白血病细胞多药耐药逆转方法的研究进展

白血病细胞耐药的产生是药物治疗中的一大阻碍,耐药细胞过表达的跨膜转运蛋白（主要为P-糖蛋白）导致胞内药物浓度降低是产生耐药的主要原因。此外,凋亡基因的异常表达、药物作用靶点的改变也产生多药耐药（MDR）。针对这些特点寻找合适的药品与化疗药合用以增加肿瘤细胞对化疗药的敏感性,或者利用高分子材料改变释药系统,以及开发新型药物是逆转白血病细胞耐药的主要手段。通过对逆转白血病MDR的方法进行探讨,旨在为白血病治疗提供新思路。     

地佐环平对边缘叶癫痫发作大鼠海马P-糖蛋白表达的影响

目的 观察兴奋性谷氨酸受体（NMDA受体）拮抗剂地佐环平（MK801）对边缘叶癫痫大鼠发作后P-糖蛋白（P—gp）表达的影响,探讨NMDA受体是否能对癫痫发作后P—gp表达进行调控。方法 制备氯化锂．匹罗卡品癫痫发作大鼠模型,60只大鼠,随机分为6组：对照组、癫痫发作终止后0、3、6、24和72h组,用实时荧光定量RT-PCR检测癫痫大鼠发作终止后不同时程海马组织P—gp mRNA的表达。另取60只大鼠随机分为对照组、癫痫持续状态发作模型（SE）组、MK801干预组,各组均分为发作终止后6h和24h时间点。用实时荧光定量RT—PCR检测癫痫发作终止后6h大鼠海马P-gp mRNA的表达,用免疫组化检测大鼠癫痫发作终止后24h海马组织P—gp蛋白的表达。结果 癫痫发作终止后72h内的所有动物海马组织多药耐药基因1a（mdr1a）的表达均显著高于对照组,SE终止即刻其表达显著升高[5．6（2．9）×10^5 mRNA拷贝数／40ng总RNA,P〈0．05],SE终止后3h时其表达进一步升高[7．6（6．3）×10^5,P〈0．01],此后至SE终止后72h,mdrla mRNA的表达一直维持在该水平;mdr1b的表达在边缘叶发作动物呈一过性增高,发作终止后3、6h[分别为（3．3±0．4）×10^4和（3．6±1．0）×10^4,为对照组的2．2、2．4倍]均明显高于对照组（P〈0．01）。另外,在发作终止后6h时,MK801组mdr1a[（4．3±0．8）×10^5]和mdr1b[（2．0±0．7）×10^4]基因表达明显低于SE组（P〈0．01）;发作终止24h时,MK801组P—gp[（26．6±5．0）个微血管／400倍视野]蛋白表达也明显低于SE组[（39．0±4．1）个微血管／400倍视野,P〈0．01]。结论 NMDA受体拮抗剂MK801下调癫痫大鼠发作后P-gp的过度表达,提示NMDA受体可能参与癫痫发作过程中P—gp表达的调控,有效抑制NMDA受体的过度活化可能有助于防止癫痫发作后P—gp的过度表达。     

裸小鼠人肝癌原位移植多药耐药模型的形态学研究

目的建立裸小鼠人肝癌原位移植多药耐药模型,探讨其生物学特征及耐药机制。方法裸小鼠人肝癌(Bel-7402)原位移植,用阿霉素腹腔注射诱导耐药,观察其病理形态学特征,采用荧光定量RT-PCR检测多药耐药基因MDR1mRNA在肝癌组织中表达。以流式细胞仪检测癌细胞MDR1基因产物P-糖蛋白(Pgp)的表达,并通过免疫组化和激光共聚焦显微成像系统对Pgp进行细胞定位及形态观察。结果移植瘤组织形态及生物学行为符合人肝癌特征,实验组MDR1mRNA表达量在1.4×103~3.6×106copy/μgRNA,阳性率达70.00%,Pgp表达为75.45%±5.67%;而对照组MDR1mRNA表达阴性,Pgp表达阳性率为4.25%±1.28%(P<0.01)。形态学观察显示Pgp大多位于细胞膜。结论裸小鼠人肝癌原位移植模型在形态及生物学特征上能高度模拟人肝癌。阿霉素在体内主要通过诱导人肝癌细胞多药耐药MDR1mRNA基因及影响功能蛋白Pgp的表达,获得耐药性。     

NF-кB活化及WT1、MDR1的表达在急性非淋巴细胞白血病中的临床意义

本研究通过观察初治与难治急性非淋巴细胞白血病中NF-κB活性及WT1、MDR1的表达水平,探讨三者在急性非淋巴细胞白血病疗效中的作用与相互关系,为寻找新的改善难治急性非淋巴细胞白血病疗效的方法提供理论依据.急性非淋巴性白血病患者45例,其中初治组20例(A组),难治组25例(分为B、C两组).以15例单纯缺铁性贫血患者作为对照组.采用凝胶电泳迁移分析(EMSA)法检测各组病人NF-κB的活化水平,用逆转录多聚酶链式反应(RT-PCR)法检测各组病人WT1及MDR1的表达水平.结果发现,在对照组未检测到NF-κB活化及WT1和MDR1表达;在初治组中NF-κB的活化水平、WT1和MDR1表达水平均明显低于难治组,而在难治B、C两组中三者无明显区别;各组病人细胞中NF-κB活化水平与WT1及MDR1的表达水平呈正相关.结论NF-κB的活化,WT1、MDR1的高表达可能是急性非淋巴细胞白血病难治的原因之一,急性非淋巴细胞白血病中NF-κB的活化水平与WT1、MDR1的表达呈正相关.     

精神分裂症患者MDR1多态性与帕利哌酮血药浓度相关性研究

目的通过测定精神分裂症患者使用帕利哌酮治疗期间其血药浓度变化及患者多药耐药1(MDR1)C3435T的基因多态性,探讨血药浓度与MDR1C3435T基因型之间的关系。方法采用液相色谱-串联质谱法,于帕利哌酮口服治疗后1周末、2周末、4周末及6周末监测61例精神分裂症患者血药浓度,同时在治疗6周末采用LDR-PCR方法测定MDR1C3435T基因型,对不同基因型及不同等位基因患者帕利哌酮血药浓度进行比较分析。结果 61例精神分裂患者中,CC、CT、TT所占比例分别为24.59%(15/61)、63.30%(38/61)、13.11%(8/61),其中CT杂合子所占比例明显高于CC和TT纯合子(P0.05),C等位基因与T等位基因所占比例分别为55.74%和44.26%(P0.05)。4个检测时间点TT基因型药物浓度均明显高于CC基因型,仅在第1周末TT基因型血药浓度明显高于CT基因型,CT基因型高于CC基因型(P0.05)。4个检测时间点带T等位基因患者的帕利哌酮血药浓度均高于C等位基因患者,但仅第1周末差异具有统计学意义(P0.05)。结论精神分裂症患者MDR1C3435T基因多态性与帕利哌酮的药物浓度具有一定的相关性。     

多元耐药相关蛋白P_(170)糖蛋白在白血病骨髓细胞中的表达

本文应用P_(170)糖蛋白的单克隆抗体MRK_(16),采用流式细胞仪法对10例正常骨髓细胞和21例白血病骨髓细胞P_(170)糖蛋白表达进行了测定。结果表明P_(170)糖蛋白仅出现于白血病骨髓细胞,其中10例难治性白血病有7例阳性表达,11例初治性白血病仅3例阳性表达。这表明 P_(170)糖蛋白与白血病相关,但 P_(170)糖蛋白与白血病疗效的关系则尚待更大宗病例的进一步研究。     

Management of multidrug resistant Gram-negative bacilli infections in solid organ transplant recipients: SET/GESITRA-SEIMC/REIPI recommendations

Solid organ transplant (SOT) recipients are especially at risk of developing infections by multidrug resistant (MDR) Gram-negative bacilli (GNB), as they are frequently exposed to antibiotics and the healthcare setting, and are regulary subject to invasive procedures. Nevertheless, no recommendations concerning prevention and treatment are available. A panel of experts revised the available evidence; this document summarizes their recommendations: (1) it is important to characterize the isolate's phenotypic and genotypic resistance profile; (2) overall, donor colonization should not constitute a contraindication to transplantation, although active infected kidney and lung grafts should be avoided; (3) recipient colonization is associated with an increased risk of infection, but is not a contraindication to transplantation; (4) different surgical prophylaxis regimens are not recommended for patients colonized with carbapenem-resistant GNB; (5) timely detection of carriers, contact isolation precautions, hand hygiene compliance and antibiotic control policies are important preventive measures; (6) there is not sufficient data to recommend intestinal decolonization; (7) colonized lung transplant recipients could benefit from prophylactic inhaled antibiotics, specially for Pseudomonas aeruginosa; (8) colonized SOT recipients should receive an empirical treatment which includes active antibiotics, and directed therapy should be adjusted according to susceptibility study results and the severity of the infection.     

Combination gene therapy using multidrug resistance (MDR1) gene shRNA and herpes simplex virus-thymidine kinase

The current study was designed to evaluate the anti-tumor effects of MDR1 shRNA in combination with herpes simplex virus-thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene therapy system. Introduction of an MDR1-targeted small hairpin RNA (shMDR) markedly enhanced the intracellular accumulation of and increased sensitivity to drugs transported by P-glycoprotein. Functional TK-eGFP fusion protein expression was confirmed by Western blot analysis and ganciclovir uptake assay. Compared with GCV or doxorubicin alone, the combination of anti-cancer drug chemotherapy with GCV administration displays additive cytotoxicity in shMDR1-TK-eGFP expressing cells. These results for the first time suggest the potential of combination gene therapy using suicide gene therapy and RNAi-based gene therapy in vitro.     

Single-step doxorubicin-selected cancer cells overexpress the ABCG2 drug transporter through epigenetic changes

Understanding the mechanisms of multidrug resistance (MDR) could improve clinical drug efficacy. Multidrug resistance is associated with ATP binding cassette (ABC) transporters, but the factors that regulate their expression at clinically relevant drug concentrations are poorly understood. We report that a single-step selection with low doses of anti-cancer agents, similar to concentrations reported in vivo, induces MDR that is mediated exclusively by ABCG2. We selected breast, ovarian and colon cancer cells (MCF-7, IGROV-1 and S-1) after exposure to 14 or 21 nM doxorubicin for only 10 days. We found that these cells overexpress ABCG2 at the mRNA and protein levels. RNA interference analysis confirmed that ABCG2 confers drug resistance. Furthermore, ABCG2 upregulation was facilitated by histone hyperacetylation due to weaker histone deacetylase 1-promoter association, indicating that these epigenetic changes elicit changes in ABCG2 gene expression. These studies indicate that the MDR phenotype arises following low-dose, single-step exposure to doxorubicin, and further suggest that ABCG2 may mediate early stages of MDR development. This is the first report to our knowledge of single-step, low-dose selection leading to overexpression of ABCG2 by epigenetic changes in multiple cancer cell lines.