RNA干扰技术逆转神经胶质瘤细胞多药耐药性

目的 探讨利用RNA干扰（RNAi）技术逆转神经胶质瘤细胞多药耐药性。方法 根据多药耐药基因1（MDR1）的碱基序列设计并合成短发夹RNA（shRNA）,构建逆转录病毒质粒载体,用阳离子脂质体法体外转染BT325细胞株,以增强型绿色荧光蛋白（EGFP）表达作为对照。采用定量PCR、Northern blot检测转染前后MDR1 mRNA的表达,Western blot检测蛋白表达;使用CCK-8试剂盒对转染后的细胞进行化疗药物敏感性试验,评价RNAi对多药耐药性的逆转作用。结果 成功构建RNAi质粒载体。共转染实验组RT-PCR定量MDR1 mRNA相对表达水平均有所下降（P〈0．05）;Northern blot表明,转染48h细胞干扰最强;Western blot显示,siRNA各转染组P-糖蛋白（P-gp）的表达分别降低12．9％、30．3％和4．8％,在48h抑制最强;而药物敏感试验显示,转染siRNA后细胞对药物的敏感性明显增强。结论 RNAi能够明显抑制神经胶质瘤细胞系MDR1 mRNA和P-gp蛋白的表达,进而对多药耐药性发挥明显的逆转作用,为基因治疗提供了一种新的手段。     

siRNA干扰与卵巢癌多药耐药的关系

化疗是综合治疗卵巢癌不可缺少的重要手段之一,但是临床上肿瘤所表现出来的耐药,却大大限制了化疗药物的疗效。多药耐药是指肿瘤细胞对多种结构与作用机制或靶位不同的化疗药物的抵抗耐受。目前,各种方法已用于逆转肿瘤的多药耐药,包括钙通道阻滞剂、环孢菌素A及其衍生物、反义核苷酸和合成转录因子,但效果不是很理想。小分子RNA干扰（small—interference RNA,siRNA）是近几年发展起来的较新技术,已广泛用于卵巢癌等肿瘤耐药的研究。     

多药耐药性逆转问题研究进展

肿瘤化疗失败的一个重要原因是由于肿瘤细胞对化疗药物产生了多药耐药性,即肿瘤细胞可耐受结构、功能及杀伤机制各异的多种化疗药物的作用。要使耐药性得以逆转,扰要阻断其产生的各个环节。逆转剂可分为P-糖蛋白功能抑制剂、MDR基因抑制剂,谷胱甘肽(GSH)合成抑制剂、谷胱甘肽转硫酶合成抑制剂及DNA修复抑制剂等。目前逆转剂应用于临床还存在一定问题,在体内要达到逆转效应所需的剂量已远远超出了人体所能耐受的剂量,目前国内外正致力于克服此缺陷的研究。     

索拉非尼体外逆转人肝癌耐药细胞BEL-7402/5-FU多药耐药性机制的研究

目的:研究索拉非尼对人肝癌耐药细胞BEL-7402/5-FU多药耐药性逆转作用及可能机制.方法:MTT法检测索拉非尼对BEL-7402/5-FU细胞的抑制率及无细胞毒性剂量的索拉非尼对化疗药物的逆转作用;流式细胞仪检测细胞内罗丹明-123和多柔比星荧光强度;RT-PCR检测MDR1、ABCG2和LRP等耐药基因mRNA表达情况;蛋白质印迹法检测P-gp表达情况.结果:2.5 ttg/mL索拉非尼对BEL-7402/5-FU细胞无细胞毒作用,对氟尿嘧啶,表柔比星和紫杉醇的逆转倍数分别为2.54、4.92和2.23倍,其相对逆转率分别为66.7%、96.5%和76.8%;流式细胞仪检测显示,BEL-7402/5-FU细胞内多柔比星(P＜0.05)和罗丹明-123(P＜0.05)浓度明显增加;RT-PCR显示,MDR1(P＜0.05)和ABCG2(P＜0.01)表达明显下降,对LRP表达无明显影响(P＞0.05);蛋白质印迹法显示,P-gp表达明显下降(P＜0.01).结论:索拉非尼具有部分逆转肝癌多药耐药的作用,可能与下调MDR1/P-gp和ABCG2的表达、抑制P-gp功能以及增加细胞内化疗药物浓度有关;而此作用可能与LRP无关.     

肿瘤多药耐药性及其逆转策略

 肿瘤细胞多药耐药性（multidrug resistance，MDR）的产生是导致肿瘤化疗失败的主要原因之一。MDR是指肿瘤细胞对一种抗肿瘤药物产生耐药性的同时，对结构和作用机制完全不同的其他抗肿瘤药物产生交叉耐药性的现象。^[1]      

逆转肿瘤多药耐药性的系列研究

多药耐药性（ｍｕｌｔｉｄｒｕｇ ｒｅｓｉｓｔａｎｃｅ, ＭＤＲ）是指肿瘤细胞接触一种来源于天然的抗肿瘤药物并产生耐药性的同时,对结构和作用机理不同的多种来源于天然的抗肿瘤药物产生交叉耐药性。多药耐药性是肿瘤化疗失败的主要原因。ＭＤＲ产生的原因复杂,但ｍｄｒｌ基因过度表达产生的Ｐ－糖蛋白（Ｐｅｒｍｅａｂｉｌｉｔｙ　 ｇｌｙｃｏｐｒｏｔｅｉｎ, Ｐ－ｇｐ）是 ＭＤＲ产生的最重要和最常见的原因。实际上,Ｐ－ｇｐ是一种能量依赖性药物转出泵,能将化疗药物从癌细胞内泵出细胞外,从而使药物在细胞内积蓄浓度降低,使…     

RNA干扰Apollon基因逆转白血病K562细胞多药耐药

背景与目的：Apollon基因在白血病等多种肿瘤内高表达。本试验通过构建高效干扰Apollon基因的短发夹RNA(short hairpin RNA,shRNA)真核表达载体,探讨RNA干扰技术能否逆转人髓系白血病K562细胞多药耐药。方法：构建靶向Apollon基因真核表达载体pGPHI-GFP-Neo-Apollon,应用Lipofectamine^TM2000转染K562细胞,G418稳定筛选。采用反转录-聚合酶链反应(RT-PCR)法、细胞免疫荧光法分别检测重组载体稳定转染K562细胞后Apollon mRNA及蛋白的表达情况;四甲基偶氮唑盐(MTT)、流式细胞术检测细胞转染前后对长春新碱(leurocristine,VCR)、足叶乙苷(etoposide,VP16)的敏感性及凋亡率的变化。结果：成功构建了pGPHI-GFP-Neo-Apollon载体并在K562细胞内稳定表达的细胞克隆,经G418筛选后,重组载体能有效沉默Apollon的表达,Apollon mRNA及蛋白表达水平明显下降。MTT结果显示,基因干扰组细胞对VCR、VP16的敏感性明显增强,其半数抑制浓度(half-inhibitory,IC₅₀)值分别为(0.144±0.018)mg/L、(17.336±3.571)mg/L,明显低于细胞对照组(P<0.05)。流式细胞术检测结果表明,基因干扰组细胞联合化疗药物后细胞凋亡率显著升高(P<0.05),而转染阴性对照组细胞凋亡率与正常对照组比较差异无统计学意义(P>0.05)。结论：pGPHI-GFP-Neo-Apollon载体能显著增强VCR和VP16对白血病K562细胞的诱导凋亡作用,提高K562细胞对化疗药物的敏感性,提示RNA干扰Apollon基因表达能一定程度逆转白血病细胞的多药耐药。     

Reversal of P-glycoprotein-mediated multidrug resistance with small interference RNA (siRNA) in leukemia cells

The multidrug resistance (MDR) mediated by P-glycoprotein (P-gp), the MDR1 gene product, is one of the major obstacles in leukemia treatment. The present study was designed to explore a MDR1-targeted small interfering RNA (si-MDR1) approach for reversal of P-gp-mediated MDR in the MDR human leukemia cell line k562/A02. It was found that si-MDR1 significantly inhibited MDR1 expression at both mRNA and protein levels. Depletion of MDR1 by si-MDR1 correlated with the increased sensitivity of the cells to cytotoxic agents and with the enhanced intracellular retention of daunorubicin (DNR). One base-pair mutated control (si-MDR1-Mut) lost the effect of si-MDR1 on both the degradation of mdr1 mRNA and the reduction of P-gp expression. These findings indicate that siRNA specifically and efficiently interferes with the expression of mdr1 and could be used as a molecularly defined therapeutic approach for MDR in the treatment of leukemia.     

Detection of multidrug resistance (MDR1) gene RNA expression in human tumors by a sensitive ribonuclease protection assay

The human MDR1 gene encoding P-glycoprotein, an energy-dependent drug-efflux pump, was initially isolated from a multidrug-resistant KB carcinoma cell. When a 3 kb genomic sequence isolated from normal human tissue including the major downstream promoter and the first and second exons of the MDR1 gene was compared to the equivalent fragment from KB cells, the MDR1 gene from KB carcinoma cells was found to have a point mutation in the first exon. Although this mutation does not affect the downstream promoter sequence or the coding sequence of the MDR1 gene, it creates a single base mismatch between the 5' KB genomic fragment previously used for RNase protection analysis of MDR1 RNA expression in normal tissues and thereby reduces the sensitivity of this assay. Using the DNA fragment from normal tissues rather than KB cells, we have reanalyzed MDR1 mRNA levels in 12 renal carcinomas and 4 colon adenocarcinomas. By this RNase protection assay, MDR1 RNA levels are as high in these tumors as in the multidrug-resistant cell line, KB-8-5. The ribonuclease protection assay indicated that the major downstream promoter was mainly used in these clinical samples including two samples of RNA from metastatic renal cancer. This assay appears to be a very sensitive and specific assay for detecting MDR1 mRNA levels and mRNA initiation sites in clinical samples.     

小细胞肺癌多药耐药细胞差异表达片段磷酸二酯酶2A(PDE2A)在多种肿瘤细胞中表达的研究

目的:检测PDE2A在多种肿瘤细胞中的表达情况,以了解PDE2A基因的表达分布特点及初步功能特征,为进一步肿瘤多药耐药相关研究奠定基础.方法:利用Northern杂交与RT-PCR方法检测PDE2A在H446、H446/CDDP、A549、A549/CDDP、SK-HEP-1、Namalwa、SGC7901等7种肿瘤细胞中的表达情况.结果:PDE2A仅在H446/CDDP中表达,而在其他细胞中无表达.结论:在所检测的7种肿瘤细胞中,PDE2A在H446/CDDP中高表达.PDE2A可能参与了H446/CDDP多药耐药性的形成.     

SOCS3对人肝癌MHCC97-H细胞上皮间质转化的影响

目的:研究细胞因子信号转导抑制因子3（suppressor of cytokine signaling 3,SOCS3）对人肝癌细胞MHCC97-H细胞上皮间质转化（epithelial-mesenchymal transition,EMT）的影响。方法:体外培养人肝癌高转移细胞株MHCC97-H,应用25 nmol/L的SOCS3 siRNA瞬时转染细胞（阳性转染组）,同时使用空质粒转染细胞（阴性对照组）。24 h后用荧光显微镜观察转染后的细胞荧光表达情况。48 h后,应用倒置显微镜观察细胞形态变化情况,采用细胞免疫荧光染色法检测MHCC97-H 细胞中EMT的上皮标志物E-cadherin和间质标志物α-SMA的表达情况。结果:与阴性对照组相比,SOCS3 siRNA成功转染的MHCC97-H细胞显示出绿色荧光。SOCS3 siRNA瞬时转染后肝癌细胞从呈现上皮样特征的鹅卵石形态,向具有间质细胞形态的纺锤形和梭形特征发生转变。免疫细胞荧光检测E-cadherin和α-SMA的表达结果显示,SOCS3 siRNA阳性转染组上皮标志物E-cadherin的免疫荧光表达明显减弱,而细胞的间质标志物α-SMA的免疫荧光表达显著增强。结论:本实验研究显示,下调肝癌细胞的SOCS3表达,其通过改变细胞的EMT表型分子及表型特征,促进肝癌的增殖,提示SOCS3可能在肝癌细胞的EMT中发挥着重要作用。调控SOCS3表达水平可以抑制肝癌的发生发展,为临床防治肝癌提供了新思路。     

Induction of apoptosis in multi-drug resistant (MDR) human glioblastoma cells by SN-38, a metabolite of the camptothecin derivative CPT-11

 The overexpression of the multidrug resistance (mdr1) gene and its product, P-glycoprotein (P-gp), is thought to limit the successful chemotherapy of human tumors. Recent studies demonstrate that SN-38, a metabolite of the camptothecin (CPT) derivative CPT-11, has antitumor effects on several tumors, but the mechanisms responsible for its cytotoxicity remain unclear. We therefore determined whether SN-38 has cytotoxic effects on MDR human glioblastoma GB-1 cells and non-MDR human glioblastoma U87-MG cells. Furthermore, we determined what role SN-38 plays in the induction of cytotoxicity in these tumor cells. In this study, we demonstrated that SN-38 had significantly stronger antitumor effects on GB-1 and U-87MG cells than did CPT (P<0.01 and P<0.05, respectively). In addition, findings obtained using a DNA fragmentation assay, Hoechst 33258 staining, in situ end-labeling and cell cycle analysis demonstrated that SN-38 induced apoptosis in these tumors. Our results suggest that SN-38 has a stronger antitumor effect on malignant glioma cells regardless of MDR expression than does CPT, and therefore can be considered a new chemotherapeutic agent potentially effective in the treatment of human primary or recurrent malignant gliomas resistant to chemotherapy. Received: 6 October 1995/Accepted 29 June 1996     

Stable and complete overcoming of MDR1/P-glycoprotein-mediated multidrug resistance in human gastric carcinoma cells by RNA interference

Multidrug resistance (MDR) is the major cause of failure of effective chemotherapeutic treatment of disseminated neoplasms. The "classical" MDR phenotype of human malignancies is mediated by drug extrusion by the adenosine triphosphate binding cassette (ABC)-transporter P-glycoprotein (MDR1/P-gp). For stable reversal of "classical" MDR by RNA interference (RNAi) technology, an H1-RNA gene promoter-driven expression vector encoding anti-MDR1/P-gp short hairpin RNA (shRNA) molecules was constructed. By introduction of anti-MDR1/P-gp shRNA expression vectors into the extremely high drug-resistant human gastric carcinoma cell line EPG85-257RDB, the MDR phenotype was completely reversed. The reversal of MDR was accompanied by a complete suppression of MDR1/P-gp expression on mRNA and protein level, and by a considerable increased intracellular anthracyline accumulation in the anti-MDR1/P-gp shRNA-treated cells. The data indicate that stable shRNA-mediated RNAi can be tremendously effective in reversing MDR1/P-gp-mediated MDR and is therefore a promising strategy for overcoming MDR by gene therapeutic applications.     

siRNA与人工半合成手型多西紫杉醇对A549多药耐药细胞的协同抑制作用

目的:探讨小分子干扰RNA (siRNA)与人工半合成多西紫杉醇对人肺腺癌细胞A549耐药细胞系(A549/CDDP)的协同抑制作用.方法:采用化学合成的方法,合成手型多西紫杉醇,设计针对多药耐药基因1(MDR1)序列的siRNA,克隆入psiRNA中(psiRNA-MDR).用半合成多西紫杉醇和转染psiRNA-MDR两种方法对培养的A549/CDDP细胞和荷瘤小鼠瘤体进行处理,观察其对A549/CDDP细胞生长的抑制作用.结果:siRNA/MDR1明显抑制A549/CDDP细胞MDR1表达;半合成多西紫杉醇可增加A549细胞凋亡,对A549组细胞、A549/CDDP组细胞和A549/CDDP-psiRNA/MDR组细胞的抑制率分别为75%、39%和87%;后两组裸鼠移植瘤体积分别为(7814±1005)mm3、(6632±1203)mm3和(540±121.4)mm3,均有显著差异.结论:siRNA与人工半合成手型多西紫杉醇对多药耐药肺癌细胞有明确的协同抑制作用.