白血病多药耐药基因和多药耐药相关蛋白基因的表达及临床研究

白血病是造血系统常见的恶性肿瘤 ,化疗是白血病治疗的主要手段 ,但白血病细胞产生的多药耐药是导致化疗失败的主要原因。多药耐药基因(ｍｄｒｌ)和多药耐药相关蛋白基因 (ＭＲＰ)的过度表达是引起白血病多药耐药发生的重要机制之一。本文应用半定量ＲＴ ＰＣＲ方法同时检测 40例急性白血病和慢性粒细胞白血病急变患者的ｍｄｒｌ和ＭＲＰ的表达水平 ,并探讨了它们与临床耐药的关系。1　对象与方法1 .1 　 研究对象40例患者均为我院 1 999年 7月～ 2 0 0 0年 5月住院患者 ,男 2 5例 ,女 1 5例 ,中位年龄 37岁 ( 4～ 68岁 )。初治组 2 7例 ,复…     

耐多药结核病患者外周血中多药耐药相关跨膜转运蛋白的表达

目的 评价耐多药结核病(MDR-TB)患者外周血中单核细胞P糖蛋白和多药耐药相关蛋白(MRP)表达水平的变化.方法选择2004年9月至2007年12月在武汉市结核病防治所住院治疗的MDR-TB患者89例为耐多药组,其中男52例,女37例;年龄17~62岁,平均(45±6)岁;平均病史3.5年;均符合MDR-TB的诊断标准.同期在武汉市结核病防治所住院的初治结核病患者55例为结核病组,其中男34例,女21例;年龄18~60岁,平均(48±8)岁;平均病史1.2年;痰涂片均为阳性.对照组为武汉市结核病防治所无肺结核史的工作人员31例,其中男19例,女12例;年龄25~62岁,平均(44±5)岁.抽取外周血,采用实时定量PCR法,分别检测单核细胞P糖蛋白和MRP的mRNA水平.多组间比较采用单因素方差分析,组间两两比较采用SNK-q检验.结果 耐多药组P糖蛋白mRNA表达的吸光度值(1.34±0.32)与结核病组(1.12±0.23)和对照组(1.05±0.16)比较,差异无统计学意义(F=0.536,P>0.05).耐多药组MRP的mRNA表达(3.45±0.43)明显高于结核病组(1.23±0.34)和对照组(1.04±0.12),3组比较,差异有统计学意义(F=24.241,P<0.05),耐多药组分别与其他2组比较,差异均有统计学意义(P<0.01).结论 MRP高表达与MDR-TB患者的多耐药存在一定的相关性.

Abstract：

Objective To evaluate the expression of P-glycoprotein (P-pg) and multidrug resistance-associated protein (MRP) mRNA levels in peripheral blood mononuclear cells from multidrug resistant tuberculosis (MDR-TB) patients. Methods The subjects of this study included 3 groups: a non-TB control group, a TB control group and a MDR-TB group. The 31 subjects in the non-TB control group were staff from Wuhan Tuberculosis Prevention and Treatment Institute. The 55 cases in the TB control group were in-patients during September 2004 to December 2007 who were diagnosed as having pulmonary tuberculosis. The 89 cases in the MDR-TB group were in-patients during the same period, but who were diagnosed as having MDR-TB. Peripheral mononuclear cells were isolated and mRNA levels of P-pg and MRP were measured by real-time PCR. Comparisons of the data between different groups were performed using one-way ANOVA, and SNK q was used for comparison between 2 groups.Results There was no significant difference in the relative P-pg mRNA levels among the MDR-TB group (1.34±0.32), the non-TB control group (1.05±0.16) and the TB control group (1.12±0.23), F=0.536, P>0.05. The relative MRP mRNA level (3.45±0.43) was the highest in the MDR-TB group (3.45±0.43), as compared to the TB control group (1.23±0.34) and the non-TB control group (1.04±0.12), F=24.241, P<0.05. Conclusion Higher expression of MRP in peripheral mononuclear cells might be related to multidrug resistance in MDR-TB patients.     

多药耐药相关蛋白-3表达与肝癌耐药的相关性

目的 探讨多药耐药相关蛋白-3(MRP-3)的表达与肝癌耐药的相关性.方法 应用RT-PCR方法检测正常肝细胞L-02、肝癌细胞BEL及肝癌耐药细胞HepG2/ADM中MRP-3 mRNA的差异表达,再将MRP-3反义及正义寡核苷酸片段分别用脂质体转染进肝癌耐药细胞HepG2/ADM中,用MTT法、流式细胞仪及RT-PCR检测转染后细胞内阿霉素(ADM)的荧光强度、耐药指数及MRP-3 mRNA的表达情况.结果 肝癌耐药细胞HepG2/ADM中MRP-3 mRNA的表达明显高于正常肝细胞L-02和肝癌细胞BEL(P<0.05).MRP-3反义转染进耐药细胞后,可明显降低细胞内ADM的耐药指数,提高细胞内ADM浓度(P<0.05),细胞内MRP-3 mRNA的表达较未转染细胞下降了62.5%(P<0.05).结论 MRP-3的高表达可能是导致肝癌多药耐药的机制之一.     

不同剂量亚砷酸钠染毒大鼠多药耐药相关蛋白2表达水平的变化

目的观察不同剂量亚砷酸钠染毒大鼠肝细胞膜转运蛋白——多药耐药相关蛋白2(multidrugresistance-associatedprotein2,MRP2)表达水平的变化及与砷代谢的关系。方法24只健康雄性Wistar大鼠随机分为4组:第1组为对照组,给予生理盐水;第2～4组为染砷组,分别给予4、10和20mg/kg体质量的亚砷酸钠溶液,隔天灌胃染毒1次,2周后将大鼠处死。原子吸收分光光度法测定胆汁、全血和肝组织中的总砷含量。蛋白印记法测定肝细胞膜上MRP2的改变。结果经方差分析检验,胆汁和肝脏中含砷量随着染砷剂量的增加而逐渐增加(P<0.05)。两两比较发现,3个染毒组的血砷与对照组之间差异有统计学意义,而3个染毒组间差异无统计学意义。随着染砷剂量增加,MRP2表达有增加的趋势,且MRP2表达量与胆汁含砷量呈正相关(r=0.986,P<0.05)。结论亚砷酸钠可以诱导MRP2的表达,随染砷剂量增加,MRP2表达量增加。MRP2在砷及其代谢产物的胆汁排泄过程中发挥了重要作用。     

肺癌多药耐药机制的研究进展

肺癌化疗失败的主要原因是肿瘤细胞多药耐药（MDR）的产生。肺癌MDR的机制十分广泛,其中以ATP结合盒结构超家族成员参与耐药机制的研究较多。本文重点综述目前研究较多的P一糖蛋白、多药耐药相关蛋白、肺耐药相关蛋白和乳腺癌耐药蛋白与肺癌MDR的关系,并对其他肺癌MDR的发生机制如肿瘤细胞内解毒物质作用增强、药物作用靶点减少、DNA损伤修复功能增强及凋亡与抗凋亡机制的失衡作一简要介绍。     

造血保护的多药耐药转基因技术研究进展

化疗药物导致的骨髓抑制，严重防碍着治疗中化疗药物剂量的增加和治疗用期的缩短，成为影响肿瘤治疗效果的重要因素之一。因此近年来人们在研究应用细胞因子及自体骨髓移植等方法处理骨髓抑制的同时，尝试将多药耐药（MDR）基因转入造血干细胞（HSC）以保护和支持造血功能，减少药物对其的损伤，从而提高肿瘤患者对大剂量化疗的耐受水平。现就有关研究进展综述如下：正实验研究1．1目的基因MDR是指由一种药物诱发，同时对其他多种给构和作用机制不同的抗癌药物产生交叉耐药。人类MDR基因家族中与耐药有关的为MDRI基因，定位于第7号染…     

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。     

多药耐药相关蛋白2的研究进展

肝脏的外分泌功能具有非常重要的作用，除了参与消化吸收以外，人体内大量化合物的代谢都需肝脏的处理并由胆汁排泄，这个外分泌过程就需要肝胆转运系统的参与，肝脏的转运系统中包含着许多的载体蛋白，这些载体蛋白与肝炎，肝内胆汁郁积，肿瘤化疗药物的耐受以及多种肝脏遗传性疾病有着密切的关系。     

肺癌多药耐药现象与生物膜表面糖蛋白

肺癌化疗前后常产生多药耐药使化疗失败,目前认为生物膜上某些糖蛋白与多药耐药有关,本文拟对这些糖蛋白的结构、功能及与肺癌的多药耐药机制加以概述。     

中药逆转恶性肿瘤多药耐药的研究进展

癌症患者的死因大约94％与肿瘤多药耐药的发生有关，目前寻求高效低毒的逆转多药耐药的方法已成为研究的热点。引起肿瘤多药耐药的机制比较复杂，中药以资源丰富、含多种逆转耐药的活性成分、作用靶点多，而引起了广泛的关注。本文就中药逆转恶性肿瘤多药耐药研究的进展情况如下综述。     

Molecular mechanisms of tumor vascularization

Tumor angiogenesis is a fast growing sub-domain of angiogenesis research and tumor biology. Basic mechanisms have been unraveled and many key players identified. For many years, tumor vascularization was explained solely by the ingrowth of new vessels into the tumor from preexisting one's. However, in recent years, additional mechanisms have been recognized. These include angioblasts recruitment, cooption, vasculogenic mimicry and mosaic vessels. These different mechanisms may exist concomitantly in the same tumor or may be selectively involved in a specific tumor type or host environment. In this article, we will review, in depth, these different mechanisms and also discuss some aspects of anti-angiogenic tumor therapy.     

VEGF-integrin interplay controls tumor growth and vascularization

Cross-talk between the major angiogenic growth factor, VEGF, and integrin cell adhesion receptors has emerged recently as a critical factor in the regulation of angiogenesis and tumor development. However, the molecular mechanisms and consequences of this intercommunication remain unclear. Here, we define a mechanism whereby integrin alpha v beta3, through activation, clustering, and signaling by means of p66 Shc (Src homology 2 domain containing), regulates the production of VEGF in tumor cells expressing this integrin. Tumors with "activatable" but not "inactive" beta3 integrin secrete high levels of VEGF, which in turn promotes extensive neovascularization and augments tumor growth in vivo. This stimulation of VEGF expression depends upon the ability of alpha v beta3 integrin to cluster and promote phosphorylation of p66 Shc. These observations identify a link between beta3 integrins and VEGF in tumor growth and angiogenesis and, therefore, may influence anti-integrin as well as anti-VEGF therapeutic strategies.     

Platelet dysfunction associated with Wilms tumor and hyaluronic acid

Acquired von Willebrand disease (AVWD) has been described in two cases of nephroblastoma. We studied a patient with nephroblastoma who presented with a coagulopathy suggestive of AVWD. The subject had undetectable levels of F.VIIIR:Ag, diminished F.VIIIR:WF (16.3%), F.VIII:C activity (37%), and lack of platelet aggregation to ADP, epinephrine, collagen, and arachidonic acid. These results were associated with abnormally high serum levels (850 mg/dl) of hyaluronic acid (HA), which made the patient's serum hyperviscous. Examination of the neoplasm revealed HA in the tumor matrix. All abnormalities of coagulation resolved after chemotherapy and extirpation of the neoplasm, which produced normal serum HA levels. To study the effects of HA on platelet function, we added HA to normal platelet-poor plasma (NPP), which rendered F.VIIIR:Ag undetectable; treatment of HA with hyaluronidase eliminated F.VIIIR:Ag assay interference caused by HA. F.VIII:C activity decreased in vitro when HA was mixed with normal platelet-poor plasma (NPP). HA reduced the initial slope of normal platelet aggregation. Partial correction of platelet aggregation occurred after hyaluronidase treatment of HA-spiked PRP. Experiments in rabbits exposed to HA (serum level 400 mg/dl) demonstrated abnormalities similar to those noted in the patient. Shear rate studies of whole blood containing HA (500 mg/dl) yielded high shear stress, 27-136 dynes/cm2 over shear rates of 10-216 sec-1. We conclude that the coagulopathy demonstrated in this case is secondary to hyperviscosity produced by elevated levels of HA, which interferes with the assay for F.VIIIR:Ag. Thus the acquired coagulopathy associated with other cases of nephroblastoma may present as spurious von Willebrand disease.     

Effects of tumor necrosis factor on sensitive and multidrug resistant human leukemia and myeloma cell lines

Two human tumor cell lines exhibiting acquired multidrug resistance (MDR) with increased expression of a cell surface glycoprotein (GP-170) were tested for their sensitivity to human recombinant tumor necrosis factor (rTNF). The drug resistant mutant lines (CEM/V, a T-cell leukemia line resistant to vinblastine, and 8226/D, a multiple myeloma line resistant to doxorubicin), were markedly more sensitive to rTNF in clonogenic assay than were their drug-sensitive parental lines (CEM, 8226). As determined by radioreceptor assay, the number of cell surface receptors for rTNF did not differ on the parental and drug-resistant lines. During the first 24 hours after addition of rTNF, there was a decrease in intracellular ATP content in the CEM/V line but not in the CEM line. No differential effect of rTNF on ATP content was observed between 8226 and 8226/D. As determined by RNA dot-blot analysis, total cellular RNA for GP-170 was increased in the 8226/D cells. After rTNF exposure, expression of total cellular RNA for GP-170 was not altered. Accumulation of radiolabeled doxorubicin by 8226/D cells was not altered by previous or coincubation with rTNF. These findings suggest that the effects of rTNF on MDR cells is not related to TNF receptor number and is mediated at a step subsequent to rTNF binding and not by either inhibition of synthesis of GP-170 or by alteration in the function of the GP-170 efflux pump.     

Role for the Wilms tumor gene in genital development?

Detailed molecular definition of the WAGR region at chromosome 11p13 has been achieved by chromosome breakpoint analysis and long-range restriction mapping. Here we describe the molecular detection of a cytogenetically invisible 1-megabase deletion in an individual with aniridia, cryptorchidism, and hypospadias but no Wilms tumor (WT). The region of overlap between this deletion and one associated with WT and similar genital anomalies but no aniridia covers a region of 350-400 kilobases, which is coincident with the extent of homozygous deletion detected in tumor tissue from a sporadic WT. A candidate WT gene located within this region has recently been isolated, suggesting nonpenetrance for tumor expression in the first individual. The inclusion within the overlap region of a gene for WT predisposition and a gene for the best-documented WT-associated genitourinary malformations leads us to suggest that both of these anomalies result from a loss-of-function mutation at the same locus. This in turn implies that the WT gene exerts pleiotropic effect on both kidney and genitourinary development, a possibility supported by the observed expression pattern of the WT candidate gene in developing kidney and gonads.     

Quantification of in vivo tumor invasion and vascularization by computerized image analysis

The matrix-inserted surface transplantation model is an in vivo assay used to analyse the kinetics of tumor-vessel interactions during different stages of skin carcinoma progression. This system allows the study of host-tumor interface, i.e. penetration of tumor cells into normal host tissue as well as infiltration of normal host cells into the tumor. In the present study, image analysis algorithms for processing and quantifying the extent of such migratory and tissue remodeling events are presented. The proposed method is non-parametric and its originality lies in its particularity to take into account the specific geometry of tumor-host interface. This methodology is validated by evaluating the contribution of matrix metalloproteases (MMPs) in skin carcinoma invasion and vascularization through pharmacological and genetic approaches.