浙江蝮蛇蛇毒成分逆转多药耐药白血病K562/ADM细胞耐药性分析

[目的]探讨浙江蝮蛇蛇毒提取物逆转K562/ADM细胞耐药的作用及机制.[方法]通过MTT法检测不同浓度浙江蝮蛇蛇毒提取物对K562/ADM细胞阿霉素(ADM)的IC50影响,分析其逆转耐药活性;通过实时定量PCR法检测浙江蝮蛇蛇毒提取物对K562/ADM细胞MDR1及GST-π耐药基因表达的影响.[结果]浙江蝮蛇蛇毒提取物呈浓度依赖性地降低K562/ADM细胞对ADM的IC50.实时定量PCR法检测结果显示,浙江蝮蛇蛇毒提取物作用后K562/ADM细胞内MDR1及GST-π基因表达水平有下调趋势,且随蛇毒提取物浓度增加下调趋势逐渐明显.[结论]浙江蝮蛇蛇毒提取物具有较强的逆转人白血病K562/ADM细胞多药耐药活性作用.     

MDR1基因短发卡样RNA表达载体逆转K562/A02人白血病细胞多药耐药的研究

目的构建MDR1基因短发卡样RNA(shRNA)真核表达载体,观察对K562/A02人白血病细胞株MDR1基因的沉默作用以及对P-糖蛋白(P-gp)表达及功能的影响。方法以基因重组技术构建表达质粒,转染重组质粒pEGFP-C1/U6/MDR1-A和pEGFP-C1/U6/MDR1-B至K562/A02细胞株,通过半定量RT-PCR和蛋白质印迹法,检测MDR1基因表达及P-gp表达水平的变化;以MTT法检测阿霉素(ADM)对K562/A02细胞的半数抑制浓度(IC_(50));高效液相色谱(HPLC)法检测细胞内ADM含量。结果构建的2种重组质粒pEGFP-C1/U6/MDR1-A和pEGFP-C1/U6/MDR1-B均明显抑制K562/A02细胞株MDR1基因表达,抑制率最高为48．2%±2．5%;同时抑制P-gp蛋白的表达,抑制率最高为50．67%。对ADM药物敏感性的相对逆转效率分别为40．8%和62．4%;同时使K562/ A02细胞内ADM含量增加。结论shRNA表达载体可明显抑制K562/A02细胞MDR1 mRNA的转录和P-gp蛋白的表达,增加K562/A02细胞内ADM含量,恢复K562/A02细胞对化疗药物的敏感性,逆转MDR1基因编码蛋白P-gp介导的多药耐药。     

mdr-1特异性核酶逆转卵巢癌的多药耐药

目的：探讨卵巢癌多药耐药的机制及应用核酶对阿霉素(ADM)引起的多药耐药(MDR)的逆转。方法应用共聚焦激光显微镜(confocal)、逆转录聚合酶链反应(RT-PCR)、蛋白免疫印迹法(Western blot)等检测卵巢癌细胞株A2780及阿霉素耐药株A2780／ADM多药耐药基因(mdr-1)及其编码的p-糖蛋白(p-gp)的表达,并比较导入mdr-1特异性核酶后A2780／ADM细胞MDR表型的改变。结果：A2780／ADM细胞对ADM的耐药性是A2780细胞的8．43倍。A2780／ADM细胞中mdr-1基因和p-gp表达较A2780细胞增高,转染mdr-1核酶基因后,A2780／ADM细胞的mdr-1 mRNA和p-gp表达明显下降。结论:ADM所致的卵巢癌细胞MDR与mdr-1基因过度表达有关,应用mdr-1核酶能在一定程度上逆转A2780／ADM细胞的MDR。     

中药提取物逆转K562/ADM细胞的耐药作用及其分子机制的探讨

Objective To investigate the reversal effect of the monomer of traditional Chinese medicine on muhidrug resistance(MDR) and its possible mechanism in K562/ADM cell line in vitro. Methods With different concentrations of baicalin, geniposide administered to K562/ADM cells, the proliferation of K562/ ADM cells was detected by the MTY assay. Expression of mdr-1 mRNA, Topo Ⅱ mRNA was measured by semi-quantitive RT-PCR. Results Thatbaicalin and geniposide could increase the sensitivity of K562/ADM cells to adriamycin, multiples of reversion were 1.95 times and 1.46 times. The proliferation of K562/ADM cells was in-hibited obviously by baicalin and geniposide, the level of mdr-1 mRNA expression was down-regulated and the Topo Ⅱ mRNA was up-regulated(P<0.01 ). Conclusion Baicalin and geniposide may reverse the multi-drag-resistance of K562/ADM cells, which was related to the down-regulation of mdr-1 expression and up-reg-ulation of Topo Ⅱ beta expression.     

中药提取物逆转K562/ADM细胞的耐药作用及其分子机制的探讨

Objective To investigate the reversal effect of the monomer of traditional Chinese medicine on muhidrug resistance(MDR) and its possible mechanism in K562/ADM cell line in vitro. Methods With different concentrations of baicalin, geniposide administered to K562/ADM cells, the proliferation of K562/ ADM cells was detected by the MTY assay. Expression of mdr-1 mRNA, Topo Ⅱ mRNA was measured by semi-quantitive RT-PCR. Results Thatbaicalin and geniposide could increase the sensitivity of K562/ADM cells to adriamycin, multiples of reversion were 1.95 times and 1.46 times. The proliferation of K562/ADM cells was in-hibited obviously by baicalin and geniposide, the level of mdr-1 mRNA expression was down-regulated and the Topo Ⅱ mRNA was up-regulated(P<0.01 ). Conclusion Baicalin and geniposide may reverse the multi-drag-resistance of K562/ADM cells, which was related to the down-regulation of mdr-1 expression and up-reg-ulation of Topo Ⅱ beta expression.     

中药提取物逆转K562/ADM细胞的耐药作用及其分子机制的探讨

Objective To investigate the reversal effect of the monomer of traditional Chinese medicine on muhidrug resistance(MDR) and its possible mechanism in K562/ADM cell line in vitro. Methods With different concentrations of baicalin, geniposide administered to K562/ADM cells, the proliferation of K562/ ADM cells was detected by the MTY assay. Expression of mdr-1 mRNA, Topo Ⅱ mRNA was measured by semi-quantitive RT-PCR. Results Thatbaicalin and geniposide could increase the sensitivity of K562/ADM cells to adriamycin, multiples of reversion were 1.95 times and 1.46 times. The proliferation of K562/ADM cells was in-hibited obviously by baicalin and geniposide, the level of mdr-1 mRNA expression was down-regulated and the Topo Ⅱ mRNA was up-regulated(P<0.01 ). Conclusion Baicalin and geniposide may reverse the multi-drag-resistance of K562/ADM cells, which was related to the down-regulation of mdr-1 expression and up-reg-ulation of Topo Ⅱ beta expression.     

中药提取物逆转K562/ADM细胞的耐药作用及其分子机制的探讨

Objective To investigate the reversal effect of the monomer of traditional Chinese medicine on muhidrug resistance(MDR) and its possible mechanism in K562/ADM cell line in vitro. Methods With different concentrations of baicalin, geniposide administered to K562/ADM cells, the proliferation of K562/ ADM cells was detected by the MTY assay. Expression of mdr-1 mRNA, Topo Ⅱ mRNA was measured by semi-quantitive RT-PCR. Results Thatbaicalin and geniposide could increase the sensitivity of K562/ADM cells to adriamycin, multiples of reversion were 1.95 times and 1.46 times. The proliferation of K562/ADM cells was in-hibited obviously by baicalin and geniposide, the level of mdr-1 mRNA expression was down-regulated and the Topo Ⅱ mRNA was up-regulated(P<0.01 ). Conclusion Baicalin and geniposide may reverse the multi-drag-resistance of K562/ADM cells, which was related to the down-regulation of mdr-1 expression and up-reg-ulation of Topo Ⅱ beta expression.     

中药提取物逆转K562/ADM细胞的耐药作用及其分子机制的探讨

Objective To investigate the reversal effect of the monomer of traditional Chinese medicine on muhidrug resistance(MDR) and its possible mechanism in K562/ADM cell line in vitro. Methods With different concentrations of baicalin, geniposide administered to K562/ADM cells, the proliferation of K562/ ADM cells was detected by the MTY assay. Expression of mdr-1 mRNA, Topo Ⅱ mRNA was measured by semi-quantitive RT-PCR. Results Thatbaicalin and geniposide could increase the sensitivity of K562/ADM cells to adriamycin, multiples of reversion were 1.95 times and 1.46 times. The proliferation of K562/ADM cells was in-hibited obviously by baicalin and geniposide, the level of mdr-1 mRNA expression was down-regulated and the Topo Ⅱ mRNA was up-regulated(P<0.01 ). Conclusion Baicalin and geniposide may reverse the multi-drag-resistance of K562/ADM cells, which was related to the down-regulation of mdr-1 expression and up-reg-ulation of Topo Ⅱ beta expression.     

中药提取物逆转K562/ADM细胞的耐药作用及其分子机制的探讨

Objective To investigate the reversal effect of the monomer of traditional Chinese medicine on muhidrug resistance(MDR) and its possible mechanism in K562/ADM cell line in vitro. Methods With different concentrations of baicalin, geniposide administered to K562/ADM cells, the proliferation of K562/ ADM cells was detected by the MTY assay. Expression of mdr-1 mRNA, Topo Ⅱ mRNA was measured by semi-quantitive RT-PCR. Results Thatbaicalin and geniposide could increase the sensitivity of K562/ADM cells to adriamycin, multiples of reversion were 1.95 times and 1.46 times. The proliferation of K562/ADM cells was in-hibited obviously by baicalin and geniposide, the level of mdr-1 mRNA expression was down-regulated and the Topo Ⅱ mRNA was up-regulated(P<0.01 ). Conclusion Baicalin and geniposide may reverse the multi-drag-resistance of K562/ADM cells, which was related to the down-regulation of mdr-1 expression and up-reg-ulation of Topo Ⅱ beta expression.     

中药提取物逆转K562/ADM细胞的耐药作用及其分子机制的探讨

Objective To investigate the reversal effect of the monomer of traditional Chinese medicine on muhidrug resistance(MDR) and its possible mechanism in K562/ADM cell line in vitro. Methods With different concentrations of baicalin, geniposide administered to K562/ADM cells, the proliferation of K562/ ADM cells was detected by the MTY assay. Expression of mdr-1 mRNA, Topo Ⅱ mRNA was measured by semi-quantitive RT-PCR. Results Thatbaicalin and geniposide could increase the sensitivity of K562/ADM cells to adriamycin, multiples of reversion were 1.95 times and 1.46 times. The proliferation of K562/ADM cells was in-hibited obviously by baicalin and geniposide, the level of mdr-1 mRNA expression was down-regulated and the Topo Ⅱ mRNA was up-regulated(P<0.01 ). Conclusion Baicalin and geniposide may reverse the multi-drag-resistance of K562/ADM cells, which was related to the down-regulation of mdr-1 expression and up-reg-ulation of Topo Ⅱ beta expression.     

小干扰RNA逆转三氧化二砷耐药的白血病K562/AS2细胞Topo Ⅱ基因表达的实验研究

 目的　研究小干扰RNA片段（shRNA）对三氧化二砷（ATO）耐药的白血病细胞株K562／AS2细胞的TopoⅡα、TopoⅡβ基因表达及其功能的影响。方法　设计并合成针对TopoⅡα和TopoⅡβ基因序列的shRNA各3对,在脂质体的介导下转染K562／AS2细胞;用荧光实时定量聚合酶链反应（PCR）分析TopoⅡα、TopoⅡβ mRNA的表达水平;流式细胞术检测TopoⅡα、TopoⅡβ蛋白表达。结果　针对TopoⅡα- shRNA、TopoⅡβ的shRNA作用于K562／AS2细胞24 h后,TopoⅡα mRNA水平和蛋白水平最大下调为（78.22±0.01）%、（31.17±1.27）%（P＜0.05）,TopoⅡβmRNA水平和蛋白水平最大下调为（57.36±0.01）%、（23.98±1.22）%（P＜0.05）。结论　转染24 h后针对TopoⅡ的shRNA可抑制对ATO耐药的白血病细胞株K562／AS2 细胞TopoⅡ基因的表达。     

三氧化二砷联合BSO对K562／ADM细胞P-糖蛋白的抑制作用研究

目的研究三氧化二砷（As2O3）联合γ-谷氨酰半胱氨酸合成酶抑制剂丁硫氨酸亚砜胺（BSO）对肿瘤多药耐药细胞K562／ADM细胞中P-糖蛋白（P—gP）的抑制作用，比较单用As2O3与两药联合的作用效果。方法As2O3组（0．5μmol／L，2．0μmol／L，5．0μmol／L）单独及联合100μmol／LBSO作用于K562／ADM细胞后，分光光度法检测K562／ADM细胞内谷胱甘肽（GSH）含量变化；流式细胞术（FCM）检测P-gp蛋白水平表达变化；反转录一聚合酶链反应（RT—PCR）方法检测mdr-1 mRNA的表达变化。结果As2O3临床剂量组（0．5μmol／L，2μmol／L）联合BSO24h内即可抑制P-gp和mdr-1mRNA的表达水平，在48h后，临床剂量联合组抑制mdr-1mRNA的作用效果要明显强于单用高剂量组，在72h后，临床剂量联合组抑制P—gP的作用效果要明显强于单用高剂量组。结论临床剂量As2O3联合BSO可有效抑制K562／ADM细胞P—gp及mdr-1 mRNA的表达。     

RNAi对白血病细胞mdr-1基因和多药耐药表型的影响

目的:探讨RNA干扰技术(RNAi)对慢性粒细胞白血病急变细胞系K562/AO2细胞mdr1基因的抑制和耐药表型的逆转作用。方法:选择合成封闭mdr-1基因的小干扰序列(si-MDR1),以1个碱基突变的si-MDR1-mut为对照序列,在脂质体介导下转染至K562/AO2细胞系。RT-PCR和Western blot检测mdr1 mRNA及P-gp蛋白水平,流式细胞术分析细胞内柔红霉素(daunorubicin,DNR)积累量,并以四甲基唑蓝快速比色法(MTT)反映K562/AO2对阿霉素、长春新碱、足叶乙甙药物敏感性的变化。结果:实验证实该序列能高效封闭K562/AO2细胞内mdr-1基因表达,增加细胞内化疗药物DNR积累量,增强K562/AO2细胞对阿霉素、长春新碱、足叶乙甙的敏感性。结论:RNAi可以通过抑制mdr1基因表达,逆转K562/AO2细胞耐药表型。     

硒酸酯多糖通过下调mdr1/P-gp表达逆转K562/ADM细胞凋亡抑制

目的:研究硒酸酯多糖(Kappa-selenocarrageenan,KSC)对多药耐药K562/ADM细胞的诱导凋亡效应及其分子机制。方法:以白血病多药耐药细胞K562/ADM为KSC作用的靶细胞,用MTT比色法检测细胞增殖活性,形态学、DNA片段化和流式细胞术(FCM)观察细胞凋亡;RT-PCR检测mdr1基因和Caspase-3基因mRNA的表达;FCM测定P-gp蛋白表达水平和Caspase-3活性。结果:KSC显著抑制K562/ADM细胞增殖,KSC诱导后K562/ADM细胞出现典型的凋亡形态学变化、DNA片段化和亚G1期细胞群等特征性改变。KSC下调K562/ADM细胞mdr1基因表达、抑制P-gp合成,并上调caspase-3基因表达、增强caspase-3活性。结论:KSC通过下调mdr1/P-gp表达逆转K562/ADM多药耐药细胞的凋亡抑制。     

马钱子碱对白血病K562/A02细胞多药耐药性的逆转作用

目的：研究马钱子碱（vauqueline）对人白血病K562/A02细胞多药耐药性的逆转作用。方法：采用噻唑蓝（MTT）法检测马钱子碱的细胞毒作用;采用半定量逆转录聚合酶链反应（RT-PCR）和免疫印迹（Western blot）分别检测非细胞毒浓度（IC10）的马钱子碱对K562/A02细胞MDR1 （multidrug resistance gene 1）、多药耐药相关蛋白（multidrug resistance-associated protein,MRP）、拓扑异构酶Ⅱ（topoisomeraseⅡ,TopoⅡ）、谷胱苷肽-S-转移酶（glutathione s-transferase,GST-π）mRNA及其蛋白表达的影响。结果：非细胞毒浓度（IC10）的马钱子碱作用后,K562/A02细胞中MDR1mRNA及P-gp表达降低（P<0.01）。而MRP、TopoⅡ、GST-π mRNA及其蛋白的表达无明显变化（P>0.05）,同时马钱子碱能增加化疗药物在白血病细胞内的积累。结论：马钱子碱能部分逆转K562/A02细胞的耐药性,其作用机制可能与下调K562/A02细胞MDR1 mRNA的表达,导致细胞膜上P-gp的表达量减少,化疗药物从细胞内溢出减少有关。     

硼替佐米提高耐药K562/ADM细胞对NK细胞杀伤的敏感性及其机制

目的:探讨硼替佐米提高耐药K562/ADM细胞对NK细胞杀伤敏感性的可能机制.方法:流式细胞术和real-time PCR检测硼替佐米处理前后K562/ADM细胞表面MHC Ⅰ类链相关分子A(major histocompatibility complex class Ⅰ chainrelated molecule A,MICA)蛋白和mRNA的表达,LDH释放法检测硼替佐米处理前后K562/ADM细胞对NK细胞的杀伤敏感性.结果:硼替佐米处理后,K562/ADM细胞表面MICA蛋白表达率上升[(17.03 ±4.94)％vs(23.77±5.26)％,P＜0.05];处理后K562/ADM细胞MICA mRNA的表达水平是处理前的(2.03±0.33)倍.效靶比为10∶1、20∶1时,NK细胞对硼替佐米处理后的K562/ADM细胞的杀伤率上升[(23.22±3.03)％、(30.30±0.74)％ vs(33.69±1.28)％、(41.40 ±1.97)％,P＜0.05].结论:硼替佐米提高耐药K562/ADM细胞对NK细胞杀伤的敏感性,其机制可能与硼替佐米上调K562/ADM细胞MICA表达有关.     

甲基莲心碱对乳腺癌MCF-7/Adr 细胞MDR逆转的研究

 目的 探讨甲基莲心碱（Neferine，Nef）对耐药人乳腺癌细胞增殖，细胞内ADM积聚浓度及mdr-1／P-gp表达的影响。方法 采用MTT法测定细胞毒作用，高效液相色谱法测定细胞内ADM积聚浓度，RT—PCR技术及蛋白质印迹技术检测mdr-1／P-gp表达。结果 10μg／ml Nef＋ADM组细胞的抑制率及细胞内ADM积聚浓度比ADM组高（P〈0.01）；20μg／ml Nef＋ADM组细胞抑制率及细胞内ADM积聚浓度较5μg／ml异博定＋ADM组及10μg／ml Nef＋ADM组均高（P〈0．01）。10μg／ml Nef＋ADM组MCF-7／Adr细胞mdr-1 mRNA及P-gp表达比ADM组明显下降（P〈0．01）。20μg／ml Nef＋ADM组细胞mdr-1mRNA及P-gp表达较10μg／ml Nef＋ADM组明显下调（P〈0．01）。结论 Nef能抑制耐药人乳腺癌细胞增殖。Nef能增加MCF-7／Adr细胞内ADM积聚浓度。Nef通过降低耐药人乳腺癌细胞mdr-1 mRNA及P-gp的表达逆转MDR。     

重组人核心蛋白聚糖基因增强多柔比星对白血病 K562细胞抑制作用的实验研究

 目的　探讨重组人核心蛋白聚糖（rhDCN）基因增强多柔比星（ADM）对人类白血病 K562细胞的作用。方法　取对数生长期的K562细胞分为0.9 % NaCl溶液组、pcDNA3.1(+)-DCN／K562组、ADM／K562组、pcDNA3.1(+)-DCN加ADM／K562组。瑞特染色观察细胞形态改变,MTT法检测细胞增殖活性,流式细胞术（FCM）分析细胞凋亡,RT-PCR分析各组TGF-β1 mRNA含量。结果　pcDNA3.1(+)-DCN加ADM／K562组细胞比单独DCN和ADM组细胞,染色呈现更显著的凋亡形态学改变;MTT法结果显示联合组细胞增殖抑制率为（61±1.32）%,明显高于单独干预组[DCN组（20±1.90）%;ADM组（47±1.04）%]（P＜0.05）;FCM检测结果显示联合组细胞凋亡指数为（61.30±0.9）%,较单独干预组[DCN组（28.25±1.3）%及ADM组（31.85±1.5）%]明显增加（P＜0.05）;RT-PCR结果显示,联合组细胞TGF-β1 mRNA的转录减少。结论　rhDCN可以明显增强ADM对K562细胞的杀伤作用,提高肿瘤细胞的凋亡率。rhDCN可能通过下调TGF-β1 mRNA的转录发挥其作用,其具体机制有待进一步研究。     

siRNA抑制K562/ADM细胞mdr1基因表达并逆转其耐药性

背景与目的：白血病耐药性是白血病治疗中的难点，RNAi技术具有特异、高效、毒性小的特点，可高效、特异地抑制特定基因的过度表达。本文研究小干扰RNA分子（siRNA）对白血病多药耐药K562／ADM细胞mdr1基因表达和耐药性的影响。方法：设计、筛选和合成针对mdr1基因的siRNAs（si—mdrl—1，si—mdr1—2），脂质体介导转染K562／ADM细胞；RT—PCR法检测mdr1 mRNA的转录；流式细胞术测定P-糖蛋白（P—gP）表达水平；MTT法检测K562／ADM细胞对多柔比星（阿霉素，ADM）的敏感性。结果：si—mdr1—1、si-mdr1-2转染24h和48h，si—mdr1—1的抑制率分别为55．5％和22．5％，而si—mdr1—2则分别为16．0％和57．6％。si—mdr1—1和si—mdr1—2作用72h时，P—gP的表达强度分别下降74％和85％。si—mdr1—1和si—mdr1—2均可提高K562／ADM细胞对多柔比星的敏感性、逆转其耐药性，逆转倍数分别为2．52倍和1．96倍。结论：siRNA可特异性地沉默mdr1基因的表达，逆转P—gP介导的白血病细胞耐药性。