流式细胞仪在膀胱癌多药耐受研究中的应用

目的：探讨流式细胞仪（ＦＣＭ）在膀胱癌多药耐受研究中的应用。方法：利用蛋白质印迹,免疫细胞化学和ＦＣＭ检测并比较膀胱癌多药耐受细胞ＥＪ／ＭＲＰ的多药耐受糖蛋白（Ｐ－ｐｇ）和多药耐受相关蛋白（ＭＲＰ）的表达;利用ＦＣＭ检测了ＥＪ／ＭＲＰ药物耐受的功能水平。结果：ＥＪ／ＭＲＰ高表达ＭＲＰ,无明显Ｐ－ｇｐ表达,ＦＣＭ的检测结果与蛋白质印迹,免疫细胞化学的结果一致,但检测更简便,功能检测显示柔红霉素（ＤＮ     

胃肠道恶性肿瘤多药耐药相关蛋白和肺耐药蛋白流式细胞术检 …

ｍｄｒ1基因编码的Ｐ糖蛋白(Ｐｇｐ)过度表达是胃肠道恶性肿瘤产生多药耐药(ｍｕｌｔｉｄｒｕｇｒｅｓｉｓｔａｎｃｅ,ＭＤＲ)的重要机理之一[1]。最近研究发现的另外两个跨膜转运蛋白———多药耐药相关蛋白(ｍｕｌｔｉｄｒｕｇｒｅｓｉｓｔａｎｃｅａｓｓｏｃｉａｔｅｄｐｒｏｔｅｉｎ,ＭＲＰ)和肺耐药蛋白(ｌｕｎｇｒｅｓｉｓｔａｎｃｅｐｒｏｔｅｉｎ,ＬＲＰ),也参与恶性肿瘤的ＭＤＲ[2,3]。我们研究ＭＲＰ和ＬＲＰ在胃肠道恶性肿瘤中的表达及其临床意义。一、对象和方法1.对象:经病理学检测确诊的胃肠道癌症患者19例,其中胃癌14例,大肠…     

三种逆转剂对人膀胱癌多药耐受相关蛋白介导的多药耐受逆转的研究

目的：观察环孢霉素Ａ（ＣｓＡ）、维拉帕米（Ｖｅｒ）、黄体酮（Ｐｒｏｇ）对膀胱癌多药耐受相关蛋白（ＭＲＰ）介导的多药面受（ＭＤＲ）的塑转作用。方法：分别采用ＭＴＴ方法和流式细胞仪检测观察ＣｓＡ、Ｖｅｒ和Ｐｒｏｇ塑转化疗药物ＶＣＲ对ＭＲＰ高表达的膀胱癌细胞的细胞毒性作用的塑转效果。以及地细胞内柔红霉素（ＤＮＲ）聚集、外排的影响。结果：ＣｓＡ和Ｖｅｒ作为多药耐受糖蛋白（Ｐｇｐ）的良好剂同样可以显著逆转ＶＣＲ对Ｍ％ＲＰ高表达的ＥＪ／ＭＲＰ细胞毒性作用,具有增加ＥＪ／ＭＲＰ细胞内ＤＮＲ的聚集百的作用。剂量越大作用越强;而Ｐｒｏｇ则无类似作用。结论：ＣｓＡ和Ｖｅｒ对人膀胱癌ＭＲＰ介导的ＭＤＲ具有良好的逆转作用。可通过增加细胞内的药物聚集和减少外排已进入细胞内的药物起作用。这种作用存在剂量依从关系。     

核酶逆转MDR研究进展

核酶是一类具有催化活性的小分子ＲＮＡ，它能特异性地与靶ＲＮＡ分子结合进行切割，阻断目的基因的表达，目前被广泛应用于基因表达调控的研究。多药耐药性（ＭｕｌｔｉｄｒｕｇＲｅｓｉｓｔａｎｃｅ，ＭＤＲ）的产生与细胞膜上一种分子量为１７０ＫＤ的跨膜蛋白的过度表达有关，应用核酶切割过度表达的ｍｄｒｌ基因可以逆转ＭＤＲ，在ＭＤＲ的治疗方面有潜在的应用价值。本文就应用核酶逆转ＭＤＲ，尤其是核酶基因治疗方面的进展作一简要综述。     

肿瘤细胞耐药性研究进展

肿瘤细胞耐药性研究进展李树奇朴炳奎中国中医研究院广安门医院（北京１０００５３）多药耐药（ｍｕｌｔｉｄｒｕｇｒｅｓｉｓｔａｎｃｅ，ＭＤＲ）是肿瘤细胞免受化疗药物攻击的最重要的细胞防御机制，ＭＤＲ是由一种药物诱发而同时对多种结构和作用机制完全不同的抗癌药...     

MRP真核表达载体的构建及其在人膀胱癌中的表达

多药耐受（ＭＤＲ）是导致化疗失败的重要原因。研究表明：ｍｄｒ１／Ｐｇｐ介导的ＭＤＲ是膀胱癌ＭＤＲ的重要机制,但不是唯一机制。新近发现的多药耐受相关蛋白（ＭＲＰ）基因是介导ＭＤＲ的一种肯定机制［１］,我们将ＭＲＰ基因转入膀胱癌细胞,并初步观察了其介导的ＭＤＲ及ＭＲＰ的表达。材料和方法　真核表达载体ｐｃＤＮＡ３．１（＋）／ＭＲＰ１的构建：含全长ＭＲＰ１ｃＤＮＡ的载体ＰＪ３Ω／ＭＲＰ１由荷兰癌症研究所的ＭａｒｃｅｌＫｏｏｌ博士赠送。用限制性内切酶ＮｈｅＩ、ＮｏｔＩ完全消化ＰＪ３Ω／ＭＲＰ１和ｐｃＤ…     

国外近期有关文献题录

［１］　ＤｅＭｅｙｅｒＪＭ,ＴｈｉｂｏＰ．Ｔｈｅｃｏｒｒｅｌａｔｉｏｎａｍｏｎｇｃａｖｅｒｎｏｕｓｐｒｅｓｓｕｒｅ,ｐｅｎｉｌｅｒｉｇｉｄｉｔｙａｄｒｅｓｉｓｔａｎｃｅｉｎｄｅｘ．ＪＵｒｏｌ１９９８,１６０（１）：６３～６．［２］　ＣｈｕａｎｇＡＴ;ＳｔｒａｕｓｓＪＤ;ＭｕｒｐｈｙＲＡ,ｅｔａｌ．Ｓｉｌｄｅｎａｆｉｌ,ａｔｙｐｅ５ＣＧＭＰｐｈｏｓｐｈｏｄｉｅｓｔｅｒａｓｅｉｎｈｉｂｉｔｏｒ,ｓｐｅｃｉｆｉｃａｌｌｙａｍｐｌｉｆｉｅｓｅｎｄｏｇｅｎｏｕｓｃＧＭＰｄｅｐｅｎｄｅｎｔｒｅｌａｘａｔ…     

多药耐药相关蛋白在肝细胞癌中的表达及其临床意义

目的 研究人肝细胞癌组织中多药耐药相关蛋白（ＭＲＰ）的表达情况并探讨其临床意义。方法 采用免疫组织化学方法检测了１１０例随访资料完整的肝细胞癌石蜡切片组织中ＭＲＰ表达情况，并与临床病理指标联系，进行统计学分析。结果 本组肝细胞癌１１０例，表达阳性５７例，其阳性率为５２％。术前行经肝动脉栓塞化疗（ＴＡＣＥ）及未行ＴＡＣＥ治疗者的ＭＲＰ阳性率分别为５１％（１９／３７）和５２％（３８／７３）。ＭＲＰ与癌     

1,25—二羟基维生素D3对小鼠骨髓细胞形成破骨细胞样细？…

目的动态观察１，２５－二羟基维生素Ｄ３「１，２５－（ＯＨ）２Ｄ３」对ＮＨ小鼠骨髓细胞培养在体外形成破骨细胞及其骨吸收的剂量效应和作用时象。方法收集ＮＨ小鼠骨髓细胞于含１０％胎牛血清的α－ＭＥＭ培养基中行体外培养，设置不同的１，２５－（ＯＨ）２Ｄ３浓度组和给药时间组，并于培养第３、６、９、１２天观察记录抗酒石酸酸性磷酸酶（ｔａｒｔｒａｔｅ－ｒｅｓｉｓｔａｎｔ ａｃｉｄ ｐｈｏｓｐｈａｔａｓｅ，ＴＲＡ     

Penetrating wounds of the heart：an analysis of 61 cases

ＧＤｅｐａｒｔｍｅｎｔｏｆＴｒａｕｍａｔｏｌｏｇｙａｎｄＣａｒｄｉｏｔｈｏｒａｃｉｃＳｕｒｇｅｒｙ,ＣｈｏｎｇｑｉｎｇＥｍｅｒｇｅｎｃｙＭｅｄｉｃａｌＣｅｎｔｅｒ,Ｃｈｏｎｇｑｉｎｇ４０００１４,Ｃｈｉｎａ（ＧａｏＪＭ）ｕｎｓｈｏｔｗｏｕｎｄｈａｓｂｅｅｎｔｈｅｃｏｍｍｏｎｅｓｔｃａｕｓｅ（ａｃｃｏｕｎｔｉｎｇｆｏｒ６０％７０％）ｏｆｐｅｎｅｔｒａｔｉｎｇｉｎｊｕｒｙｏｆｔｈｅｈｅａｒｔｉｎｔｈｅＵｎｉｔｅｄＳｔａｔｅｓｏｆＡｍｅｒｉｃａｉｎｔｈｅｐａｓｔ２０ｙｅａｒｓ．１４Ｉｔｃａｕｓｅｓ…     

Multifactorial Involvement of Multidrug Resistance Protein, DNA Topoisomerase II and Glutathione/Glutathione-S-Transferase in NonP- Glycoprotein-Mediated Multidrug Resistance in Human Bladder Cancer Cells

Background :
Multiple mechanisms are important in multidrug resistance in urothelial cancers. We investigated the acquisition of a multidrug resistance phenotype in human bladder cancer cells exposed to doxorubicin.
Methods :
Human bladder cancer cell line 5637 and 2 doxorubicin drug-resistant sublines (5637/DR5.5 and 5637/DR50) were used. Measurements were made of the steady state mRNA levels of the multidrug resistance gene ( mdr 1), multidrug resistance-associated protein (MRP), glutathione-S-transferase-π and DNA topoisomerase II (topo II) genes, P-glycoprotein (PgP) and MRP expression, glutathione (CSH) and GSH enzyme activity, and topo II catalytic activity. The pharmacokinetics were compared between the parent and the drug-resistant sublines.
Results :
5637/DR5.5 and 5637/DR50 cells were 7.6- and 1 6.2-fold more resistant to doxorubicin and 16.7-and 48.3-fold more resistant to etoposide, respectively, compared with 5637 cells. A dose escalation of doxorubicin increased the MRP expression, CSH levels and glutathione-S-transferase (GST) activity, although no PgP expression was observed in any cell line. Resistance was brought about by decreased drug accumulation through drug efflux, although intracellular daunorubicin concentrations were similar between DR5.5 and DR50 cells. Topo II catalytic activity was undetectable in DR50 cells, but maintained in both the parent and DR5.5 cells.
Conclusion :
Reduced drug accumulation in doxorubicin-resistant cells was mediated by MRP instead of PgP indicating that MRP-mediated drug efflux functions in a limited manner for drug resistance. An increase in drug efflux via MRP, reduced topo II activity, and increased GSH levels/GSH-related enzyme activities may play major roles in nonPgP-mediated multidrug resistance in urothelial cancers treated with anthracyclines.     

多药耐药相关蛋白在阿霉素诱导人肝癌细胞SMMC—7721耐药性产生中的作用及

目的 动态观察阿霉素（ＡＤＭ）诱导肝癌细胞ＳＭＭＣ－７７２１耐药性的产生，了解多药耐药相关蛋白（ＴＲＰ）在其耐药机理中的作用。方法 分别用逐步提高培养基中ＡＤＭ的浓度诱导ＳＭＭＣ－７７２１细胞和用含不用ＡＤＭ浓度的培养基直接短期培养ＳＭＭＣ－７７２１细胞的方法，诱导细胞产生耐药性，绘制剂量反应曲线，确定细胞耐药倍数，ＲＴ－ＰＣＲ法测定细胞ＭＲＰｍＲＮＡ的表达水平，流式细胞仪检测细胞内柔红霉素（ＤＮＲ）的浓度。结果 随着培养基中ＡＤＭ浓度的逐步提高，ＭＲＰｍＲＮＡ的表达也逐渐增强，细胞内ＤＮＲ浓度明显下降，ＳＭＭＣ－７７２１细胞的耐性逐渐增加；用含不同ＡＤＭ浓度的培养基直接培养亲代细胞后，虽然ＭＲＰｍＲＮＡ表达明显升高，但细胞内ＤＮＲ浓度仍维持较高水平，并且绝大部分细胞短期内死亡。结论 ＡＤＭ可以逐步诱导肝癌细胞     

人肝癌多药耐药细胞模型的建立

为研究肝癌ＭＤＲ机制,采用５种抗癌药物通过不同的诱导方式建立一组人肝涪ＭＤＲ细胞模型。对其耐药机理的研究结果表明,ＳＭＭＣ７７２１／ＤＯＸ亚系由ｍｄｒ１基因介导耐药,ＳＭＭＣ７７２１／ＶＣＲ亚系由ＭＲＰ基因介导耐药,ＳＭＭＣ７７２１／ＣＢＰ亚系由ＬＰＲ基因介导耐药,ＳＭＭＣ７７２１／ＶＰ１６亚系由ＴｏｐｏⅡα基因介导耐药,ＳＭＭＣ７７２１／ＭＭＣ亚系由ＧＳＴｐ１基因介导耐药,多重ＭＤＲ亚系ＳＭＭＣ     

单基因人肝癌耐药细胞株(HepG2/MRP1)的构建及其生物学意义

目的 对抗肿瘤药的天然耐药和化疗过程中产生的继发性耐药是肝癌化疗敏感性差的重要原因之一,为探讨体外逆转肝癌多药耐药(muhidrug resistance,MDR)的新方法,笔者建立了单因素人肝癌细胞多药耐药模型HepG2/mrp1,并研究其牛物学特性.方法 双酶切克隆质粒pGEMmrp1获得人全长多药耐药相关蛋白基因(mrp1),并将其插入哺乳动物表达载体pCI-neo的多克隆位点,构建重组载体,利用质脂体法将重组载体转入人肝癌细胞HepG2,建立单基因耐药细胞株HepG2/mrp1.观察细胞的生长规律;MTT法检测其多药耐药性;流式细胞仪检测细胞表面多药耐药相关蛋白(multidrug resistance-associated protein,MRP)的表达;RT-PCR检测MRP1 mRNA表达量.结果 与HepG2细胞相比较,HepG2/mrp1细胞的倍增时间延长30.86 h.该细胞对多种抗肿瘤药耐药,HepG2/mrp1对阿霉素和柔红霉素的耐药指数比亲本细胞增加了11.4倍和8倍,细胞表面MRPl的表达明显增加,mrp1 mRNA明显增加.结论 HepG2/mrp1细胞稳定高表达MRP1,具有多药耐药性,为进一步研究肝癌的多药耐药构建了一个技术平台.     

RNA干扰逆转肝癌多药耐药

目的 研究siRNA对肝癌耐药细胞系HepG2/mrp1中多药耐药相关蛋白基因(multidrug-resistant-associated 1,mrp1)及其蛋白产物MRP1表达的抑制作用和逆转其耐药性的效果.方法 设计合成针对mrp1启动子区域的RNA干扰小片段,转染肝癌耐药细胞HepG2/mrp1.通过RT-PCR和流式细胞仪,在mRNA和蛋白质水平评估RNA干扰对mrp1表达的影响;MTT法检测RNA干扰逆转HepG2/mrp1细胞多药耐药性的变化,按照实验组和亲本组细胞的半数抑制剂量(ICso)评估RNA干扰对细胞耐药倍数的改变.结果 在耐药细胞HepG2/mrp1中,RNA干扰明显抑制了 mrp1 mRNA和蛋白产物MRP1的表达水平,在相同浓度药物作用下实验组细胞耐药性显著下降.结论 我们设计的siRNA对肝癌耐药细胞HepG2/mrp1中的mrp1基因有显著的抑制作用,具有良好的逆转多药耐药性的效果.

Abstract：

Objective To investigate the suppression of mrp1 and MRP1 induced by small interfering RNA and the restoration of sensitivity to chemotherapeutic drugs in the multidrug-resistant hepatocellular carcinoma cell lines HepG2/mrp1. Methods mrp1-targeted small interfering RNA duplexes were designed and composed and introduced into multidrug-resistant hepatocellular carcinoma cell lines HepG2/mrp1. The suppression of mrp1 mRNA and its gene product MRP1 was examined by RT-PCR and flow cytometry (FCM), respectively. MTT assay was performed to measure the reverse effect of small interfering RNA based on the results of ICs0. Results The overexpression of mrp1 mRNA and MRP1 was effectively suppressed by small interfering RNAs. The level of mrp1 mRNA in the transfected HepG2/mrp1 cells was reduced to (86.36±2.76)% and MRP1 to (89.38±3.76)%compared with those of the controls. The resistance to ADR was reversed five-fold, which indicated the restoration of sensitivity to drugs. Conclusion Small interfering RNA can inhibit mrp1 expression effectively and reverse the multidrug resistance mediated by MRP1.     

反义寡核苷酸降低细胞株SMMC—7721／ADM之MRP基因表达的研究

目的 研究反义硫代磷酸寡核苷酸（AS－ODN）抑制人肝癌细胞耐药株（SMMC－7721／ADM）MRP基因表达的作用。方法 用人工合成互补于MRP基因mRNA特定片断的反义硫代磷酸寡核苷酸，以脂质体Lipofectamine为载体转染SMMC－7721／ADM细胞株，用RT－PCR测定mRNA表达，流式细胞技术测定细胞膜MRP1蛋白P190表达及MTTI地检测细胞对柔红霉素（DNR）和阿霉素（ADM）的敏感性。结果 AS－ODN能抑制AMR mRNA和其蛋白P190表达，提高细胞对DNR和ADM的敏感性。结论 （1）AS－ODN能降低MRP基因表达;(2)MRP介导的多药耐药（MDR）是SMMC－7721／ADM耐药的重要机理之一。     

Prostanoid transport by multidrug resistance protein 4 (MRP4/ABCC4) localized in tissues of the human urogenital tract

PURPOSE: The seminal vesicles are the major source of prostaglandins in seminal fluid. For prostanoid action on cell surfaces they must be released from synthesizing cells. MRP4/ABCC4 (multidrug resistance protein 4 adenosine triphosphate-binding cassette, subfamily C, member 4) is an adenosine triphosphate dependent export pump for organic anions that may mediate prostanoid transport across the plasma membranes. Therefore, we analyzed whether MRP4 is expressed in the seminal vesicles and other tissues of the human urogenital tract, whether MRP4 and prostanoid synthesizing enzymes are co-expressed in the same cell type and whether MRP4 functions as a prostanoid export pump. MATERIALS AND METHODS: The expression and localization of MRP4 and prostanoid synthesizing enzymes were investigated in several tissues of the male human urogenital tract by immunoblot and immunofluorescence analyses. Prostanoid transport was measured into inside-out membrane vesicles from cells expressing recombinant human MRP4. RESULTS: MRP4 and prostanoid synthesizing enzymes were co-expressed in the epithelial cells of human seminal vesicles. Moreover, MRP4 was localized in the plasma membrane of epithelial cells of the ureter, in the basolateral membrane of glandular epithelial cells of the prostate, and in smooth muscle cells of the bladder and corpus cavernosum. Transport studies established MRP4 as an efflux pump for prostaglandin E2 (Michaelis constant [Km] 3.5 muM), thromboxane B2 (Km 9.9 muM) and prostaglandin F2alpha (Km 12.6 muM). CONCLUSIONS: The co-expression of prostanoid synthesizing enzymes and MRP4 in epithelial cells of the human seminal vesicles and the function of MRP4 as a prostanoid efflux pump indicate that MRP4 mediates prostanoid transport from these cells, which are the main prostanoid synthesizing cells in the male urogenital tract.