KBv200细胞株耐药逆转前后HLA、B7抗原的表达

目的 探讨肿瘤多药抗性细胞的免疫逃避机制。方法 利用特异性切割mdr1的核酶为工具,以表达Mdr1的耐药细胞株KBv200为靶细胞,采用脂质体转染技术,将含核酶的质粒pHβApr-1neo/5mR3及空载体pHβApr-1neo导入KBv200及其亲本KB细胞内,运用Northern-blotting、免疫组化方法观察核酶对mdr1 mRNA及P-gp的影响,运用流式细胞仪技术检测各种不同细胞亚株HLA-Ⅰ、HLA-Ⅱ、B7-1、B7-2的表达。 结果 含核酶的质粒pHβApr-1neo/5mR3及空载体pHβApr-1neo可以在KB、KBv200细胞中稳定表达,核酶可以特异性地切割mdr1,导致KBv200/5mR3的mdr1 mRNA含量下降,P 糖蛋白（P-gp）表达减低,各种不同细胞亚株均表达较强的HLA-Ⅰ类抗原,而HLA-Ⅱ、B7-1、B7-2的表达较低。各亚株HLA-Ⅰ表达无明显差异,但HLA-Ⅱ、B7-1、B7-2的表达变化较大,KB的HLA-Ⅱ、B7-1、B7-2的表达较KBv200强,经化疗药物作用后KB的HLA-Ⅱ、B7-2进一步表达增强,核酶逆转后,KBv200/5mR3 HLA-Ⅱ、B7-1、B7-2的表达趋于接近KB水平。 结论 mdr1-核酶在细胞内具有一定的逆转肿瘤多药抗性的生物学效应;多药耐药细胞和敏感株细胞有着不同的免疫逃避特点,与敏感株相比,KBv200较易逃避机体的免疫反应。     

小分子干扰RNA片段逆转KBv200细胞多药耐药的实验研究

目的：研究小分子干扰RNA片段（siRNA）对舌癌多药耐药（MDR）细胞系KBv200细胞的MDR1基因表达及其功能的影响，探讨siRNA作为未来化疗增敏剂的可能性。方法：运用针对MDR1基因序列的siRNA（MDR1-siRNA）在脂质体的介导下转染KBv200细胞；用RT—PCR分析MDR1 mRNA的表达水平；流式细胞术检测P-糖蛋白（P-gP）的表达；荧光分光光度法检测细胞内多柔比星（Dox）的积蓄；MTT法检测KBv200细胞对长春新碱（VCR）和Dox的敏感性变化。结果：MDR1-siRNA作用24和48h能抑制MDR1基因的表达（P〈0．05），其mRNA水平和P-gP水平均明显下降，同时使KBv200细胞对VCR和Dox的敏感性显著增加（P〈0．01），并显著增加细胞内Dox的蓄积（P〈0．01）。结论：MDR1-siRNA能显著抑制KBv200细胞内MDR1基因介导的MDR。     

苦参碱与6种抗肿瘤药联用对KBV200耐药细胞株P-糖蛋白表达的研究

目的探讨6种常用抗肿瘤药物长春新碱、阿霉素、平阳霉素、顺铂、5-Fu、环磷酰胺等与苦参碱联合对KBV200耐药细胞株P-糖蛋白（P-gp）表达的影响。方法以喉癌KBV200耐药细胞株为研究对象，通过流式细胞仪（FCM）检测6种抗肿瘤药物作用于KBV200耐药细胞株后p-Pg表达以及分别联合苦参碱后对p-Pg表达的影响。结果苦参碱分别联合长春新碱、阿霉素作用后，KBV200耐药细胞株P—Pg表达水平下降。结论苦参碱分别与长春新碱和阿霉素联合作用后，可使KBV200耐药细胞株P-pg的表达水平下降，从而逆转其耐药性。     

STI571 combined with vincristine greatly suppressed the tumor formation of multidrug-resistant K562 cells in a human-nude mice xenograft model

Multidrug resistance (MDR), often associated with decreased intracellular drug accumulation in patient's tumor cells resulting from enhanced drug efflux,1 is a major obstacle to successful chemo- therapy in leukemia. It is related to the overexpression of a membrane protein, P- glycoprotein (P-170), thereby reducing drug cytotoxicity. For example, Philadelphia-chromo- some-positive (Ph+) chronic myelogenous leukemia (CML) is often resistant to chemotherapy in advanced phases because of the…     

TRAIL抑制胃癌多药耐药基因MDR1/P-gp的表达

目的观察肿瘤坏死因子相关凋亡诱导配体(TRAIL)对胃癌耐药细胞株SGC-7901/VCR多药耐药基因MDR1及其编码P-gp蛋白表达的影响,探讨以TRAIL为靶点逆转胃癌多药耐药的机制。方法 SGC-7901/VCR细胞株经不同浓度的TRAIL处理48 h后,RT-PCR检测各组胃癌细胞株中多药耐药MDR1 mRNA的表达情况,同时用ELISA法检测各组胃癌细胞株中P-gp表达的含量。结果不同浓度TRAIL(50、100、200、400μg/L)作用于细胞后,胃癌耐药细胞株SGC-7901/VCR的MDR1/P-gp表达受不同程度抑制,与对照组相比差异均具有统计学意义(P<0.05)。400μg/L与200μg/L组相比其MDR1/P-gp抑制程度并不明显,其余各组间差异均有统计学意义(P<0.05)。结论 TRAIL可抑制SGC-7901/VCR MDR1/P-gp的表达,且呈量效关系。TRAIL可能通过降低耐药基因MDR1的表达逆转胃癌的多药耐药。     

乳腺癌

目的探讨肿瘤多药抗性（ｍｄｒ１）的基因调控及其特异的凋亡机制。方法在建立的ｍｄｒ１肿瘤裸鼠的瘤组织中,采用ＡＢＣ免疫组化法,检测Ｐ－１７０、ｃ－ｍｙｃ、野生型ｐ５３、ｐ１６、Ｆａｓ和Ｂｃ１－２等蛋白的表达。结果发现ｍｄｒ１肿瘤细胞的ｃ－ｍｙｃ表达增强;经抗ｍｄｒ１－ｒｉｂｏｚｙｍｅ部分逆转ＭＤＲ表型后,ｍｄｒ１肿瘤细胞的ｐ５３表达有所升高。结论ｃ－ｍｙｃ、ｐ５３可能参与了对ｍｄｒ１基因表达的调控,其中ｃ－ｍｙｃ可能起着激活ｍｄｒ１基因转录的作用。实验还发现ｍｄｒ１肿瘤细胞的Ｆａｓ蛋白缺失、Ｂｃ１－２表达升高,提示ｍｄｒ１肿瘤可能拥有自己特异的凋亡调节机制。     

五味子乙素对转染多药耐药1基因的MCF-7细胞的多药耐药逆转作用

目的 探讨五味子乙素(SchB)对转染多药耐药1基因(MDR1)的人乳腺癌细胞MCF-7的多药耐药逆转作用及相关机制。方法 将人MDR1基因导入MCF-7细胞,形成耐药细胞株MCF-7／MDR1;用该细胞株为模型评价SchB的体外逆转多药耐药作用,用MTT法进行化疗药物单独或与SchB联合作用时对耐药细胞的IC50比较,计算逆转倍数。结果 转染细胞MCF-7／MDR表现为P糖蛋白高表达,对阿霉素、长春新碱、紫杉醇、高三尖杉酯的抗药性均增加;SchB(25μmol／L)显著减少阿霉素、长春新碱、紫杉醇和高三尖杉酯对MCF-7／MDR细胞的IC50,逆转倍数达6．03-23．94倍;SchB(25μmol／L)使MCF-7／MDR细胞对若丹明123的胞内积聚增加约5倍。效果与维拉帕米10μmol／L浓度时相当;但SchB(25μmol／L)不影响MCF-7／MDR细胞的P-糖蛋白表达。结论 SchB能有效逆转转染MDR1的MCF-7细胞的多药耐药,其机制可能是抑制了P-糖蛋白的药物外排生物学活性。     

Expression of the human multidrug resistance gene mdr1 in leukemic cells and its application in studying P-glycoprotein antagonists

Objective To explore the role of endothelin (ET) in the pathogenesis of exercise-induced asthma (EIA), we investigated the effects of ET(B) receptor antagonists, ET-1 (11-21)fragment and N-cis-2,6-dimethylpi-peridinocardonyl-L-γ-methylleucyl-D-1-methoxycarbonyl tryptophanyl-D-norleucine (BQ788) on broncho-constriction elicited by isocapnic hyperpnea in guinea pigs. Methods Eighteen pathogen-free Hartley guinea pigs were randomly divided into three groups. A: normal saline (NS) inhalation control group (n=6), B: BQ788 group (n=6), and C: ET-1(11-21) fragment group (n=6). Guinea pigs were anesthetized with pentobarbital sodium. After measuring the basal value of lung resistance (R[L]) and dynamic compliance of the respiratory system (Cdyn), NS (0.96 ml), BQ788 (9 nmol) and ET-1(11-21)fragment (9 nmol) were inhaled. A rodent respirator with a dry 5%CO(2)-95%O(2) mixture at room temperature provided mechanical ventilation (V[T] 8 ml/animal, 100 breaths/min) for 5 min. R[L] and Cdyn of the 3 groups were measured again after isocapnic hyperpnea challenge. Results In the control group, isocapnic hyperpnea of dry gas elicited a marked increase in R[L] and decrease in Cdyn. R[L] and Cdyn of the guinea pigs from BQ788 group and ET-1(11-21)fragment group did not change significantly. Conclusion It was demonstrated that selective ET(B) receptor antagonists, ET-1(11-21) fragment and BQ788, inhibited the bronchoconstriction induced by isocapnic hyperpnea in guinea pigs. The data showed that ETs are potent constrictors of guinea pig airway smooth muscle via a direct effect on ET receptors. It was suggested that ET receptor antagonists, especially ET(B) receptor antagonist, might be beneficial in preventing EIA.     

蛋白激酶C活性、亚细胞分布与KBV200细胞多药耐药性的相关研究

OBJECTIVE: To investigate the relationship between protein kinase C (PKC) and multidrug resistance (MDR) in cancer cell line KBV200. METHODS: MTT assay was used to evaluate the IC50 of vincristine (VCR) and adriamycin (ADR) in KB cell line and its VCR-resistant derivative KBV200 cells. PKC activities in the 2 cell lines were assayed by measuring the incorporation of (32)P from [gamma-(32)P] ATP into the peptide substrates. RESULTS: The IC50 values of VCR and ADR in KBV200 cells was 64.03 and 18.8 folds greater than those in KB cells. PKC activities of the membrane and cytosol fraction in KBV200 cells were increased compared with that in KB cells, with the total PKC activity 1.12-fold higher. Phorbol-12-myristate-13 -acetate increased PKC activity of the membrane fraction and IC50 values of KBV200 cells, while staurosporine worked to the opposite effects. CONCLUSION: PKC may contribute to MDR mechanism in KBV200 cells.     

逆转录病毒转染法建立兔VX-2多药耐药株

目的：建立VX-2多药耐药(MDR)细胞(VX-2mdrI)。方法：利用逆转录病毒转染法将带有mdrI cDNA全序列的逆转录病毒载体pHaMDR1转入到兔VX-2细胞中，MTT法检测细胞在不同化疗药物作用下的存活率；免疫组化检测细胞的P-糖蛋白(P-gp)表达，RT-PCR检测细胞内mdrI mRNA的表达量．结果：转基因的细胞对吡柔比星、秋水仙素的耐药性分别提高10和25倍，免疫组化见转基因细胞中P-gp表达增加，RT-PCR示细胞内mdrI mRNA的表达量增加。结论：利用逆转录病毒转染法构建了兔VX-2 MDR细胞。     

逆转mdr1引起的多药耐药性的策略

多药耐药基因1（mdr1）编码的P-糖蛋白表达的增加是肿瘤细胞多药耐药性最常见的发生机制之一。近年来以mdr1/P-糖蛋白为靶点,在蛋白质水平和基因水平上采用多种策略逆转MDR,这些研究均有较大进展,本文就目前逆转多药耐药性的研究状况进行综述。     

Reversal of P-glycoprotein-mediated multidrug resistance in SGC7901/VCR cells by PPARγ activation by troglitazone

Over-expression of P-glycoprotein(P-gp),an ATP-dependent drug efflux pump,represents one of the major mechanisms that contribute to multidrug resistance(MDR) in cancer cells.This study examined the effects of troglitazone,a ligand of peroxisome proliferator-activated receptor gamma(PPARγ),on P-gp-mediated MDR in SGC7901/VCR cells(a vincristine-resistant human gastric cancer cell line).The expression of P-gp was detected by RT-PCR and Western blotting,respectively.The SGC7901/VCR cells were treated with 0.1 ...     

蛋白激酶C活性、表达及亚细胞分布与KBV200细胞多药耐药相关性的研究

目的研究多药耐药肿瘤细胞PKC活性、PKC亚型的表达和亚细胞分布与KBV200细胞多药耐药的关系。方法MTT法检测敏感株KB和耐药株KBV200细胞的耐药性；印掺入法测定PKC的活性；Western blot法检测KBV200及其亲本KB细胞株PKC亚型的表达和亚细胞分布。结果长春新碱（VCK）和阿霉素（ADK）对KBV200细胞的IC50值分别高于KB细胞（P〈0．01）；耐药指数（RI）分别为65．03和19．8。KBV200细胞的膜组分、浆组分的PKC活性均较KB细胞高,KBV200细胞的总PKC活性是KB细胞的2．12倍。Western blot结果发现KBV200和KB细胞膜组分及浆组分均有PKCq的表达，且KBV200细胞的表达较KB明显增强。PMA可升高KBV200细胞的PKC总活性和膜组分PKC活性，降低浆组分PKC活性、表达（P〈0．01）；PMA可升高VCR、ADK对KBV200细胞的IC50值（P〈0．01）。SP可降低PKC活性；SP预孵育使PKCα膜组分和浆组分的表达均降低，可降低VCR、ADK对KBV200细胞的IC50值（P〈0．01）。结论KBV200细胞的PKC活性、表达、亚细胞分布与KB细胞有明显差别，这种差别可能与KBV200耐药性的变化密切相关，在PKC亚型中，PKCα的表达明显高于其他亚型，且高于亲本株，提示KBV200细胞中PKCα与多药耐药相关。     

RNA干扰逆转白血病细胞耐药性的研究

目的 用RNA干扰技术下调白血病耐药细胞系K562/A02多药耐药基因mdr1的表达以逆转白血病对化学药物的耐药性.方法 针对mdr1基因已知mRNA序列不同位点,选择两条靶序列,构建靶向mdr1 shRNA真核表达载体,脂质体介导转染白血病耐药细胞株K562/A02.实时荧光定量RT-PCR检测mRNA表达,Western blot检测细胞膜P-gp表达,柔红霉素泵出试验检测P-gp外排泵功能,MTT法检测细胞对阿霉素的敏感性.结果 构建了二条针对mdr1基因的shRNA真核表达载体,分别下调mdr1 mRNA的表达89.74%和87.18%,降低细胞膜P-gp表达,使膜外泵功能明显下降,柔红霉素在细胞内贮留明显增多,60min 时柔红泵出率为13.16%、22.02%,对照组为40.44%、45.31%,对阿霉素药物敏感性的相对逆转率为84.36%和76.69%.结论 RNA干扰技术可有效逆转mdr1所致耐药.     

高温合并化疗对耐药性大鼠肝癌CBRH-7919细胞多药耐药基因的影响

【目的】观察热化疗对多药耐药性CBRH-7919细胞MDR基因的影响。【方法】采用单纯化疗,单纯热疗及热疗联合化疗,分别作用于CBRH-7919及CBRH-7919mdr 1细胞。观察不同处理组细胞的存活率、mdr1mRNA及P-gp的表达情况。【结果】热化疗组细胞的存活率明显低于单纯加热组及单纯化疗组,同时热化疗组细胞mdr1mRNA的表达明显减低或消失,P-gp表达明显减低。【结论】热化疗可以使多药耐药细胞mdr1mRNA表达降低或消失,使P-gp表达降低,从而逆转了肿瘤细胞的多药耐药性,提高了化疗的敏感性。     

实时荧光定量PCR检测乳腺癌5种多药耐药基因的表达强度

目的 :检测 5种多药耐药基因在乳腺癌细胞株 MCF- 7和 MCF- 7/ADR里的表达强度变化 ,为乳腺癌多药耐药逆转研究提供新的思路。方法 :通过实时荧光定量 PCR技术分别检测乳腺癌敏感细胞株 MCF- 7和耐药细胞株 MCF- 7/ADR里的 mdr1、MRP、L RP、GST- π、TOPO α基因表达强度。结果 :MCF- 7/ADR里的 mdr1、MRP、LRP、GST- π基因表达强度分别比它们在 MCF- 7里的表达强度相对增加了 1 2 5 .39、5 5 .5 3、1 .2 4、3.38倍 ,TOPO α则降低了 0 .43倍 ,其中 mdr1、MRP表达强度的相对增加倍数均显著高于 L RP、GST- π、TOPO α的增加 (或降低 )倍数 ( P<0 .0 0 1 )。结论 :除了 mdr1之外 ,MRP也可能在乳腺癌的多药耐药产生机制里起重要作用。因此 MRP基因可以成为乳腺癌耐药逆转研究的新作用靶点     

用逆转录多聚酶链反应法检测多药耐药基因（mdr1）的表达

以长春新碱诱导的耐药细胞Ｋ５６２／ＶＣＲ和阿霉素诱导的耐药细胞Ｋ５６２／ＤＯＸ及敏感细胞Ｋ５６２作为检测对象，采用逆转录多聚酶链反应法，并且以β２－微球蛋白（β２ｍ）基因作为内参照，检测和比较了上述细胞的多药耐药基因ｍｄｒ１表达。经过３０个循环扩增，耐药细胞检测出ｍｄｒ１和β２ｍ基因的扩增产物，而敏感细胞无ｍｄｒ１扩增产物。结果提示，本方法可用以区分耐药和敏感细胞，有希望成为临床个体化疗敏感性预测的重要指标之一。     

Upregulation of drug sensitivity of multidrug-resistant SGC79 01/VCR human gastric cancer cells by bax gene transduction

Objective To investigate the role of bax in a vincristine （VCR）-induced multidrug-resi stant （MDR） human gastric cancer cell line, SGC7901/VCR, in which the Bax prot ein expression level was significantly lower compared with that in parent cells .Methods A bax eukaryotic expression vector was constructed and transfected into SGC7901/ VCR cells by lipofectamine, and resistant clones were selected by G418. Western blotting detected Bax expression in transfectants. Tetrazolium blue (MTT) assa y evaluated the differences in drug sensitivity and cell cycle changes of transf ectants were analyzed using flowcytometry (FCM). Results The bax eukaryotic expression vector was constructed and transfected into SGC790 1/VCR cells. Through G418 selection, resistant clones were obtained. Western b lotting demonstrated that the expression of Bax protein was markedly increased i n bax transduced cells. These cells were more sensitive to adriamycin (ADR) and VCR than mock vector transducted cells. Moreover, bax transfection enhanced AD R-induced apoptosis and VCR-induced G(2)/M phase arrest of SGC7901/VCR cells. Conclusion Bax was involved in the MDR of SGC7901/VCR cells.     

环氧合酶-2抑制剂联合苦参碱逆转K562/AO2细胞多药耐药的研究

目的:研究塞来昔布单独及联合应用苦参碱对K562/AO2细胞多药耐药逆转作用的影响,以及探讨他们互相作用的机理.方法:采用四甲基偶氮唑盐(MTT)法检测阿霉素(ADM)的半数抑制量;流式细胞术检测细胞凋亡;RT-PCR方法检测多药耐药基因(MDR)和环氧合酶-2(COX-2)mRNA的表达水平;Western blotting方法检测人p-糖蛋白(P-gp)和COX-2蛋白的表达水平.结果:ADM对K562、K562/AO2细胞的IC50值分别为0.398、33.31 μg·ml-1;苦参碱(200 μg·ml-1)、塞来昔布(7.5 μg·ml-1)单独及联合应用处理细胞时,ADM对K562/AO2细胞的IC50值分别为9.44、12.84、2.71 μg·ml-1;苦参碱、塞来昔布单独及联合应用于K562/AO2细胞后凋亡率明显增加,MDR1和COX-2 mRNA以及P-gp、COX-2蛋白的表达明显下调,且两药联合时下调更明显.结论:苦参碱和塞来昔布均有逆转K562/AO2细胞多药耐药的作用,两药联合应用时效果更加明显,P-gp的高表达可能与COX-2的表达上调有关.