伤寒减毒活疫苗Ty21a免疫前后小鼠肠细胞差异表达的基因

以基因表达谱芯片对Ty2 1a免疫前后小鼠肠细胞 (包括肠粘膜上皮细胞和肠上皮间淋巴细胞 )基因表达的差异性进行研究比较。将 490条经抑制消减杂交法筛选出的与小鼠Ty2 1a免疫相关的cDNA制备成表达谱芯片 ;利用免疫前后小鼠肠细胞的mRNA通过逆转录方法 ,将Cy3和Cy5两种荧光分别标记到两种组织的cDNA上 ,制备成cDNA探针 ,并与表达谱芯片进行杂交及扫描 ,单点重复 2次实验 ,通过计算机数据处理判定基因是否在上述两种细胞群中有表达差异。筛选出差异表达基因共 98条 ,其中 92条为表达上调基因 ,6条为表达降低基因。提示 ,基因表达谱芯片技术是高通量进行基因表达模式研究的方法 ,可同时定量研究大量的基因表达水平 ,从而鉴定可能参与免疫的基因。     

急性髓细胞白血病患者CD56抗原表达与MDR1基因表达量的关系研究

目的 研究初治急性髓细胞白血病(AML)患者CD56抗原表达与多药耐药(MDR)1基因表达量的关系,探讨CD56抗原表达与MDR1基因表达量在AML耐药中的作用及相互关系.方法 采用流式细胞术(FCM)和建立实时荧光定量PCR技术分别检测79例AML患者CD56抗原表达及MDR1基因表达水平并分析两者之间的关系及临床意义.结果 24.1%AML患者表达CD56抗原,FAB亚型中M5 AML患者表达阳性率高于其他亚型.遗传学危险度分级高危组患者CD56抗原表达阳性率显著高于中危组(P<0.01),伴t(8:21)AML患者CD56抗原表达阳性率(57.1%)显著高于其他低危组AML(P<0.05).CD56抗原表达阳性初治AML患者MDR1基因表达水平显著高于表达阴性患者(P<0.001).MDR1基因高表达且CD56抗原阳性AML组的CR率(58.8%)显著低于MDR1基因低表达且CD56表达阴性组(89.2%,P<0.01).结论 AML患者CD56抗原表达与MDR1基因表达水平存在相关性;同时定量检测MDR1基因表达及CD56抗原表达更有助于判断预后.     

苯妥英钠耐药鼠神经元保护基因的表达

目的：探索神经元自身保护基因在苯妥英钠耐药鼠和非耐药鼠中的差异表达。方法：选择成年Wistar大鼠用电刺激杏仁核方法建立耐PHT和非耐药癫痢大鼠模型，应用含有4096条人类全长基因的cDNA表达谱芯片，检测耐药与非耐药组神经元保护基因表达谱的差异。结果：发现10条神经元自身保护基因的表达在两组间存在差异。结论：神经元自我保护机制的降低可能是难治性癫痫形成的原因之一。     

实验性肺纤维化相关基因的表达谱研究

目的：通过研究小鼠肺纤维化组织和正常肺组织基因的差异表达，寻找与纤维化相关的基因。方法：以包含4 096个cDNA基因表达谱芯片研究不同时间肺纤维化的基因表达谱。结果：共筛选出差异表达基因271条，包括高表达的炎性基因43条和与细胞外基质代谢有关的基因46条，112条基因表达下降。结论：基于cDNA微矩阵技术的肺纤维化基因表达谱能够高通量筛选与肺纤维化发生密切相关的基因。     

应用基因芯片技术对Graves病免疫相关基因的研究

目的 :应用基因芯片技术研究Graves病 (GD)和正常成人甲状腺组织免疫相关基因的差异表达。方法 :分别用Cy5和Cy3两种不同的荧光染料通过逆转录反应将GD组和对照组甲状腺组织的mRNA分别标记成探针 ,并与载有一组靶基因的基因表达谱芯片进行杂交。通过扫描荧光强度 ,计算机软件分析 ,寻找两组差异表达基因。结果 :GD组和正常对照组之间共筛选出80条差异表达基因 ,其中表达增加的基因有 31条 (2 0倍以上 ) ,表达降低的基因有 4 9条 (0 5倍以下 )。结论 :基因表达谱芯片筛选GD与正常成人甲状腺组织差异表达基因具有样品用量少、高速度、…     

SFRP5基因甲基化通过激活Wnt/β-catenin通路调节白血病细胞MDR1/P-gp的表达

 目的:研究分泌型卷曲相关蛋白5（SFRP5）基因甲基化对耐药白血病细胞多药耐药蛋白1/P-糖蛋白（MDR1/P-gp）表达的调节及其可能的信号通路。方法:采用甲基化特异性PCR（MSP）检测白血病患者及白血病细胞系中SFRP5基因启动子区甲基化状态。采用Western blotting检测非磷酸化β-catenin（NP-β-catenin）在SFRP5基因甲基化白血病患者及细胞系中表达。使用去甲基化试剂5-氮杂-2′-脱氧胞嘧啶（DAC）恢复SFRP5表达,采用RT-PCR和real-time PCR检测处理前后MDR1 mRNA表达,采用Western blotting检测P-gp表达。结果:5/12例白血病患者标本及4种白血病细胞系存在SFRP5基因完全甲基化。在5例患者标本中,有3例（L2、L7和L8）NP-β-catenin水平高于其它标本,在4种白血病细胞系中,KG1a中NP-β-catenin水平高于另外3个细胞系。存在NP-β-catenin高水平表达的3例患者（L2、L7和L8）中检出MDR1 mRNA及P-gp表达。在4种细胞系中,KG1a存在MDR1 mRNA及P-gp表达。使用去甲基化试剂DAC处理L2、L7、L8患者标本和KG1a细胞,恢复SFRP5表达,L2、L7、L8和KG1a中MDR1 mRNA水平均显著下降（P<0.01）,P-gp表达水平亦被下调。结论: SFRP5基因甲基化可导致Wnt/β-catenin信号通路被激活,活化的β-catenin激活下游靶基因MDR1转录,编码的药物外排泵P-gp表达增加,促进白血病多药耐药的形成。     

人退变椎间盘组织的基因表达谱

目的：研究人类退变椎间盘的基因表达谱，分析人退变椎间盘基因表达水平的变化。方法：按条件优化的一步法抽提人退变及正常椎间盘组织的总RNA各3例，分别用cy3，cy5荧光标记，获得2组椎间盘cDNA的探针，与含有4096条人类全长基因的cDNA表达谱芯片杂交，扫描芯片荧光信号图像，对所获得的基因进行生物信息学分析。结果：在4096条基因中，有差异表达的基因706条，358条基因表达量明显下降，298条基因表达量明显上升。在有差异表达的基因中，细胞凋亡相关类蛋白8条，其中上皮细胞膜蛋白(EMP-1)基因的表达上调明显。结论：退变椎间盘的基因表达发生变化，细胞凋亡增加可能是椎间盘退变发生和发展的因素之一。     

应用基因表达谱芯片观察小鼠心肌缺血后基因表达的变化以及四逆汤对其影响

目的:应用基因表达谱芯片观察小鼠心肌缺血后基因表达的变化以及四逆汤对其影响。方法:昆明种小鼠,随机分为对照组、缺血组、缺血加四逆汤组。提取各组心肌组织总RNA,纯化mRNA,与含有2304条小鼠基因的cDNA表达谱芯片进行杂交。结果:小鼠缺血后有33条基因表达下调,70条基因表达上调;服用四逆汤后,相对单纯缺血组而言,有23条基因表达下调,52条基因表达上调。结论:运用基因芯片技术能快速地检测出缺血心肌及四逆汤治疗后心肌基因表达谱的改变,对差异表达基因的研究将有助于进一步了解心肌缺血及四逆汤治疗的分子机制。     

基因芯片检测自噬与凋亡的基因表达谱改变

目的:利用基因芯片研究自噬与凋亡关系,阐明两者间的分子调控机制.方法:采用4096条人类基因的cDNA芯片检测3-MA自噬抑制剂在诱导凋亡的人肝细胞株L02基因表达谱的改变,以探讨在自噬被抑制的情况下,凋亡的发生及其相关基因的变化.结果:L02细胞株cisplain凋亡诱导16小时的实验模型中,实验组在诱导凋亡前加入3-MA自噬抑制剂,诱导前后对照组与实验组相比,差异表达基因共151条.其中上调基因74条;下调基因77条.APG5基因无变化.结论:用基因表达芯片对3-MA自噬抑制剂在凋亡诱导的L02细胞株基因表达谱的改变进行高通量分析,能够有效地观察出凋亡基因及相关基因的表达.自噬被抑制的状态下,凋亡相关基因同样表达,说明3-MA自噬抑制剂并不能阻止凋亡的发生.     

转染肿瘤坏死因子-α及MDR1反义RNA逆转乳腺癌多药耐药的研究

目的：转染肿瘤坏死因子-α(TNF-α)cDNA和多药耐药基因(MDR1)的反义RNA到乳腺癌耐药细胞株MCF-7/ADR中进行表达，并观察它们在乳腺癌耐药逆转中的作用。 方法：应用RT-PCR和DNA重组技术构建反义绿色荧光蛋白pEGFP-MDR1融合蛋白表达载体和红色荧光蛋白pDsRed2-TNF-α融合蛋白表达载体，分别和同时导入乳腺癌耐药细胞株MCF-7/ADR中进行表达，检测转染前后细胞的生长曲线、细胞凋亡程度、MDR1-mRNA和P糖蛋白（P-gp）表达情况及对ADR敏感性的变化。 结果：转染后的细胞生长明显减慢，凋亡率显著增加，MDR1-mRNA和P糖蛋白（P-gp）表达明显降低，对ADR的耐药性明显下降，敏感性增加。 结论：联合运用不同的逆转耐药机制，将TNF-α cDNA及MDR1反义RNA分别或同时导入乳腺癌耐药细胞中，能有效达到逆转耐药的目的。     

Prognostic significance of multidrug resistance gene 1 (MDR1), multidrug resistance-related protein (MRP) and lung resistance protein (LRP) mRNA expression in acute leukemia

The prognostic significance of multidrug resistance (MDR) gene expression is controversial. We investigated whether multidrug resistance gene 1 (MDR1), multidrug resistance-related protein (MRP) and lung resistance protein (LRP) mRNA expression are associated with outcomes in acute leukemia patients. At diagnosis we examined MDR1, MRP and LRP mRNA expression in bone marrow samples from 71 acute leukemia patients (39 myeloid, 32 lymphoblastic) using nested RT-PCR. The expression of each of these genes was then expressed as a ratio in relation to beta-actin gene expression, and the three genes were categorized as being either 0, 1+, 2+ or 3+. MDR1, MRP and LRP mRNA expression was detected in 23.9%, 83.1% and 45.1 %, respectively. LRP mRNA expression was significantly associated with resistance to induction chemotherapy in acute leukemia patients, and in the AML proportion (p=0.02 and p=0.03, respectively). MRP and high MDR1 mRNA expression was associated with poorer 2-yr survival (p=0.049 and p=0.04, respectively). Patients expressing both MRP and LRP mRNA had poorer outcomes and had worse 2-yr survival. The present data suggest that MDR expression affects complete remission and survival rates in acute leukemia patients. Thus, determination of MDR gene expression at diagnosis appears likely to provide useful prognostic information for acute leukemia patients.     

本芴醇衍生物LY980503对人乳腺癌多药耐药细胞株MCF/DOX基因表达谱的影响

目的:应用基因表达谱芯片探讨本芴醇衍生物LY980503对肿瘤多药耐药逆转机制。方法:提取多药耐药细胞株MCF/DOX总RNA, 与含有320人基因的cDNA芯片杂交。结果:有9条基因表达下调, 1条基因表达上调。结论:LY980503在体外能通过调节多种基因的表达而逆转MCF/DOX的耐药性。     

Multi-drug resistance (MDR1) gene expression in de novo acute leukemia cells: correlations with CD surface markers and treatment outcome.

One important mechanism of drug resistance in acute leukemia is the overexpression of the multi-drug resistance (MDR1) gene that encodes a 170-kDa membrane protein called P-glycoprotein. To estimate the incidence and role of MDR1 gene expression in patients with acute leukemia, we investigated the expression of MDR1 by using the RT-PCR method in blast cells from 40 cases of de novo acute leukemia. We found a high frequency of MDR1 gene expression: 10 out of 20 with de novo acute myeloid leukemia (AML), 8 out of 17 with de novo acute lymphoblastic leukemia (ALL), and none of the 3 with de novo acute mixed leukemia, were MDR1 mRNA-positive. No correlation between cluster designation (CD) surface markers (CD19, CD7, CD13, CD33, CD34, CD14, HLA-DR) and MDR1 gene expression in AML was found. The complete remission rate was correlated with MDR1 gene expression. Among 40 evaluable patients examined, 17% (3 of 18) with MDR1 mRNA-positive reached complete remission versus 77% (17 of 22) with MDR1 mRNA-negative (p=0.044). These results suggest that MDR1 gene expression can be used as a prognostic factor and may be helpful in determining chemotherapeutic protocol for patients with acute leukemia.     

Expression of P-glycoprotein (multidrug-resistance gene product) in haematological tumors

The fact that cancer cell acquires multidrug resistance to carcinostatics at cancer treatment is a very important subject clinically. The mode of multidrug-resistance is complicated, but the gene associated with multidrug resistance (MDR 1) has been isolated. It has become evident that MDR 1 gene carries membrane glycoprotein (P-glycoprotein) which occurs in the cell acquired drug-resistance. Assessment has been made this time regarding the occurrence of P-glycoprotein in the tumorous cells and tissues by the use of monoclonal antibody (C 219) to P-glycoprotein. Occurrence of P-glycoprotein in malignant lymphoma exhibited positivity in 9 cases out of 36 immunohistologically. 170 KD P-glycoprotein was detected in 4 cases out of 10 at Western blotting analysis of the protein isolated from the nuclear cell in the peripheral blood in the patients with leukemia. Further, P-glycoprotein positive cases were all progressive cases clinically and showed resistance to treatment. From these results, it has been clarified that occurrence of P-glycoprotein in haematological tumors is related to multidrug resistance.     

多药耐药基因修饰的小鼠骨髓细胞对造血功能的保护作用

目的:将体外转染了人多药耐药基因(MDR1)的小鼠骨髓细胞,移植给经致死剂量照射的受体小鼠,观察该基因对小鼠造血功能的保护作用。方法:分离经5-Fu预处理的供体小鼠骨髓有核细胞,体外转染由逆转录病毒介导的人多药耐药基因MDR1,然后移植给经85Gy致死剂量照射的同系受体小鼠,以紫杉醇(Taxol)、长春新碱(VCR)、柔红霉素(DNR)筛选,观察小鼠血象、生存期和生存率变化,应用聚合酶链反应(PCR)和流式细胞仪(FCM)分析人多药耐药基因在小鼠中的整合与表达。结果:致死剂量辐照后,移植组小鼠造血功能逐渐恢复,未移植组15d内全部死亡。腹腔注射紫杉醇或静脉注射长春新碱、柔红霉素后,实验组生存率和生存期明显高于对照组(P<005)。表明MDR1基因能保护骨髓细胞,并且具有体内选择和富集作用,PCR分析提示,实验组外周血、骨髓、肝、脾组织中均检测到原病毒整合,RT-PCR与FCM检测到MDR1基因表达。结论:人MDR1基因修饰的小鼠骨髓细胞,能有效重建经致死剂量照射的受体小鼠造血功能,一定程度上保护骨髓免受化疗药物所致的细胞毒性作用。     

高三尖杉酯碱白血病多药耐药细胞系K562/HHT的诱导及米非司酮逆转耐药的研究

目的: 研究高三尖杉酯碱（HHT）诱导的白血病多药耐药细胞株耐药机制以及米非司酮(RU486)逆转其耐药效应。方法：采用反复短期暴露并逐渐增加HHT浓度的方法建立白血病多药耐药细胞系K562/HHT，MTT法检测K562/HHT细胞对化疗药物的敏感性及RU486对该细胞的细胞毒效应，RT-PCR法检测细胞MDR1基因、葡萄糖神经酰胺合成酶（GCS）基因的表达，流式细胞术检测细胞P-糖蛋白的表达和柔红霉素积累量，免疫组化法检测细胞Bcl-2、Bax、caspase-3的表达状况。结果： 成功诱导出多药耐药细胞系K562/HHT，K562/HHT较其亲代细胞K562对HHT、阿霉素、长春新碱、依托泊苷的耐药性分别提高462.6、2.8、1 183.4、2.6倍，K562/HHT细胞MDR1基因、GCS基因、P-糖蛋白和Bcl-2/Bax比值明显高于K562细胞（P<0.05），caspase-3表达、柔红霉素积累量明显低于K562细胞（P<0.05）。10 μmol/L的RU486对K562/HHT细胞无明显杀伤（存活率94.67%±2.48%），该浓度RU486可不同程度地逆转K562/HHT细胞对上述化疗药物的耐药性，RU486作用后K562/HHT细胞caspase-3表达、柔红霉素积累量明显高于作用前（P<0.05），Bcl-2/Bax比值明显低于作用前（P<0.05）。结论： 白血病细胞系K562在HHT的作用下可形成多药耐药细胞系K562/HHT，其耐药性的形成与P-糖蛋白、GCS、Bcl-2/Bax比值及caspase-3等机制有关，RU486可通过提高细胞内药物积累、调节Bcl-2/Bax比值和caspase-3的表达逆转此细胞的耐药性。     

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.     

反义寡聚脱氧核苷酸抑制卵巢癌细胞株SKOV3／mdr1中MDR1基因的表达

多药耐药基因（MDR1）编码的P－糖蛋白(Pgp,P-170)是导致卵巢癌化疗失败的重要因素之一。本研究将针对MDR1基因的硫代反义寡聚脱氧核苷酸导入卵巢癌耐药细胞株SKOV3/mdr1,观察其对该细胞株P170表达的影响。反义寡聚脱氧核苷酸（MDR1－AS）通过阳离子脂质体介导或单纯转染的方式导入SKOV3／mdr1 细胞,将正义寡聚脱氧核苷酸（MDR1－S)同样导入细胞为对照。经以脂质体介导的1．6μmol/L MDR1-AS转染后,Pgp阳性的细胞百分数从100％降至48．7％,经过16和10μmol／L两个浓度的单纯MDR1－AS转染后,Pgp阳性细胞的百分数也从100％分别降至52．6％和86．7％。同时,利用RT－PCR检测了MDR1mRNA水平的变化,以β-actin作为内对照,发现MDR1mRNA水平在MDR1－AS转染后降至内对照的60％左右,而用MDR1－S转染的细胞与SKOV3／mdr1细胞比无明显变化。所以导入MDR1基因硫代反义寡聚脱氧核苷酸可以部分抑制SKOV3／mdr1细胞中MDR1基因的表达。