胶质瘤多药耐药细胞系C6／adr的建立及其耐药性逆转的研究

目的 研究胶质瘤多药耐药（ＭＤＲ）的发生机理及逆转方法。方法 建立了Ｃ６／ａｄｒ ＭＤＲ细胞系,经ＲＴ－ＰＣＲ及免疫组化染色分别研究了ｍｄｒ－１基因及Ｐ－糖蛋白（Ｐ－ｇｐ）的表达;采用ＭＴＴ药敏试验及ＨＰＬＣＡ测定细胞内阿霉素浓度的方法研究了异搏定、红霉素、潘生丁、Ｐ－ｇｐ单克隆抗体、复方丹参对ＭＤＲ的逆转作用。结果 Ｃ６／ａｄｒ ＭＤＲ细胞系ｍｄｒ－１基因阳性,Ｐ－ｇｐ高度表达。异搏定（２～６μ     

马桑内酯致痫大鼠大脑皮层,海马NMDA受体亚单位1（NMDAR1） …

目的 探讨癫痫发病的发子机制。方法 采用原位杂交技术，研究了马桑内酯致痫大鼠大脑皮层、海马Ｎ－甲基－Ｄ－天安门冬氨酸受体亚单位１（ＮＭＤＡＲ１）ｍＲＮＡ表达的变化。结果 马桑内酶致痫大鼠顶叶大脑皮层及海马齿回ＮＭＤＡＲ１ｍＲＮＡＡ水平显著高于生理盐水对照组（Ｐ〈０．０１，Ｐ〈０．０５）。结论 马桑内酯上调脑组织内ＮＭＤＡＲ１ｍＲＮＡ水平，此可能是其致痫及惊痫及惊厥易感性增加的分子机制之一。     

胶质瘤细胞PDGF BB自分泌环活怀与肿瘤细胞egr—1基因表 …

目的 探讨胶质瘤细胞血小板源生长因子（ＰＤＧＦ）ＢＢ自分泌环活性与肿瘤细胞ｅｇｒ－１基因表达的关系,以及它们在胶质瘤发生、发展中的作用。方法 用ｍＲＮＡ原位杂交和免疫组化染色观察了７３例２ 脑胶质瘤组织。结果 胶质瘤细胞ＰＤＧＦＢ ｍＲＮＡ表达水平随肿瘤恶性程度增加而升高。ｅｇｒ－１ｍＲＮＡ、ＥＧＲ－１蛋白、ＰＤＧＦα受体和β受体及酷氨酸磷酸化蛋白阳性肿瘤细胞密度彼此间均呈显著性正相关,并均随肿瘤     

人脑胶质瘤细胞多药耐药性的形成及其耐药特性的研究

目的探讨人脑胶质瘤细胞多药耐药性（ＭＤＲ）的形成规律及其耐药特性。方法采用长春新碱（ＶＣＲ）在体外对人脑胶质瘤ＢＴ３２５细胞持续诱导获得了表现为ＭＤＲ特征的人脑胶质瘤细胞模型．以ＭＴＴ法测定胶质瘤细胞增殖，透射电镜行超微结构研究，通过流式细胞仪，检测培养的胶质瘤细胞与３２例术后胶质瘤新鲜组织标本中Ｐ－糖蛋白的表达。结果耐药的胶质瘤细胞ＢＴ３２５／ＶＣＲ对长春新碱的耐受程度为其亲本细胞的２４倍，对阿霉素、威猛交叉耐药，对５－氟脲嘧啶敏感。透射电镜下见ＢＴ３２５／ＶＣＲ细胞常染色质明显，线粒体丰富，粗面内质网增多。研究表明ＢＴ３２５／ＶＣＲ表现为Ｐ－糖蛋白介导的典型ＭＤＲ。胶质瘤新鲜组织标本中，Ｐ－糖蛋白表达阳性率为３７５％（１２／３２），同肿瘤的恶性程度呈正相关（Ｐ＜０．０１）。结论长春新碱能够诱导人脑胶质瘤细胞产生Ｐ－糖蛋白介导的典型ＭＤＲ。胶质瘤中多药耐药基因产物Ｐ－糖蛋白表达的阳性率同肿瘤的恶性程度呈正相关。应用流式细胞仅能早期发现耐药细胞，具有临床推广价值。     

马桑内酯致痫大鼠大脑皮质,海马NMDA受体亚单位1mRNA表达的 …

ＮＭＤＡ受体与惊厥和癫痫易感性的形成密切相关。为了探讨癫痫发病的分子机制，采用原位杂交技术，研究了马桑内酯致痫大鼠大脑皮质、海马ＮＭＤＡ受体亚单位１（ＮＭＤＡＲ１）ｍＲＮＡ表达的动态变化。结果显示，马桑内酯致痫大鼠顶叶大脑皮质及海马齿状回ＮＭＤＡＲ１ ｍＲＮＡ水平显著高于生理盐水对照组（Ｐ〈０．０１，Ｐ〈０．０５）。提示：马桑内酯上调脑组织内ＮＭＤＡＲ１亚单位ｍＲＮＡ水平，可能是其致痫及使惊厥易感     

阿欠茨海默病的Aβ表达与APOE,PS1基因的相关分析

目的 研究阿尔茨海默病的ＡＰＰ基因中Ａβ表达量与载脂蛋白Ｅ（ＡＰＯＥ）基因和早老素１（ＰＳ１）基因间相互关系。方法 应用竞争ＲＴ－ＰＣＲ技术,测定５２例ＡＤ患者,２８例血管性痴呆（ＶＤ）患者及６０名健康老人的外周单核血细胞中Ａβ相对半定量表达量,以及采用ＰＣＲ－ＲＦＬＰ方法检测所有研究对象的ＡＰＯＥ基因和ＰＳ１基因的多态性分布。疾病诊断按ＤＳＭⅢ－Ｒ标准。结果 ＡＰＯＥε４等位基因和ＰＳ１的各种     

药物难治性癫痫患者脑内多种耐药基因表达的初步研究

目的研究药物难治性癫痫患者与正常对照组脑内多种药物耐药基因（ＭＤＲ１）表达的情况。方法　选择１５例药物难治性癫痫患者和５例对照组患者的脑组织标本,用逆转录聚合酶链反应（ＲＴ－ＰＣＲ）技术,以Ｂ－ａｃｔｉｎ为内参照对ＭＤＲ１基因进行扩增,经琼脂糖凝胶电泳分别得到２２６ｂｐ和５４８ｂｐ的条带,通过照相、扫描、计算ＭＤＲ１／ｂ－ａｃｔｉｎ比值进行半定量分析。结果统计学处理提示,药物难治性癫痫患者脑内ＭＤＲ１的表达明显高于对照组（Ｐ＜０．０１）。结论　Ｐ－糖蛋白为ＭＤＲ１的产物,能将药物从脑内排入血循环而降低脑内药物浓度。药物难治性癫痫患者因脑内ＭＤＲ１的高表达,导致脑内抗癫痫药物（ＡＥＤ）浓度较低而耐药。故应用ＭＤＲ１抑制剂也许可以提高ＡＥＤ在脑内的药物浓度,使癫痫的药物治疗效果更令人满意。     

逆转录－聚合酶链反应、狭缝印迹检测重症肌无力患者IL－2mRNA表达水平

为了探讨重症肌无力的发病机制利用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）、狭缝印迹技术和非放射性地高辛配基（ＤＩＧ）ＤＮＡ标记和检测手段，采用分子生物学技术和免疫学反应相结合，定量检测了重症肌无力（ＭＧ）患者和健康对照外周血单个核细胞（ＰＢＭＣ）的白细胞介素－２信使核糖核酸（ＩＬ－２ｍＲＮＡ）表达水平。结果表明：ＲＴ－ＰＣＲ方法特异而灵敏，可检测出１００ｆｇ含量的ＤＮＡ。重症肌无力患者（ｎ＝３５）与健康对照（ｎ＝１５）的ＩＬ－２ｍＲＮＡ表达水平无明显差异（Ｐ＝０．１７）。用ＰＨＡ刺激ＰＢＭＣ，患者和对照的ＩＬ－２ｍＲＮＡ表达均明显增多。摘除胸腺或（和）肾上腺皮质激素治疗，可明显抑制ＩＬ－２ｍＲＮＡ表达水平     

HLA-DPB_1等位基因对广西汉族重症肌无力的遗传易感性研究

目的探讨ＨＬＡ－ＤＰＢ１等位基因对广西汉族重症肌无力（ＭＧ）的遗传易感性。方法用限制性片段长度多态性（ＰＣＲ－ＲＦＬＰ）多酶共同消化法对ＨＬＡ－ＤＰＢ１的３１个等位基因进行分型。结果发现广西汉族ＭＧ患者的ＨＬＡ－ＤＰＢ１０５０１及３６０１的频率明显高于正常人。ＤＰＢ１２７０１在全身型ＭＧ明显增多。ＤＰＢ１易感基因为纯合子时，ＭＧ发病早，并与全身型ＭＧ密切相关。早发病ＭＧ与晚发病ＭＧ、男性ＭＧ与女性ＭＧ、眼肌型ＭＧ与全身型ＭＧ的ＨＬＡ－ＤＰＢ１遗传背景有所不同。结论广西汉族ＭＧ的ＨＬＡ－ＤＰＢ１易感基因为０５０１、３６０１及２７０１，其中前两者对ＭＧ发病最重要，后者见于全身型病人     

人脑胶质瘤WAF1／CIP1表达的研究

目的检测抑癌基因ＷＡＦ１／ＣＩＰ１在人脑胶质瘤中的表达。方法取４６例胶质瘤组织及１２例脑外伤组织，提取其ｍＲＮＡ，用ＷＡＦ１／ＣＩＰ１作为模板进行逆转录ＰＣＲ（ＲＴ－ＰＣＲ）。结果４６例胶质瘤中有１４例有ＷＡＦ１／ＣＩＰ１蛋白表达（１４／４６），表达率为３０．４％，１２例脑外伤标本全部表达（１２／１２），表达率为１００％，两组比较有显著差异（Ｐ＜０．０１）。结论ＷＡＦ１／ＣＩＰ１的缺失是脑胶质瘤发生及进展的原因之一。     

Association of ABCB1 genetic variants 3435C>T and 2677G>T to ABCB1 mRNA and protein expression in brain tissue from refractory epilepsy patients

Purpose: There is evidence from studies in rodents that P‐glycoprotein (P‐gp) overexpression is implicated in the causation of refractory epilepsy. Genetic variants in the human ABCB1 (MDR1) gene were shown to affect the expression levels of the transporter in various tissues and to be associated with refractory epilepsy. However, the effect of the genetic variants on the P‐gp level in epileptogenic brain tissue is poorly investigated. In the present study, we examined the impact of putatively functional polymorphisms 3435C>T and 2677G>T in the ABCB1 gene on the ABCB1 mRNA expression and P‐gp content in human brain tissue from epileptogenic foci of the patients with refractory epilepsy. Methods: Fresh brain tissue specimens were obtained from therapy‐refractory epilepsy patients during neurosurgery of the epileptogenic focus. We determined the ABCB1 mRNA expression in 23 samples using 5′ exonuclease‐based real‐time polymerase chain reaction (PCR) as well as the P‐gp content in 32 samples determined by immunohistochemistry, genotyping was performed by PCR/restriction fragment length polymorphism (RFLP). Results: There was lack of association of 3435C>T and 2677G>T as well as diplotype configurations on ABCB1 mRNA expression and P‐gp content in epileptogenic brain tissues. Conclusions: We cannot exclude an association of ABCB1 variants on P‐gp function, but our results suggest that brain ABCB1 mRNA and protein expression is not substantially influenced by major ABCB1 genetic variants thus explaining in part results from case‐control studies obtaining lack of association of ABCB1 polymorphisms to the risk of refractory epilepsy.     

Reversion of multidrug resistance in human glioma by RNA interference

OBJECTIVE: To explore whether the vector construction of short hairpin in vivo could make human glioma cell line BT325 produce RNA interference (RNAi) duplexes and reverse the expression of the MDR1 gene. METHODS: Three 62nt oligonucleotide fragments (shRNA) were constructed according to GeneBank MDR1 sequence and were cloned to the retrovirus-delivered vectors. After these vectors were transfected directly to the BT325 cell by Lipofectamine 2000 with enhanced green fluorescent protein (EGFP) co-transfection, the MDR1 gene silence effects were detected by the changing levels of mRNA and P-glycoprotein (P-gp) including real-time PCR (RT-PCR), Northern blot and Western blot analysis. For assessing multidrug resistance against doxorubicin (DOX) and vincristine (VCR), cell proliferation assays were performed by cell counting kit-8 and IC50 was calculated. RESULTS: The RNAi plasmid vectors were constructed successfully. The gene silence became the strongest after 48 hour transfection from Northern blot; Western blot analysis demonstrated that P-gp expression reduced to 12.9, 30.3 and 4.8%. The chemosensitivity assays showed that the transfected cell could increase the sensitivity of DOX and VCR. Based on the value of IC50, BT325 cells increased sensitivity to drugs obviously. The sequence specific RNAi could inhibit MDR1 mRNA and P-gp expression of the glioma cell line. And it may reverse multidrug resistance phenotype, which may provide promising therapeutic modalities in the treatment of human glioma.     

P16基因改变与脑胶质瘤生物学特性相关性的研究

目的 研究Ｐ１６基因改变与脑胶质瘤恶性程度分级及肿瘤细胞增殖活性之间的关系。方法 检测４１例不同级别脑胶质瘤标本和７例正常脑组织Ｐ１６基因改变和ＰＣＮＡ蛋白表达情况。结果 实验组Ｐ１６基因缺失和突变２２例（５４％），ｍＲＮＡ和蛋白表达缺失分别为２３例（５６％）和２７例（６６％），三者改变非一对一关系。Ｐ１６基因改变和ＰＣＮＡ指数随脑胶质瘤恶性程度级别增高呈上升趋势（Ｐ〈０．０５）。Ｐ１６基因改变与     

脑肿瘤中P53基因突变与突变型P53蛋白表达的研究

采用聚合酶链反应－单链构象多态性（ＰＣＲ－ＳＳＣＰ）法对４１例脑肿瘤进行Ｐ５３基因突变的检测，并与抗突变型Ｐ５３蛋白单抗的免疫组化染色结果相对照，结果显示脑肿瘤中Ｐ５３基因突变率为３４．１％（１４／４１），胶质瘤为２８％（１０／３６）。Ｐ５３基因突变与突变型Ｐ５３蛋白表达具有高度一致性，且二者均与脑肿瘤的分化程度有关。Ｐ５３基因突变及蛋白的表达不仅出现在高恶度胶质瘤及转移癌，而且可出现在低恶度胶质瘤中，表明Ｐ５３基因的突变在脑瘤的发生、发展过程中起重要作用。     

MDR1 shRNA对人脑胶质瘤干细胞多药耐药性实验研究

目的构建多药耐药基因（MDR1）的短发卡RNA表达质粒，并检测其对胶质瘤干细胞药物敏感性的作用。方法培养脑胶质瘤干细胞；根据MDR1的DNA序列设计shRNA，用Siportxp-1脂质体法转染胶质瘤干细胞。分别采用定量聚合酶链反应（PCR）和Westernblot检测转染前后MDR1mRNA和蛋白表达情况；使用活细胞计数试剂盒（CCK-8）对转染后细胞进行药物敏感性试验，评价shRNA对多药耐药性的逆转作用。结果成功构建MDR1shRNA表达质粒，转染组MDR1mRNA表达水平有所下降（P〈0．05），转染5d组下降最明显；RT—PCR结果显示MDR1mRNA水平降低，第3、5、7d抑制率分别为78．46％±14．17％，82．02％±11．87％，79．5％±13．27％。Westernblot结果显示：shRNA转染组P-糖蛋白（P-gp）的表达降低，第3、5、7d抑制率分别为58．1％±6．5％，39．5％±5．2％，45．8％±8．6％；而药敏实验显示：阿霉素、长春新碱对转染MDRIshRNA的胶质瘤干细胞的半数抑制浓度（IC50）均有不同程度降低，且出现凋亡峰（P〈0．05）。结论MDR1短发卡RNA可在转录后水平对多药耐药进行调节，下调MDR1基因表达，提高药物敏感性，诱导细胞凋亡。     

DLC1基因在人脑胶质瘤组织中的表达

目的 探讨抑癌基因DLC1在人脑胶质瘤组织中的表达和功能.方法 收集广州医学院第二附属医院神经外科自2007年1月至2009年6月手术切除的胶质瘤标本39例及同期行颅脑减压术治疗的颅脑损伤患者的正常脑组织10例,实时荧光定量PCR检测DLC1 mRNA的表达;将质粒pCS2-DLC1转染体外常规培养的人胶质瘤细胞株U251,同时用空载体pCS2-MT转染作为对照组,转染后36 h应用Western blot检测DLC1表达标签蛋白Mvc,应用MTT检测U251细胞增殖能力的变化.结果实时荧光定量PCR结果显示胶质瘤组织中DLC1 mRNA表达水平高于对照组,差异有统计学意义(P=0.000),且Ⅰ级胶质瘤组织DLC1 mRNA的表达水平高于Ⅱ、Ⅲ、Ⅳ级,差异有统计学意义(平均秩次分别为44.20、30.23、32.25、31.0,H=18.818,P=0.001);Western blot检测结果显示DLC1标签蛋白Mvc在pCS2-DLCI转染组细胞呈过表达,在pCS2-MT转染组细胞无明显表达;MTT检测显示pCS2-DLC1转染组U251细胞吸光度值高于pCS2-MT转染组,差异有统计学意义(P=0.002).结论 高表达的DLC1可能促进胶质瘤的形成和发展.     

血脑/血瘤屏障体外模型的构建、形态与功能特性

目的构建并观测血脑/血瘤屏障体外模型的形态与功能特性,为中枢神经系统药物跨血脑和（或）血瘤屏障研究提供体外实验模型.方法将分离的Balb/C小鼠脑微血管内皮细胞（BMEC）在铺有明胶的微孔膜上单层培养或与大鼠胶质瘤细胞C6双室共培养,分别建立血脑屏障（BBB）和血瘤屏障（BTB）模型,采用光镜和扫描电镜观察BMEC形态,采用Millicell-ERS系统检测屏障中的跨内皮电阻（TEERs）,免疫细胞化学检测P-糖蛋白（P-gp）表达,并比较BBB和BTB中BMEC的形态和功能特征.结果两组模型中的BMEC均形成单层生长和良好的细胞间紧密连接,产生较高的TEERs值,并检测到P-gp表达.瘤细胞诱导使BMEC间出现间隙,同时相点TEERs值降低,P-gp表达减弱.结论两种体外模型可用于研究药物跨血脑和（或）血瘤屏障特性,其中BTB模型可能更适合抗肿瘤药物的研究.     

Gli1 is a potential target for alleviating multidrug resistance of gliomas

Although chemotherapy plays an important role in combined treatment of human gliomas, it fails to bring about satisfactory outcomes in a number of patients. One of the major obstacles in this treatment is the development of multidrug resistance (MDR) during treatment. Regulation of MDR in the context of gliomas is poorly understood, and clinical efforts to inhibit it have not been fruitful. Activation of the Hedgehog (HH) pathway has been shown to contribute to the growth, maintenance and relapse of various cancers. As data were not available on the role of Gli1 expression in glioma progression, we analyzed the correlation between Gli1 expression and tumor recurrence after chemotherapy in 60 glioma samples. The effects of inhibiting or activating the HH pathway on sensitizing or rendering glioma cell lines resistant to VCR, VP16, CDDP and ACNU, which are widely used in clinical chemotherapy, were determined. Additionally, the impact of the HH pathway on the expressions of MDR1, MRP1, MVP, MGMT, Bcl-2 and Survivin genes was determined. Our results indicate that overexpression of Gli1 is correlated with glioma recurrence after chemotherapy. We further show that the HH pathway activity can promote clonogenic survival of glioma cell lines in chemotherapy. Additionally, we found that blocking the HH pathway enhanced cytotoxicity of chemotherapeutic agents in glioma cells, through down-regulating the expressions of MDR1, MRP1, MVP, MGMT, Bcl-2 and Survivin genes. Taken together, these results suggest that Gli1 plays a dominant role in chemoresistance of glioma cells, and that suppression of Gli1 expression might be a valid therapeutic option for overcoming MDR and for increasing the success of chemotherapy.     

小儿颅内肿瘤P-糖蛋白表达的定位检测及临床意义

目的 为了探讨小儿颅内肿瘤化疗失败的原因，本实验对２０例小儿颅内肿瘤Ｐ－糖蛋白的表达情况进行研究。方法 用单克隆抗体ＪＳＢ１及免疫组化法检测Ｐ－糖蛋白。结果 小儿颅内肿瘤Ｐ－糖蛋白表达很低，２０例中８例表达于血管内皮细胞，７例表达于纤维细胞，５例表达于浸润的淋巴细胞，肿瘤细胞只１例表达。结论 ８小儿颅内肿瘤化疗失败的原因是患儿血脑屏障Ｐ－糖蛋白表达，表现为对抗癌药物较强耐受而非肿瘤细胞表达Ｐ－糖蛋     

Intestinal expression of cytochrome P450 enzymes and ABC transporters and carbamazepine and phenytoin disposition

OBJECTIVES: Interindividual variability in intestinal absorption and bioavailability might contribute to inadequate control of seizures under treatment with carbamazepine and phenytoin. We therefore correlated intestinal expression levels and genetics of CYP3A4, CYP2C9/19, MDR1 and MRP2 with dose requirement and plasma levels of carbamazepine and phenytoin. MATERIALS AND METHODS: Epileptic patients on carbamazepine (n = 29) or phenytoin (n = 15) were stratified into a 'high'-dose (carbamazepine > or =800 mg/day, phenytoin > or =300 mg/day) and a 'low'-dose group (carbamazepine < or =600 mg/day, phenytoin < or =200 mg/day). Duodenal biopsies and DNA were obtained for Western blotting and genotyping studies. RESULTS: Low carbamazepine plasma levels showed a trend towards higher intestinal MDR1 expression (P = 0.06). Furthermore, carbamazepine dose was positively correlated with MRP2 expression (P = 0.1). Moreover, MDR1 expression and carbamazepine and phenytoin dose requirement was influenced by the genotype in position 2677 and 3435 of the MDR1 gene. CONCLUSION: Differences in intestinal MDR1 and MRP2 expression may influence carbamazepine and phenytoin disposition and may account for interindividual pharmacokinetic variability.