性的作用及机制．方法:MTT法检测As2O3的细胞毒作用和处理前后耐药细胞对化疗药物的敏感性,用流式细胞仪检测细胞内阿霉素浓度,通过RT-RCR检测MDR1基因的表达．结果:As2O3在0．25 mg／L剂量以下时对HepG2和HepG2／ADM耐药细胞株的抑制率均小于15％,半数抑制率(IC50)分别为1．02和1．34 mg／L,无细胞毒剂量0．2 mg/L的As2O3能部分逆转HepG2／ADM细胞对阿霉素、顺铂(CDDP)、丝裂霉素(MMC)、5-氟尿嘧啶(5-FU)的耐药性,逆转倍数分别为2．92,3．09,2．13,2．60倍．同时无细胞毒剂量0．2 mg／L的As2O3能使HepG2／ADM细胞内阿霉素浓度明显增加,MDR1表达下降．结论:As2O3具有体外逆转人肝癌细胞多药耐药性的作用,可能与下调MDR1表达、增加细胞内药物积累有关．     

三磷酸腺苷结合盒转运子基因R219K多态与脑梗死的关系

目的探讨脂代谢相关基因三磷酸腺苷结合盒转运子(ABCA1)的R219K多态与脑梗死及血脂的关系。方法收集国内8家医院神经内科收治的脑梗死患者177例(脑梗死组),另选健康体检者234例(对照组)。全部患者经过头颅MRI或CT确诊,生化指标经全自动生化仪统一测定,用PCR-RFLP方法分析R219K多态。结果ABCA1基因R219K基因型分布为RR 20.20%、RK 52.55%、KK 27.25%。ABCA1基因R219K多态R等位基因频率为46.47%,K等位基因频率为53.53%。ABCA1基因的R219K多态RR型及R等位基因在脑梗死组分布较对照组多,但差异无统计学意义(P>0.05)。性别分层后比较发现,男性R219K多态RR型及R等位基因在脑梗死组分布较对照组明显增多,差异有统计学意义(P<0.05);男性脑梗死患者R219K多态RR型及R等位基因较女性脑梗死患者明显增多,差异有统计学意义(P<0.05);这种差异在脑梗死组女性与对照组女性中不存在。ABCA1基因的R219K多态在人群中和脑梗死患者中各基因型间血脂水平无统计学差异。结论ABCA1基因的R219K多态RR型可能是男性患脑梗死的易感基因,其机制可能与血脂水平无关。     

多药耐药基因反义寡核苷酸逆转肝癌细胞耐药的研究

目的 观察反义硫代磷酸酯寡核昔酸(AsODN)联合逆转耐药肝癌细胞多药耐药基因1(MDR1)和多药耐药相关蛋白基因(MRP)的作用。 方法 用人工合成互补于MDR1基因及MRP基因的反义20聚硫代磷酸寡核苷酸,以脂质体作载体,转染入耐阿霉素(ADM)肝癌细胞SMMC-7721/ADM,四甲基偶氮唑蓝法测定细胞对化学疗法药物的敏感性,流式细胞仪分析细胞相对荧光强度,激光扫描共聚焦显微镜测定细胞内Rhdaming123(Rh123)及柔红霉素(DNR)潴留以反映蛋白质p170和p190功能。 结果 ASODN/MDR1 MRP联合转染SMMC-7721/ADM细胞,能更大程度增加细胞对ADM(47.8倍)和DNR(21.6倍)的敏感性。ASODN/MDR1 MRP联合转染SMMC-7721/ADM细胞,与单独任一种ASODN转染相比,对p170或p190表达的抑制并不增加(q值分别为3.23、3.24,P>0.05)。 结论 针对MDR1 MRP的ASODN联合转染SMMC-7721/ADM细胞,能更大程度逆转肝癌细胞的耐药性。     

ATP binding cassette transporters and drug resistance in breast cancer

Resistance to chemotherapy is a critical issue in the management of breast cancer patients. The nature of clinical drug resistance is likely to be multifactorial. However, in the last decade considerable attention has been dedicated to the role played by membrane transporter proteins belonging to the ATP binding cassette protein superfamily, and in particular by the MDR1 product P-glycoprotein (Pgp) and the multidrug resistance protein (MRP1). Heterogeneity of results is a common feature of studies evaluating the expression and prognostic role of these proteins, due to both methodological and biological factors. Nonetheless, Pgp and MRP1 are detected in a significant proportion of untreated breast cancers (on average 40 and 50% respectively, by immunohistochemistry), without a clear and consistent association with cancer stage. Exposure to chemotherapy increases the expression of both proteins. In vitro studies on primary cultures of breast cancer cells obtained at surgery consistently show an association between Pgp (protein) or MDR1 (mRNA) expression and resistance to chemotherapy. However, the correlation with clinical drug resistance is not as well defined. A stronger association of Pgp/MDR1 with response rates has been observed when expression or an increase in expression are detected immediately following chemotherapy. Correlations with prognosis appear more evident in studies using immunohistochemistry, in adjuvant and neoadjuvant settings. Evidence of clinical reversal of drug resistance by verapamil suggests a functional role of Pgp in drug resistance, although the significance of the evidence is generally weakened by poor trial designs. Future studies should take into account the multifactorial nature of drug resistance in breast cancer and use standardized approaches with adequate controls. Expression studies should be complemented by well-designed trials of drug-resistance reversal using target-specific chemosensitizing agents, and relating the results to the levels of expression of the target proteins.     

ABC转运蛋白在大鼠肝脏卵圆细胞中的表达

目的研究ABC转运蛋白基因家族的三个主要成员MDR1、MRP1和Bcrp1基因在大鼠肝脏卵圆细胞中的表达及意义。方法建立大鼠2-乙酰氨基芴／三分之二肝切除模型，两步胶原酶灌注结合Percoll密度梯度离心分离大鼠肝脏卯圆细胞和肝细胞，采用免疫组织化学染色检测大鼠肝脏组织中MDR1、MRP1、Bcrp1转运蛋白的表达；采用荧光定量PCR方法检测MDR1、MRP1和Bcrp1基因mRNA在卵圆细胞和肝细胞中的表达水平。结果免疫组织化学染色显示大鼠肝脏组织中MDR1表达位于门静脉区附近，呈放射状分布，Bcrp1表达定位在细胞膜上。大鼠肝脏卵圆细胞MDR1、MRP1和Bcrp1基因mRNA的表达水平分别是肝细胞的9倍、1．5倍和13．8倍。结论卵圆细胞表达高水平的ABC转运蛋白，后者参与卵圆细胞免受外源性化学物质损伤的自我保护机制。     

ATP binding cassette transporter G1 (ABCG1) is an intracellular sterol transporter

Four members of the mammalian ATP binding cassette (ABC) transporter G subfamily are thought to be involved in transmembrane (TM) transport of sterols. The residues responsible for this transport are unknown. The mechanism of action of ABCG1 is controversial and it has been proposed to act at the plasma membrane to facilitate the efflux of cellular sterols to exogenous high-density lipoprotein (HDL). Here we show that ABCG1 function is dependent on localization to intracellular endosomes. Importantly, localization to the endosome pathway distinguishes ABCG1 and/or ABCG4 from all other mammalian members of this superfamily, including other sterol transporters. We have identified critical residues within the TM domains of ABCG1 that are both essential for sterol transport and conserved in some other members of the ABCG subfamily and/or the insulin-induced gene 2 (INSIG-2). Our conclusions are based on studies in which (i) biotinylation of peritoneal macrophages showed that endogenous ABCG1 is intracellular and undetectable at the cell surface, (ii) a chimeric protein containing the TM of ABCG1 and the cytoplasmic domains of the nonsterol transporter ABCG2 is both targeted to endosomes and functional, and (iii) ABCG1 colocalizes with multiple proteins that mark late endosomes and recycling endosomes. Mutagenesis studies identify critical residues in the TM domains that are important for ABCG1 to alter sterol efflux, induce sterol regulatory element binding protein-2 (SREBP-2) processing, and selectively attenuate the oxysterol-mediated repression of SREBP-2 processing. Our data demonstrate that ABCG1 is an intracellular sterol transporter that localizes to endocytic vesicles to facilitate the redistribution of specific intracellular sterols away from the endoplasmic reticulum (ER).     

Multidrug resistance reversal in human gastric carcinoma cells by neferine

AIIVI: To investigate the reversal effect of neferine on multidrug resistance in human gastric carcinoma cell line. METHODS: Cells of a human gastric cancer cells line, SGC7901, and its vincristine (VCR) -resistant variant, SGC7901/VCR, were cultivated with or without neferine and/or VCR. The cytotoxic effect of VCR was evaluated by the MTT assay. Cell apoptosis induced by VCR was determined by flow cytometry(FCM). The expression of P-glycoprotein (P-gp) and a multidrug-resistance-associated protein (MRP) in cells was examined by immunofluorescence and FCM. RESULTS: Neferine at the concentration from 2.5 μmol/L to 10 μmol/L had no cytotoxicity to SGC7901 cells, and its variant SGC7901/VCR cells. The ICso of VCR against SGC7901 and SGC7901/VCR cells was 0.059 μg/mL and 2.32 μg/mL, respectively, indicating that SGC7901/VCR cells were 39 times more resistant to VCR than its parent SGC7 901 cells. After treatment with neferine at concentrations of 2.5, 5 and 10 μmol/L, the IC50 of VCR to SGC7901/VCR cell line decreased to 0.340, 0.128 and 0.053 μg/mL, respectively,thus, increased the chemosensitivity by 6.8-, 18.1- and 43.8-fold, respectively. SGC7901/VCR cells were apoptosis resistant to VCR. Neferine (2.5, 5 and 10 μmol/L) promoted the VCR-induced apoptosis of SGC7901/VCR cells in a dosedependent manner. The expressions of P-gp and MRP were strongly positive in SGC7901/VCR cells, which were significantly down-regulated after treatment with neferine (10 μmol/L)for 24 h. CONCLUSION: Neferine reverses multidrug resistance of human gastric carcinoma SGC7901/VCR cells, which may be associated with the down-regulations of P-gp and MRP expression in SGC701/VCR cells.     

Ghrelin对THP-1源性泡沫细胞ATP结合盒转运子G1的调控作用及途径

目的 探讨Ghrelin对人单核细胞系(THP-1)源性泡沫细胞ATP结合盒转运子G1(ABCG1)表达的调控作用及其可能途径.方法 体外培养THP-1,由佛波酯(PMA)作用使其分化为巨噬细胞,继而在氧化低密度脂蛋白(ox-LDL)存在条件下进一步转变为泡沫细胞,油红O染色法鉴定泡沫细胞形成.运用RT-PCR法和Western印迹法分别检测Ghrelin干预后及封闭过氧化物酶体增生物激活受体γ(PPARγ)后ABCG1 mRNA与蛋白表达.采用酶法,通过荧光分光光度计检测胆固醇含量及胆固醇流出率.结果 Ghrelin可明显减少细胞内脂滴的形成,增加胆固醇流出,并能显著增加单核/巨噬细胞泡沫化过程中ABCG1 mRNA水平和蛋白表达,此作用呈浓度依赖性.封闭PPARγ后,Ghrelin抑制泡沫细胞形成的效应被阻断,且PPARγ拮抗剂浓度越高,该阻断作用越明显.结论 Ghrelin可能通过上调ABCG1转录翻译水平延缓动脉粥样硬化的发生,且该效应可能通过PPARγ途径发挥作用.     

抑制HSP70—2基因表达诱导肝癌细胞G2／M期阻滞的机制研究

目的探讨HSP70—2在肝癌组织中的表达，以及抑制HSP70—2表达对肝癌细胞生长和周期影响的详细作用机制。方法免疫组织化学检测HSP70—2在45例肝癌组织和癌旁组织中的表达，Westernblot检测HSP70—2在5种肝癌细胞株中的表达。设计合成针对HSP70—2基因的特异性shRNA，构建真核表达载体HSP70-2shRNA1和HSP70—2shRNA2。转染肝癌细胞后，MTT法检测细胞增殖，流式细胞仪检测细胞周期，Westernblot检测HSP70—2表达及细胞周期相关蛋白p-Cdc2（Tyr15）、Cdc25C、Cdc2和cyclinB1的表达变化。结果免疫组织化学检测显示，45例肝癌组织中有33例（73．3％）HSP70—2蛋白表达阳性，45例癌旁组织中仅4例（8．9％）表达阳性，表达差异有显著性（P〈0．05）。HSP70—2蛋白在HepG2、Bel-402、Huh-7、Hep3B、SMMC-7721肝癌细胞中的表达明显高于其在L02细胞中的表达。HSP70—2shRNA1和shRNA2均能有效抑制肝癌细胞HepG2和Bel-7402中HSP70-2的表达。与controlshRNA组相比，转染HSP70-2shRNA1和shRNA2组HepG2和Bel-7402细胞生长增殖速度均明显减慢，细胞周期表现为处于G1／M期的细胞比例显著增加。Westernblot检测发现，转染HSP70-2shRNA1和shRNA2后肝癌细胞中磷酸化的p-Cdc2（Tyr15）表达增加，Cdc25C、Cdc2和cyclinB1表达显著减少。结论HSPT0-2在肝癌中过表达，抑制HSP70．2表达可以显著抑制肝癌细胞的增殖，诱导细胞周期阻滞，其诱导G，／M期阻滞的机制是通过影响Cdc25C-Cdc2／cyclinB1通路来实现的。     

Torin2 overcomes sorafenib resistance via suppressing mTORC2-AKT-BAD pathway in hepatocellular carcinoma cells

BackgroundSorafenib is an oral multi-kinase inhibitor that was approved by the US Food and Drug Administration for the treatment of patients with advanced hepatocellular carcinoma (HCC). However, resistance to sorafenib is an urgent problem to be resolved to improve the therapeutic efficacy of sorafenib. As the activation of AKT/mTOR played a pivotal role in sorafenib resistance, we evaluated the effect of a dual mTOR complex 1/2 inhibitor Torin2 on overcoming the sorafenib resistance in HCC cells.MethodsThe sorafenib-resistant Huh7 and Hep3B cell lines were established from their parental cell lines. The synergistic effect of sorafenib and Torin2 on these cells was measured by cell viability assay and quantified using the Chou-Talalay method. Apoptosis induced by the combination of sorafenib and Torin2 and the alteration in the specific signaling pathways of interest were detected by Western blotting.ResultsSorafenib treatment inversely inhibited AKT in parental but activated AKT in sorafenib-resistant Huh7 and Hep3B HCC cells, which underscores the significance of AKT activation. Torin2 and sorafenib synergistically suppressed the viability of sorafenib-resistant cells via apoptosis induction. Torin2 successfully suppressed the sorafenib-activated mTORC2-AKT axis, leading to the dephosphorylation of Ser136 in BAD protein, and increased the expression of total BAD, which contributed to the apoptosis in sorafenib-resistant HCC cells.ConclusionsIn this study, Torin2 and sorafenib showed synergistic cytostatic capacity in sorafenib-resistant HCC cells, via the suppression of mTORC2-AKT-BAD pathway. Our results suggest a novel strategy of drug combination for overcoming sorafenib resistance in HCC.     

The structure of Escherichia coli BtuF and binding to its cognate ATP binding cassette transporter

Bacterial binding protein-dependent ATP binding cassette (ABC) transporters facilitate uptake of essential nutrients. The crystal structure of Escherichia coli BtuF, the protein that binds vitamin B12 and delivers it to the periplasmic surface of the ABC transporter BtuCD, reveals a bi-lobed fold resembling that of the ferrichrome binding protein FhuD. B12 is bound in the "base-on" conformation in a deep cleft formed at the interface between the two lobes of BtuF. A stable complex between BtuF and BtuCD (with the stoichiometry BtuC2D2F) is demonstrated to form in vitro and was modeled using the individual crystal structures. Two surface glutamates from BtuF may interact with arginine residues on the periplasmic surface of the BtuCD transporter. These glutamate and arginine residues are conserved among binding proteins and ABC transporters mediating iron and B12 uptake, suggesting that they may have a role in docking and the transmission of conformational changes.