【作者】 赵红兵；

【导师】 张明智；

【作者基本信息】 郑州大学 ， 临床医学博士（专业学位）， 2015， 博士

【摘要】 【背景与目的】淋巴瘤(Malignant Lymphoma,ML)是一组源自造血干/祖细胞的恶性克隆性疾病,淋巴结或结外淋巴组织或器官原发,国人多见NHL(淋巴瘤的两种分类之一,非霍奇金淋巴瘤)。NK/T细胞淋巴瘤(NK/T cell Lymphomas)是来源于T细胞的NHL,该病侵袭性高、病程短、疗效差、死亡率高,以放化疗为主,但对阿霉素(Adriamycin,ADM)为基础的传统CHOP方案疗效不敏感。核苷类似药物吉西他滨(gemcitabine)自问世以来一直是各种类型实体肿瘤(如胰腺癌、胆管癌和乳腺癌等)化疗方案中的基础药,已取得不凡的临床疗效。近年来,以吉西他滨为基础的化疗方案应用于NK/T细胞淋巴瘤病人的治疗后,临床治疗效果明显优于无吉西他滨药物的化疗方案,但仍有部分NK/T细胞淋巴瘤病人出现耐药现象。但无论在实体肿瘤还是淋巴瘤临床研究中,吉西他滨药物敏感性和耐药性的决定因素尚未明晰。细胞内代谢是核苷类似药物发挥其毒性作用的必要步骤,但前提是药物必须首先完成其跨膜转运,这一步骤对于核苷类似药物来说,是实现其药效必不可少的。故近年来对膜转运蛋白的研究越来越多,研究认为,药物疗效的发挥离不开膜转运蛋白的介导,其转运效率直接决定药物的治疗效果,甚至将其作为药物疗效和肿瘤病人存活长短的生物标志物。人均衡型核苷转运蛋白1(human equilibrium nucleoside transporter 1,hENT1)蛋白是近来膜转运蛋白研究的热点之一,其研究方向主要在其介导抗肿瘤药物和抗病毒药物方面。目前,实体瘤研究认为,吉西他滨的跨膜转运是一个几乎完全由转运蛋白hENT1介导的过程,该蛋白是决定吉西他滨药物敏感度的决定因素,但对阿霉素没有相关研究。hENT1(SLC29A1)是核苷转运家族SLC29(the solute carrier 29)发现的第一个成员,hENT1是承担机体生理性核苷和多种抗癌、抗病毒核苷类似药物的细胞吸收和(或)细胞排出的主要跨膜载体。根据细胞对NBTI/NBMPR的敏感特性,hENT1蛋白被分为NBMPR敏感型(es)和NBMPR不敏感型(ei)两类。近5年来,关于hENT1与抗癌药物治疗关系的临床研究大多集中在:肿瘤组织标本免疫组化检测hENT1蛋白高表达与癌症病人的长期生存有关;检测hENT1蛋白表达程度来评估肿瘤病人对核苷类似药物的敏感性和耐药性;hENT1蛋白能否作为核苷类似药物的治疗靶点;大多数实体瘤研究数据显示,核苷类似药物(如吉西他滨)通过hENT1蛋白介导实现了高效的药物跨膜转运,能作为核苷类似药物治疗癌症病人的生物标志物之一;但部分研究对此持不同意见;故hENT1蛋白在核苷类似药物治疗实体瘤病人的生物标志物价值仍保持着争议。在PubMed检索中,hENT1蛋白水平、基因水平研究的文献不多,基础研究多于临床研究,临床研究多集中在胰腺癌和胆管壶腹癌;在人NK/T淋巴瘤细胞株YTS上hENT1蛋白与吉西他滨、阿霉素的相关功能、蛋白水平和基因水平的研究,在PubMed、中国知网和万方数据中未检索到相关文献。本实验拟采用CCK-8技术、实时荧光定量PCR技术和Western Blot技术,检测膜转运蛋白hENT1与抗癌药物吉西他滨、阿霉素对人NK/T细胞淋巴瘤细胞株YTS的细胞毒作用的相关性,探讨在NK/T细胞淋巴瘤细胞株YTS中,膜转运蛋白hENT1是否为介导吉西他滨、阿霉素进入癌细胞的主要通道蛋白及其功能特性。【材料与方法】1.研究对象人NK/T细胞淋巴瘤细胞株YTS膜均衡型核苷转运蛋白1(hENT1,SLC29A1)。2.研究方法本研究采用CCK-8技术、实时荧光定量PCR技术(Q-rt-PCR)和Western Blot技术,通过膜转运蛋白hENT1与抗癌药物吉西他滨、阿霉素对人NK/T细胞淋巴瘤细胞株YTS的细胞毒作用的相关性检测,运用统计学方法,对hENT1进行功能、基因和蛋白水平上的研究。3.CCK-8检测采用CCK-8技术,结合hENT1的小分子特异抑制剂NBMPR,通过hENT1与抗癌药物吉西他滨、阿霉素对人NK/T细胞淋巴瘤细胞株YTS的细胞毒作用的相关性,检测人NK/T细胞淋巴瘤细胞株YTS膜转运蛋白hENT1的功能。4.实时荧光定量PCR检测采用实时荧光定量PCR技术,通过hENT1与抗癌药物吉西他滨、阿霉素对人NK/T细胞淋巴瘤细胞株YTS的细胞毒作用的相关性,运用统计学方法,研究人NK/T细胞淋巴瘤细胞株YTS膜转运蛋白hENT1在基因水平上的变化情况。并运用该技术检测了淋巴瘤细胞株YTS、SNK-6、Jeko-1、ly1、Raji、Karpas和Jurket中的hENT1在基因水平上的表达。5.Western Blot检测采用Western Blot技术,检测了淋巴瘤细胞株YTS、SNK-6、Jeko-1、ly1、Raji、Karpas和Jurket中的hENT1在蛋白水平上的表达。6.统计学处理采用SPSS17.0统计软件,两组比较采用t检验;检验水准为α=0.05。【结果】1.膜转运蛋白hENT1在细胞株YTS、SNK-6、Jeko-1、ly1、Raji、Karpas和Jurket中的表达研究Q-RT-PCR检测结果显示:hENT1在细胞株YTS、SNK-6、Jeko-1、ly1、Raji、Karpas和Jurket中均有表达,表达量不均,YTS、Jeko-1表达相对低,其余表达较高。Western Blot检测结果显示:hENT1蛋白在细胞株YTS、SNK-6、Jeko-1、ly1、Raji、Karpas和Jurket中均有表达,表达量不均。2.人NK/T细胞淋巴瘤细胞株YTS膜转运蛋白hENT1功能的检测在吉西他滨、阿霉素药物作用组,随药物作用时间延长,其细胞毒性明显增强,即两种药物对YTS的细胞毒作用存在时间依赖性;但预先加入NBMPR作用后,首先细胞毒作用明显减弱,甚至不起作用,其次时间依赖性不存在。先NBMPR作用+药物作用组与单纯药物作用组,无论吉西他滨、阿霉素,两组间比较差异均有统计学意义(P<0.05)。低浓度NBMPR(5nM/L、10nM/L和20nM/L)即可明显减弱吉西他滨、阿霉素对YTS的细胞毒作用。3.人NK/T细胞淋巴瘤细胞株YTS膜转运蛋白hENT1与吉西他滨细胞毒作用的相关性研究CCK-8结果显示:1)吉西他滨对YTS细胞的IC50值为25.63nM/L;2)吉西他滨对YTS的细胞毒作用随作用时间的延长和药物浓度加大而明显增强。不同时间和不同浓度组间比较差异均有统计学意义(P<0.05,P<0.05),即吉西他滨对YTS的细胞毒作用存在时间和浓度依赖性。3)加NBMPR作用后,吉西他滨的毒性作用改变明显;随作用时间延长,其细胞毒性几乎不显现。NBMPR作用+药物作用组与单纯药物作用组,组间比较差异有统计学意义(P<0.05)。Q-RT-PCR检测结果显示了吉西他滨作用YTS细胞后hENT1在mRNA水平的变化情况。不同浓度吉西他滨作用YTS细胞后3小时、6小时后hENT1基因相对表达量2-△△Ct值,组间比较差异有统计学意义(P<0.05);但3小时、12小时组间比较差异无统计学意义(P>0.05);即随吉西他滨对淋巴瘤细胞株YTS的作用的时间延长、浓度增加,6小时左右,hENT1基因显著增加;但随后的增加出现下降趋势。4.人NK/T细胞淋巴瘤细胞株YTS膜转运蛋白hENT1与阿霉素细胞毒作用的相关性研究CCK-8结果显示:1)阿霉素对YTS细胞的IC50值为400nM/L;2)阿霉素对YTS的细胞毒作用随作用时间的延长和药物浓度加大而明显增强。不同时间和不同浓度组间比较差异均有统计学意义(P<0.05,P<0.05),即阿霉素对YTS的细胞毒作用存在时间和浓度依赖性。3)加NBMPR作用后,阿霉素的毒性作用改变明显;随作用时间延长,其细胞毒性几乎不显现。NBMPR作用+药物作用组与单纯药物作用组,组间比较差异有统计学意义(P<0.05)。Q-RT-PCR检测结果显示了阿霉素作用YTS细胞后hENT1在mRNA水平的变化情况。不同浓度阿霉素作用YTS细胞后3小时、6小时之间,3小时、12小时之间,hENT1基因相对表达量2-△△Ct值组间比较差异均有统计学意义(P<0.05,P<0.05);即3、6、12小时,hENT1基因随阿霉素对淋巴瘤细胞株YTS的作用的时间延长、浓度增加而增加。【结论】1.在功能、基因水平证实NK/T细胞淋巴瘤细胞株YTS膜转运蛋白hENT1可能是介导吉西他滨、阿霉素进入的主要通道蛋白。2.在吉西他滨作用时,YTS细胞的hENT1 mRNA随时间延长、浓度增加而增加,上升趋势在6小时左右出现下降;在阿霉素作用时,YTS细胞的hENT1 mRNA随时间延长、浓度增加而增加,hENT1 mRNA在12小时左右仍处上升趋势。3.低浓度hENT1的抑制剂NBMPR可明显抑制吉西他滨、阿霉素对YTS细胞的细胞毒作用,YTS细胞的hENT1为NBMPR敏感型。4.膜转运蛋白hENT1在淋巴瘤细胞株YTS、SNK-6、Jeko-1、ly1、Raji、Karpas和Jurket中均存在表达,但表达不均,可能作为淋巴瘤治疗的新靶点。更多还原

【Abstract】 Background and ObjectiveMalignant Lymphoma(ML),including Hodgkin’s Lymphoma(HD)and non-hodgkin’s Lymphoma(NHL),is a set of derived from the hematopoietic stem/progenitor cells malignant clone disease,come on lymph nodes or outside lymph nodes of lymphatic tissue or organ primarily,the Chinese more occur NHL.NK/T cell lymphoma is derived from T cell NHL,it has high-grade invasive,short term of the disease,poor curative effect,high mortality,it is given priority to with radiation and chemotherapy,but Adriamycin(ADM)on the basis of CHOP scheme is not sensitive for NK/T cell lymphoma treatment.Gemcitabine(nucleoside-derived drugs)is chemotherapy regimens of basic medicine in the treatment of all kinds of solid tumors(such as pancreatic cancer and bile duct cancer and breast cancer,etc.),and have remarkable clinical curative effect.In recent years,on the basis of gemcitabine chemotherapy scheme applied in NK/T cell lymphoma in patients,the clinical treatment effect is significantly better than no gemcitabine chemotherapy regimens,but there are still a part of NK/T cell lymphoma patients appear drug resistance phenomenon.But in solid tumors and lymphoma in clinical research,the determinants of sensitivity and/or resistance to gemcitabine is not yet clear.Cell metabolism is the steps necessary to play a role of the toxicity ofnucleoside-derived drugs,but the premise is a drug must first complete its transport across the membrane,this step is essential to achieve its effectiveness for nucleosidederived drugs.Therefore,the study of membrane transporters is becoming more and more in recent years,research datas suggest that drug effects play without protein mediated drug transport,its transfer efficiency directly determine the therapeutic effect of drugs.hENT1(human equilibrium nucleoside transporter 1)is one of research focuses of membrane transporters in recent years,the main research direction locates in the mediated antitumor and antiviral drugs.At present,the solid tumor research thought,gemcitabine across plasma membrane is an almost entirely mediated process by hENT1,and it is determinants of sensitivity to gemcitabine,but no related research to adriamycin.hENT1(SLC29A1)is the first member found in SLC29 family(the solute carrier 29)in nucleoside transport.hENT1 is the main carrier across the membrane to cells absorb and(or)output of physiological nucleosides and a variety of anticancer and antiviral drugs.According to the NBTI/NBMPR characteristics,hENT1 is divided into NBMPR sensitive type(es)and NBMPR insensitive type(ei).In recent five years,the relationship between hENT1 and anti-cancer drug mostly focused on: a high expression of hENT1 in tumor tissue specimens is associated with the long-term survival of cancer patients;assess the sensitivity and resistance of nucleoside-derived drugs through hENT1 expression in the tumor patients;hENT1 maybe act as therapeutic targets in the tumor patients.most solid tumor research data shows that nucleoside-derived drugs(e.g.,gemcitabine)mediated by hENT1 implement effective drug transport across the membrane,maybe act as one of the biomarkers of cancer patients;but some studies have hold different views on this.So the value of hENT1 as biomarkers in the treatment of solid tumor patients in nucleoside-derived drugs remains controversial.Pub Med search results show the study of hENT1 in protein level and gene level is not much,basic research are more than clinical research,clinical research more concentrated in pancreatic cancer and bile duct ampulla carcinoma.Pub Med,CNKI and Wanfang data failed to retrieve relevant literature data on hENT1 associated with gemcitabine and adriamycin in NK/T lymphoma YTS cell lines.Using the CCK 8 technology,real-time fluorescent quantitative PCR and Western Blot technique,the experiments in this paper explored functional properties of hENT1 in NK/T cell lymphoma YTS cell lines,and tested the correlation between hENT1 and cytotoxic effect of gemcitabine and adriamycin.Materials and Methods1.SubjectsHuman equilibrium nucleoside transporter 1(hENT1,SLC29A1)in NK/T cell lymphoma YTS cell lines.2.Research methodsThis study use CCK 8 technology,real-time fluorescent quantitative PCR and Western Blot technique,detecting the correlation between membrane transporters hENT1 and the cytotoxic effects of gemcitabine and adriamycin in NK/T cell lymphoma YTS cell lines,study functional properties of hENT1 in the level of gene and protein.3.CCK-8 detectionUsing CCK 8 technology,combined with NBMPR(specific small molecule inhibitors of hENT1),study correlation between hENT1 and cytotoxic effects of gemcitabine and adriamycin in NK/T cell lymphoma YTS cell lines,and explore functional properties of hENT1.4.Real-time fluorescent quantitative PCR detectionBy real-time fluorescent quantitative PCR technology,with the correlation between hENT1 and cytotoxic effects of gemcitabine and adriamycin in NK/T cell lymphoma YTS cell lines,study the expression changes of human NK/T cell lymphoma YTS cell lines membrane transporters hENT1 at the genetic level.And apply the techniques tested the expression of hENT1 at the level of gene in lymphoma YTS,SNK-6,Jeko-1,ly1,Raji,Karpas and Jurket cell lines.5.Western Blot assaysUsing Western Blot technique,detect expression of hENT1 at the level of protein in lymphoma YTS,SNK-6,1,ly1,Raji,Jeko-the Karpas and Jurket cell lines.6.Statistics analysisStatistical package(SPSS 17.0)was used for processing data.Continuous normally distributed variables were presented as mean ± standard deviation(SD),and were compared using either the t test or One-way analysis of variance.Non-normally distributed variables were presented as medians(ranges),and were compared using rank sum test.Chi-square test was used to verify Hardy-Weinberg equilibrium.The classified variables were analyzed using Chi-square test,Fisher exact test or rank sum test.The detection level is considered statistically α=0.05.Results1.Expression of hENT1 in YTS、SNK-6、Jeko-1、ly1、Raji、Karpas and Jurkat cell linesQ-RT-PCR detection results showed that expression of hENT1 in YTS,SNK-6,Jeko-1,ly-1,Raji,Karpas and Jurkat cell lines exist,lower expression exist in YTS and Jeko-1,expression of the rest is higher.Western Blot test results show that: hENT1 in YTS,SNK-6,Jeko-1,ly1,Raji,Karpas and Jurkat cell lines,expression are inequality.2.hENT1 functional detection in NK/T cell lymphoma YTS cell linesIn gemcitabine,adriamycin group,with the extension of drug effect time their cytotoxicity enhanced obviously,the cytotoxic effects of gemcitabine and adriamycin on time dependence exists;but after joining NBMPR in advance,firstly,the cytotoxic effects of gemcitabine and adriamycin decreased significantly,even doesn’t work,secondly,the cytotoxic effects of gemcitabine and adriamycin on time dependence do not exist.NBMPR+drug group and simple drug group,regardless of gemcitabine or adriamycin,comparing differences between the two groups have statisticalsignificance(P<0.05).Low concentration NBMPR(5 nm/L,10 nm/L and 20 nm/L)can be diminished significantly the cytotoxic effects of gemcitabine and adriamycin.3.The correlation research between hENT1 and cytotoxic effects of gemcitabine in NK/T cell lymphoma YTS cell linesCCK 8 results showed: 1)IC50 value of gemcitabine for YTS cells is 25.63nM/L;2)cytotoxic effects of gemcitabine for YTS cells enhance with the extension of time and increased drug concentration obviously.Comparing differences between different times and different concentration groups were statistically significant(P<0.05,P<0.05);3)after add NBMPR,the toxic effects of gemcitabine change significantly;With the extension of action time,its cytotoxicity hardly appeared.NBMPR+drug group and simple drug group,comparing differences between groups was statistically significant(P<0.05).Q-rt-PCR test results shows the gene expression of hENT1 in YTS cells.Different concentrations of gemcitabine after 3 hours,6 hours,compare the difference between groups was statistically significant(P<0.05);But the 3 hours,12 hours comparative differences between groups have no statistical significance(P>0.05);Namely,with gemcitabine to extend the time,increase concentration,hENT1 gene significantly increased in 6 hours;but then the increasing trend began to decline.4.The correlation research between hENT1 and cytotoxic effects of adriamycin in NK/T cell lymphoma YTS cell linesCCK 8 results showed: 1)IC50 value of adriamycin for YTS cells is 400 nM/L;2)cytotoxic effects of adriamycin for YTS cells enhance with the extension of time and increased drug concentration obviously.Comparing differences between different times and different concentration groups were statistically significant(P<0.05,P<0.05).3)after add NBMPR,the toxic effects of adriamycin change significantly;With the extension of action time,its cytotoxicity hardly appeared.NBMPR+drug group and simple drug group,comparing differences between groups was statistically significant(P<0.05).Q-rt-PCR test results shows the gene expression of hENT1 in YTS cells.Different concentrations of adriamycin after 3 hours,6 hours and 12 hours,comparing the difference between groups was statistically significant(P<0.05,P<0.05);Namely,the gene expression of hENT1 increased with extended time and increased concentration of adriamycin.Conclusions1.hENT1 may be the main channel of protein mediated gemcitabine,adriamycin into YTS cell lines.2.When gemcitabine role in YTS cell lines,expression of hENT1 at the level of gene increased with the extension of time,increased concentration;rising trend in 6 hours began to decline;When adriamycin effects in YTS cell lines,expression of hENT1 at the level of gene increased with the extension of time,increased concentration;rising trend in 12 hours or so are still rising.3.Low concentration of NBMPR can obviously inhibit cytotoxic effects of gemcitabine,doxorubicin in YTS cell lines,hENT1 is NBMPR sensitive type.4.Expression of hENT1 in lymphoma YTS,SNK-6,Jeko-1,ly1,Raji,Karpas and Jurkat cell lines exist,but uneven expression,probably as a new target of lymphoma treatment.更多还原