难治性癫痫患者脑组织胶质原纤维酸性蛋白和多药耐药基因蛋白的免疫电镜观察

目的在电镜水平上观察多药耐药基因蛋白(MDR1蛋白)和胶质原纤维酸性蛋白(GFAP)在难治性癫痫患者脑组织的表达，确定其在细胞超微结构中的定位。方法临床难治性癫痫患者手术切除的病理组织标本，固定、冷冻超薄切片，以免疫胶体金(PAG)标记技术标记检测MDR-1和GFAP蛋白，在电镜下观察其表达。结果MDR-1蛋白与GFAP均在胶质细胞内有阳性表达，见于胶质细胞膜及其胞浆的广泛区域。在神经元和毛细血管内皮细胞中未见有GFAP和MDR-1蛋白的阳性表达。结论在难治性癫痫患者脑组织中，MDR1蛋白与GFAP的高表达现象只存在于胶质细胞胞膜和胞浆内。     

细胞色素P4503A基因表达调控及与抗癫痫药物的关系

细胞色素P4503A酶(Cytochrome P450 3A enzymes)在外源性化合物尤其是药物代谢中起重要作用,临床中约有50%的药物经由CYP3A代谢,其中包括多种抗癫痫药物.本文概述了CYP3A的基因家系的结构、分布和调控表达的异同,CYP3A与难治性癫痫患者药物难治性的可能机制--多药耐药基因和P-糖蛋白的关系,CYP3A4基因表达上的差别与药物作用强度和持续时间、急性毒性的相关性.     

难治性癫痫与多药耐药基因表达上调

癫痫是神经系统常见、多发病症。在过去20年中，有多个新型抗癫痫药物（AED）问世并应用于临床，但仍有1／3的癫痫患者发作不能得到满意控制，药物治疗效果不良，称为难治性或耐药性癫痫。难治性癫痫耐药现象的细胞分子机制尚不完全清楚，可能为：①AED的作用靶点如钠通道或GABA受体消失或改变；②由于血脑屏障上药物转体等的变化，药物进入脑实质细胞外液量减少，这样可能血药浓度在治疗范围，但脑细胞外液中浓度不够而达不到治疗作用；③神经细胞膜上药物转运体的改变，致使细胞内液中药物量不足，药物不能有效通过细胞内机制发挥抗癫痫作用。目前已有研究显示多药耐药基因（MDR1）表达上调，其表达产物P-糖蛋白（p-gp）增多与顽固性癫痫耐药现象有关，现就这方面的研究作一综述。     

细胞色素P4503A基因表达调控及与抗癫痫药物的关系

细胞色素P4503A酶（Cytochrome P450 3A enzymes）在外源性化合物尤其是药物代谢中起重要作用，临床中约有50％的药物经由CYP3A代谢，其中包括多种抗癫痫药物．本文概述了CYP3A的基因家系的结构、分布和调控表达的异同，CYP3A与难治性癫痫患者药物难治性的可能机制——多药耐药基因和P－糖蛋白的关系，CYP3A4基因表达上的差别与药物作用强度和持续时间、急性毒性的相关性．     

多药耐受基因家族与难治性癫痫

大约25%的癫痫患者由于对抗抗癫痫药物作用而成为难治性癫痫,多药耐受(MDR)蛋白、MDR相关蛋白以及其它一些能引起多药耐受的转运子如MVP等过表达与难治性癫痫密切相关。正常情况下,几乎只有血管内皮细胞少量表达MDR基因,但难治性癫痫患者脑组织、杏仁核点燃模型和海人酸致癫大鼠模型脑组织的血管内皮细胞、星形胶质细胞、神经元等细胞MDR的表达明显高于正常脑组织。     

长春新碱降低由转染A20 siRNA促进的弥漫大B细胞淋巴瘤细胞系增殖

目的研究A20小分子干扰RNA片段(siRNA)对弥漫大B细胞淋巴瘤细胞(OCI-LY1)增殖和多药耐药基因(MDR1)表达的影响。方法分对照组、转染组、长春新碱干预组(VCR)组和转染+VCR组。用脂质体转染法将A20 siRNA转入OCI-LY1细胞,MTT法检测细胞对VCR的敏感性;流式细胞计量术检测细胞凋亡;用real-time PCR检测A20及MDR1 mRNA;用Western blot检测细胞内A20、Pgp蛋白及核蛋白NF-κB的表达。结果 1)A20 siRNA转染后,细胞增殖能力增强(P0.05),VCR刺激后转染细胞的增殖曲线下降缓慢。2)转染细胞凋亡率明显低于对照组,VCR刺激24 h后OCI-LY1细胞凋亡率较加药前明显增加(P0.05),VCR组凋亡率比转染+VCR组增高的幅度大(P0.05)。3)转染后,A20 mRNA及蛋白表达明显降低(P0.001),NF-κB蛋白(P0.001)、MDR1mRNA(P0.001)和Pgp(P0.001)表达都明显增高;但MDR1 mRNA和Pgp在药物刺激后表达降低(P0.05),且VCR组较转染+VCR组降低的幅度大(P0.01)。结论 A20 siRNA能有效的增强OCI-LY1细胞内NF-κB的表达,使得MDR1基因及编码蛋白Pgp蛋白增强,从而抑制细胞凋亡、促进细胞增殖;VCR刺激后细胞内MDR1 mRNA和Pgp的表达明显降低,A20 siRNA则减弱了VCR的这一作用。     

迷走神经刺激对戊四氮致痫大鼠丘脑网状核NMDAR1 mRNA和GABAARα1 mRNA的影响

为探讨迷走神经刺激（Vagus nerve stimulation,VNS)抗癫痫的作用机制。应用原位杂交组织化学及图像分析方法研究戊四氮（Pentylenetetrazol,PTZ)致痫大鼠丘脑网状核谷氨酸受体NMDAR1mRNA和γ-氨基丁酸A受体（GABAAR）α1亚单位mRNA的变化。结果显示，PTZ致痫组大鼠丘脑网状核NMDAR1mRNA表达明显高于正常对照组，而VNS抗癫痫组明显低于PTZ致癫痫组，与之相反，PTZ致癫痫组GABAARα1mRNA的表达明显低于正常对照组，VNS抗癫痫组明显高于PTZ致癫痫组，上述结果表明，VNS可能通过抑制丘脑网状核兴奋的神经递质受体NM-DAR的活动和增强抑制性神经递质受体γ-氨基丁酸受体GABAAR的活动，降低大脑皮层的兴奋性，抑制癫痫的发生和发展。     

难治性癫痫发生机制研究进展

目前,大约三分之一的癫痫患者对抗癫痫药物耐药。药物难治性瘢痫是指无中枢神经系统进行性疾病或占位性疾病,但临床迁延,经两年以上正规抗癫痫治疗,单独或合用主要抗癫痫药,并达到患者能耐受最大剂量且血药浓度达到有效范围,仍不能控制,且影响日常生活的癫痫发作。     

多药耐药逆转剂药物开发药理实验对临床研究指导作用的重要性

多药耐药性(multidrugresistance,MDR)的产生已构成了肿瘤化疗失败的一个重要原因。MDR产生的原因很复杂．但MDR基因过度表达产生的P糖蛋白(Pglycoprotein,Pgp)是MDR产生的最重要原因。然而．伴随研究的深入,不断发现许多耐药现象与已知的耐药机制不相关或相关性很低的矛盾。事实表明还存在其他目前尚不认识的机制,提示肿瘤细胞抗性的调控。在很大程度上没有解决。目前研究对象主要是抗肿瘤药物。     

Potentiation of anti-epileptic drugs effectiveness by pyronaridine in refractory epilepsy

One of the major neurobiological mechanisms proposed in drug resistant epilepsy is removal of anti-epileptic drugs (AEDs) from the epileptogenic tissue through excessive expression of multi-drug efflux transporters such as P-glycoprotein (P-gp). P-gp, the encoded product of the human multi-drug resistance-1 (MDR-1; ABCB1) gene, is of particular clinical relevance in the emergence of multi-drug resistance (MDR), which may play an important role in preventing treatment response of some tumors and infectious diseases to chemotherapeutic agents and antibiotics. It has been shown that MDR-1 is over-expressed in brain tissue (hippocampal neurons) in patients with refractory temporal lobe epilepsy. For direct evidence that drug transporters such as P-gp are responsible for drug resistance, an experiment can be conducted to determine whether seizure control is improved when P-gp inhibitors are administered in addition to existing AEDs in patients with medically refractory epilepsy. In comparison with first and second-generation of P-gp inhibitors, third-generation inhibitors such as pyronaridine (PND) have advantages, such as higher potency and specificity for P-gp, lack of non-specific cytotoxicity, relatively long duration of action with reversibility, and good oral bioavailability. We suggest that a pilot study be conducted to determine whether adding of PND to existing AEDs decreases seizure frequency in patients with drug resistant epilepsy, and should this show promise, that a double-blind randomized controlled trial be designed to test the efficacy of PND.     

Significance of MDR1 and multiple drug resistance in refractory human epileptic brain

Background  

The multiple drug resistance protein (MDR1/P-glycoprotein) is overexpressed in glia and blood-brain barrier (BBB) endothelium in drug refractory human epileptic tissue. Since various antiepileptic drugs (AEDs) can act as substrates for MDR1, the enhanced expression/function of this protein may increase their active extrusion from the brain, resulting in decreased responsiveness to AEDs.     

Restoration of chemosensitivity in cancer cells with MDR phenotype by deoxyribozyme,compared with ribozyme

One of the main mechanisms for multidrug resistance (MDR) involves multidrug resistance gene 1 (MDR1) which encodes P-glycoprotein (Pgp). Pgp acts as a drug efflux pump and exports chemotherapeutic agents from cancer cells. Specific inhibition of Pgp expression by gene therapy is considered a well-respective strategy having less innate toxicities. At present, the investigation of DRz in reversal MDR is scarce. In the study, phosphorothioate DRz that targets to the translation initiation codon AUG was synthesized and transfected into breast cancer cells and leukemia cells with MDR phenotype. ASODN (antisense oligonucleotide) and ribozyme targets to the same region were also synthesized for comparison analysis. Alterations in MDR1 mRNA and Pgp were determined by RT-PCR, Northern blot, flow cytometry and Rh123 retention tests. Chemosensitivity of the treated cells was determined by MTT assay. The results showed that DRz could significantly suppress expression of MDR1 mRNA and inhibit synthesis of Pgp. The efflux activity of Pgp was inhibited accordingly. Chemosensitivity assay showed that a 21-fold reduction in drug resistance for Adriamycin and a 45-fold reduction in drug resistance for Vinblastine were found in the treated cells 36 h after transfection. These data suggest that DRz targeted to the translation initiation codon AUG can reverse MDR phenotype in cancer cells and restore their chemosensitivity. Moreover, the reversal efficiency of DRz is better than that of ribozyme and ASODN targets to the same region of MDR1 mRNA.     

颞叶癫痫患者颞叶皮层和海马组织内多药耐药相关蛋白1和胶质原纤维酸性蛋白共表达

目的 观察颞叶癫痫病人多耐药基因1（MDR1）、胶质原纤维酸性蛋白（GFAP）和神经元特异性烯醇化酶（NSE）在颞叶和海马组织内的表达。方法 癫痫组样本来自12例颞叶癫痫病例的手术切除标本,对照组为4例非癫痫病的尸检脑组织。应用双重免疫荧光组织化学方法结合激光共聚焦显微镜技术显示MDR1、GFAP和NSE在颞叶和海马组织内的表达。结果 对照组颞叶皮质和海马齿状回内均可见到许多GFAP表达阳性的星形胶质细胞和NSE表达阳性的神经元,未见到表达MDR1的细胞。癫痫组颞叶皮质和海马齿状回内GFAP阳性星形胶质细胞高于对照组。颞叶皮层和海马组织内可见星形胶质细胞MDR1 和GFAP 共表达现象。结论 颞叶难治性癫痫可能与星形胶质细胞的多药耐药性有关。     

MDR-1-overexpression in HT 29 colon cancer cells grown in SCID mice

The multidrug-resistance 1 (MDR-1) P-glycoprotein (Pgp) is a transmembrane transporter system, which actively pumps cytotoxic drugs out of the cell. MDR-1 acquired in vitro differs from MDR-1 acquired in vivo, but has important consequences on the cellular phenotype and metastatic behavior. Here we report that the human colonic cancer cell line HT29 (MDR-1 negative) is more malignant than its MDR-1 overexpressing variant (HT29 MDR-1 positive). HT29 MDR-1 negative cells produce undifferentiated signet ring carcinomas when implanted subcutaneously into SCID mice, while HT29 MDR-1 positive cells form tumors with tubular structures, but without signet ring cells. Immunohistochemical proliferation marker analysis revealed that the MDR-1 positive cells proliferate much more slowly than the MDR-1 negative cells. MDR-1 overexpression results in a less differentiated phenotype at the cellular level (absence of mucin producing cells) but in a more differentiated phenotype at the tissue level (tubule formation). In addition, lectin binding patterns including that of Helix pomatia agglutinin (HPA), an indicator of metastatic potential, differed between the two cell lines. HT29 MDR-1 positive cells had less HPA binding sites than HT29 MDR-1 negative counterparts and metastasized less frequently in SCID mice. As slow proliferation, low degree of differentiation and multidrug-resistance is a hallmark of cancer stem cells and all were present in MDR-1 positive tumors, it is attractive to speculate that they represent a stem cell rich tumor. As shown by global gene expression analyses, genes involved, e.g. in cell adhesion, glycosylation and signal transduction, were deregulated in MDR-1 positive tumors compared to MDR-negative tumors. Overexpression of E-cadherin and carcinoembryonic antigen-related cell adhesion molecules 1 (CEACAM1) may provide clues to the mechanisms responsible for the reduced metastatic potential of MDR-1 overexpressing tumors. Since drug treatment shifted the cells towards a less metastatic phenotype in this in vivo model, it seems conceivable to achieve this using drug treatment also in a clinical situation.     

High expression of MDR-1 gene and P-glycoprotein in initial and re-biopsy specimens of relapsed B-cell lymphoma

AIMS: Multidrug resistance (MDR) is a major obstacle in the treatment of lymphoma. The expression of MDR-1 mRNA and P-glycoprotein (MDR-1/P-gp) has been linked to MDR. We aimed to investigate the expression of MDR-1/P-gp in B-cell lymphoma. METHODS AND RESULTS: Samples at diagnosis and relapse from 10 patients with B-cell lymphoma were obtained. We also obtained 14 unselected control cases of B-cell lymphoma at diagnosis. The expression of mRNA and protein were determined semiquantitatively by RT-PCR and immunohistochemistry. High MDR-1 and P-gp expressions were found in seven and seven of 10 samples obtained at diagnosis, eight and eight of 10 samples obtained at relapse, and three and four of 14 control cases at diagnosis, respectively. The results of RT-PCR paralleled those of immunohistochemistry. Concordance of high MDR-1/P-gp expression was noted in 27 of 34 samples (r = 0.73, P = 0.001). There were no significant changes in MDR-1/P-gp expression in all cases at relapse and during the clinical course following chemotherapy. In the 14 control cases, the average survival time was 12.7 months in MDR-1/P-gp positive cases and 29.0 months in the MDR-1/P-gp negative cases (P = 0.20). CONCLUSIONS: Our results showed that at least some B-cell lymphomas express MDR-1/P-gp, which could be detected by different methods, and suggested that high MDR-1/P-gp expression in tumour cells may be associated with a high probability of relapse and poor prognosis.     

Neuronal expression of the drug efflux transporter P-glycoprotein in the rat hippocampus after limbic seizures

In the brain, the efflux transporter P-glycoprotein (Pgp) is predominantly located on the luminal membrane of endothelial cells lining brain microvessels and forming the blood-brain barrier. Many lipophilic drugs, including antiepileptic drugs, are potential substrates for Pgp. Overexpression of Pgp in endothelial cells of the blood-brain barrier has been determined in patients with drug resistant forms of epilepsy such as temporal lobe epilepsy and rodent models of temporal lobe epilepsy and suggested to lead to reduced penetration of antiepileptic drugs into the brain. Expression of Pgp after seizures has also been described in astrocytes, whereas it is not clear whether neurons can express Pgp. In the present study, Pgp expression was studied by immunohistochemistry in rats 24 h after a status epilepticus induced by either pilocarpine or kainate, widely used models of temporal lobe epilepsy. Unexpectedly, in addition to endothelial Pgp staining, intense Pgp staining was found in neurons in the CA3c/CA4 sectors and hilus of the hippocampus formation, but not in other brain regions examined. The neuronal Pgp staining was confirmed by two different Pgp antibodies. Double immunolabeling and confocal microscopy showed that Pgp was colocalized with the neuronal marker neuronal nuclear antigen, but not with the glial marker glial fibrillary acidic protein. No neuronal Pgp staining was seen in control rats. The expression of Pgp in neurons after limbic seizures was substantiated by determining Pgp encoding genes (mdr1a, mdr1b) in neurons by real time quantitative RT-PCR. Increased Pgp expression in hippocampal neurons is likely to affect the action of drugs with intraneuronal targets and, in view of recent evidence from other cell types, could be associated with prevention of apoptosis which is involved in neuronal damage developing after seizures such as produced by pilocarpine.     

Genetic diagnosis for drug resistance in cancers

The failure of chemotherapy to eradicate tumor cells is often due to the development of drug resistance. MDR(multidrug resistance) whose one form of resistance results from a decreased intracellular accumulation of the drugs, most often mediated by the overexpression of P-glycoprotein. MRP also related to pump function of cell membrane in acute leukemia. We have developed the new quantitative assay based on real-time PCR to measure expression of drug-resistance related genes such as MDR-1 and MRP in clinical samples. These results indicates that real-time PCR system is a reliable method to quantitatively determine drug resistant genes expression, it may be to predict responsiveness to chemotherapy by using this technique.     

Immunohistochemical detection of P-glycoprotein in endometrial adenocarcinoma.

P-glycoprotein (Pgp) has emerged as the central mediator in classic multidrug resistance in model systems in vitro. High levels of Pgp also have been detected in many normal human tissues and tumors; and its role in clinical drug resistance is currently under investigation. Recently significant levels of Pgp were localized to gravid and secretory endometrium; and it was demonstrated that the combination of estrogen and progesterone is sufficient to induce high levels of both Pgp mRNA and Pgp in uterine secretory epithelium. These findings suggest that increased Pgp expression also may be present in hormone-responsive malignancies such as endometrial adenocarcinoma. To determine whether Pgp is expressed in endometrial adenocarcinoma, 36 endometrial adenocarcinomas (grade I [n = 17]; grade II [n = 6]; grade III [n = 13]) were investigated retrospectively by the avidin-biotin-complex immunohistochemical procedure using three murine monoclonal antibodies (MAb) MAb C219, MAb C494, and MAb JSB-1, which recognize spatially distinct cytoplasmic epitopes of Pgp. Seventy-two percent of the tumors showed positive immunostaining with at least one MAb; 67% showed immunostaining with MAb C219, 50% with MAb C494, and 62% with MAb JSB-1. Forty-six percent of tumors were immunoreactive to two and 29% to all three antibodies. Membranous and Golgi/paranuclear type staining patterns were observed. Overall the intensity of immunostaining varied from one sample to another for a given tumor type, and considerable heterogeneity of expression was commonly seen within a given tumor. Strong to moderate immunoreactivity was seen in diffusely infiltrating, adenosquamous, and serous papillary carcinomas. In general, immunoreactivity to MAb C494 was weaker than MAb C219 or MAb JSB-1. Adenomatous and non-neoplastic endometrium adjacent to the tumors displayed strong membranous immunostaining with MAb JSB-1. Endometrial capillaries showed weak-to-moderate immunostaining to all three antibodies. It is concluded that Pgp is commonly expressed in endometrial adenocarcinoma and may be a significant factor responsible for their drug-resistant nature subject to modulation by progesterone.