Reversal of multi-drug resistance by vector-based-ShRNA-Mdr1 <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis>

In order to investigate the effects of vector-based hairpin small interference RNA （shRNA） on the reversal of multi-drug resistance （mdr） of A2780/Taxol cells, a novel vector pEGFP-HI/mdrl containing mdrl-shRNA targeting at position 2943-2963 of mdrl was designed and synthesized. Subsequently, A2780/Taxol cells were transfected with pEGFP-H1/rndrl, and the expression ofmdrl mRNA and P-gp was detected by using RT-PCR and Western blot respectively. MTT was used to measure the 50% inhibition concentration （IC50） of Taxol to A2780/Taxol cells. The results showed that at the 24th and 48th h after transfection, the expression of mdrl mRNA was decreased to （52.1±1.0）% and （0.01±1.7）%, and that of P-gp decreased to （88.3±2.1）% and 0%, respectively. At the 48th h after transfection, the relative reversal rate of A2780/Taxol cells to Taxol was 69.54%. In vivo, the nude mice xenografts were injected with pEGFP-H1/mdrl, and then administrated Taxol. The tumor volume in pEGFP-H1/mdrl-transfected group was significantly reduced as compared with that in blank control group or pEGFP-Hl-transfected group （807.20±103.16 vs 1563.78±210.54 or 1480.78±241.24 mm^3, both P〈0.01）. These results suggested that transfection of pEGFP-HI/mdrl could efficiently down-regulate the expression of mdrl mRNA and P-gp in A2780/Taxol cells, and effectively restore the sensitivity of A2780/Taxol ceils to Taxol both in vitro and in vivo.     

急性髓系白血病细胞多药耐药相关基因的筛选

目的筛选急性髓系白血病细胞多药耐药相关基因。方法在建立HL-60／DNR多药耐药（MDR）细胞系的基础上，采用抑制消减杂交的方法结合基因芯片技术建立HL-60／DNR抑制消减杂交文库，并挑选其中部分克隆进行序列测定和同源性分析。结果HL-60／DNR对多种化疗药物均产生了不同程度的耐受。在所构建的HL-60／DNR抑制消减杂交文库中，经过基因芯片筛选出明显差异表达克隆共有14个；进一步分析发现这些基因在白血病细胞多药耐药中的作用大多未见报道。结论多种基因（已知的或未知的）均参与了急性髓系白血病细胞多药耐药的调节,大规模筛选与克隆这些基因为进一步研究急性髓系白血病细胞多药耐药产生的机制奠定了必要的理论基础.     

Reversal of MDR1 gene-dependent multidrug resistance using short hairpin RNA expression vectors

Background RNA interference using short hairpin RNA (shRNA) can mediate sequence-specific inhibition of gene expression in mammalian cells. A vector-based approach for synthesizing shRNA has been developed recently. Overexpression of P-glycoprotein (P-gp), the MDR1 gene product, confers multidrug resistance (MDR) to cancer cells. In this study, we reversed MDR using shRNA expression vectors in a multidrug-resistant human breast cancer cell line (MCF-7/AdrR). Methods The two shRNA expression vectors were constructed and introduced into MCF-7/AdrR cells. Expression of MDR1 mRNA was assessed by RT-PCR, and P-gp expression was determined by Western Blot and immunocytochemistry. Apoptosis and sensitization of the breast cancer cells to doxorubicin were quantified by flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. Cellular daunorubicin accumulation was assayed by laser confocal scanning microscopy (LCSM). Statistical significance of differences in mean values was evaluated by Student’s t tests. P&lt;0.05 was considered statistically significant.Results In MCF-7/AdrA cells transfected with MDR1-A and MDR1-B shRNA expression vectors, RT-PCR showed that MDR1 mRNA expression was reduced by 40.9% (P&lt;0.05), 30.1% (P&lt;0.01) (transient transfection) and 37.6 % (P&lt;0.05), 28.0% (P&lt;0.01) (stable transfection), respectively. Western Blot and immunocytochemistry showed that P-gp expression was significantly and specifically inhibited. Resistance against doxorubicin was decreased from 162-fold to 109-fold (P&lt;0.05), 54-fold (P&lt;0.01) (transient transfection) and to 108-fold (P&lt;0.05), 50-fold (P&lt;0.01) (stable transfection). Furthermore, shRNA vectors significantly enhanced the cellular daunorubicin accumulation. The combination of shRNA vectors and doxorubicin significantly induced apoptosis in MCF-7/AdrR cells. Conclusions shRNA expression vectors effectively reduce MDR expression in a sustained fashion and can restore the sensitivity of drug-resistant cancer cells to conventional chemotherapeutic agents.     

RNA干扰逆转肝细胞癌多药耐药

目的 筛选高效dsRNA/mdr1,以备研究RNA干扰逆转肝细胞癌多药耐药之用.方法 首先根据siRNA设计原则,以多药耐药基因(mdr1)为靶基因,设计并选择4～5条siRNA/mdr1,经BLAST后体外转录法合成dsRNA/mdr1,用Oligofectamine试剂分别转染HepG2/mdr1,然后从mRNA、蛋白(P-gp)表达水平和细胞功能变化评价HepG2/mdr1耐药性被逆转的程度,比较各个dsRNA/mdr1的逆转效率,筛选出有效的siRNA/mdr1.结果 成功合成5条dsRNA/mdr1(其中1条为阴性对照),dsRNA/mdr1-4 mRNA表达(18.73±1.33)%、蛋白表达变化(79.1±1.6)%～(16.8±0.4)%与其他各组细胞比较,有显著性差异;细胞内柔红霉素(DNR)累积量也较其他组明显增加(平均荧光强度79.58,阳性率84.25%,P＜0.05).结论 体外转录法结合脂质体转染适用于筛选高效siRNA,肯定了siRNA干扰序列能够有效阻抑mdr1基因编码蛋白p170的功能.     

一株携带多种耐药基因的铜绿假单胞菌研究

目的了解铜绿假单胞菌的多重耐药情况和有关机理。方法聚合酶链反应（PCR）法对多重耐药的铜绿假单胞菌进行β-内酰胺酶基因、氨基糖苷类修饰酶（AMEs）基因、耐消毒剂和磺胺基因（qacE△1—sul1）的检测。并对CARB基因进行了测序。结果PCR显示CARB、TEM型β-内酰胺酶基因，aac（6’）-Ⅰ、aac（6’）-Ⅱ、ant（2″）-Ⅰ、ant（3″）-Ⅰ AMEs基因、qacE△1-sul1耐消毒剂基因和耐磺胺基因扩增阳性，oprD2基因扩增检测呈缺失型。CARB基因扩增产物测序，经BLAST程序分析（WWW．ncbi．nlm．nih．gov）为CARB-3型。结论铜绿假单胞菌存在多重耐药基因，管壁涂有季胺类、双胍类消毒剂和磺胺的Ⅱ代导管的抑菌效果需重新评价。     

RNA干扰逆转白血病细胞耐药性的研究

目的 用RNA干扰技术下调白血病耐药细胞系K562/A02多药耐药基因mdr1的表达以逆转白血病对化学药物的耐药性.方法 针对mdr1基因已知mRNA序列不同位点,选择两条靶序列,构建靶向mdr1 shRNA真核表达载体,脂质体介导转染白血病耐药细胞株K562/A02.实时荧光定量RT-PCR检测mRNA表达,Western blot检测细胞膜P-gp表达,柔红霉素泵出试验检测P-gp外排泵功能,MTT法检测细胞对阿霉素的敏感性.结果 构建了二条针对mdr1基因的shRNA真核表达载体,分别下调mdr1 mRNA的表达89.74%和87.18%,降低细胞膜P-gp表达,使膜外泵功能明显下降,柔红霉素在细胞内贮留明显增多,60min 时柔红泵出率为13.16%、22.02%,对照组为40.44%、45.31%,对阿霉素药物敏感性的相对逆转率为84.36%和76.69%.结论 RNA干扰技术可有效逆转mdr1所致耐药.     

腺病毒介导的靶向自杀基因对卵巢癌耐药细胞株的杀伤作用

目的研究多药耐药基因1（mdr1）调控的胞嘧啶脱氨酶-尿嘧啶磷酸核糖转移酶融合基因（CD∷UPP）联合5-氟胞嘧啶（5-FC）后对卵巢癌紫杉醇耐药细胞株生长的影响。方法以复制缺陷型重组腺病毒为载体，将mdr1-CD∷UPP基因分别转染2对人卵巢癌紫杉醇耐药细胞株A2780/Taxol、SKOV3/Taxol及非耐药细胞株A2780和SKOV3，加入含5-FC的培养液培养，5 d后噻唑蓝（MTT）法检测细胞存活率，观察旁观者效应。结果5-FC对转基因耐药细胞的生长抑制作用明显高于转基因的非耐药细胞，随着5-FC浓度增加，抑制作用增强；通过旁观者效应5-FC可杀伤周围未转基因的耐药细胞。结论mdr1-CD∷UPP靶向自杀基因联合5-FC后对紫杉醇耐药细胞具有显著的特异性杀伤作用。     

mdr1介导的多药耐药及其逆转的研究进展

目的介绍mdr1介导的多药耐药及其逆转的研究进展。方法采用文献回顾的方式对国内外相关文献进行分析整理,对mdr1基因的结构功能、表达调控以及逆转多药耐药的方法进行综述。结果mdr1基因的过度表达是导致肿瘤多药耐药的重要原因,mdr1表达调控网络主要由转录、转录后水平和表观遗传调控构成,各种机制通常同时在细胞中起作用,逆转多药耐药的研究集中在药物和基因治疗两个方面。结论多种方法联合应用有助于逆转多药耐药。     

RNA干扰技术沉默MDR1基因逆转大肠癌耐药细胞株LoVo/5-Fu的耐药性

目的  探讨多药耐药（MDR1）基因沉默对大肠癌耐药细胞系LoVo/5-Fu耐药性的逆转作用。方法  构建靶向MDR1的短发夹RNA(EASY-shRNA-MDR1)干扰质粒转染LoVo/5 Fu,实时荧光定量PCR（Realtime-PCR）在mRNA水平检测抑制效果,噻唑蓝（MTT）法检测细胞对5-Fu的敏感性,流式细胞仪检测细胞凋亡情况。结果  与未转染组相比,EASY-shRNA-MDR1组细胞MDR1mRNA表达明显下调（P<0.05）;细胞对5-Fu的IC50及RI显著降低（P<0.05）,敏感性的相对逆转率为74.7%;凋亡率明显升高(P<0.05)。结论  EASY-shRNA-MDR1可有效抑制大肠癌耐药细胞中MDR1的表达,进而降低了大肠癌耐药株对5-Fu的耐药性。     

结核分枝杆菌rpoB基因突变与多重耐药及利福平耐药程度的相关性

目的 研究结核分枝杆菌，rpoB基因突变与多重耐药及利福平耐药程度强弱之间的关系。方法应用绝对浓度法测定58株结核分枝杆菌对4种－线抗结核药物的药敏特性，对耐利福平结核分枝杆菌的耐药谱进行分析。同时对含有核心突变区的结核分枝杆菌，rpoB基因片段进行PCR扩增和DNA测序，分析rpoB基因突变与多重耐药的关系，以及突变位点和突变性质与利福平耐药强弱的相关性。结果58株结核分枝杆菌中，利福平耐药株36株，敏感株22株。36株耐药株均存在，rpoB基因突变，其中88．9％(32／36)至少对利福平和异烟肼同时耐药。90．O％的利福平高度耐药株(18/20)与rpoB基因531位和526位密码子突变相关。密码子531位和526位各有1株双碱基突变，且均为多重耐药株。结论结核分枝杆菌，rpoB基因突变所致的利福平耐药株大部分对异烟肼耐药，因此单独检测rpoB基因突变即可初步筛选多重耐药的结核分枝杆菌；而rpoB基因的突变位点与利福平耐药程度之间具有一定的相关性。     

Reversal of HCC Drug Resistance by Using Hammerhead Ribozymes against Multidrug Resistance 1 Gene

To reverse multidrug resistance（MDR） of HepG2 by anti-MDR1 hammerhead ribozyme, an anti-MDR1 hammerhead ribozyme was developed and delivered to P-gp-overproducing human hepatocarcinoma cell line HepG2 by a retroviral vector containing RNA polymerase Ⅲ promoter. The expression of mdrl/Pgp and Rz was detected in HepG2, HepG2 muhidrug-resistant cell line and HepG2 Rz-transfected cells by semi-quantitative RT-PCR and Western blot methods. Moreover, MTT assay was employed to detect the sensitivity of these ribozyme-transfected cells, and Rhodamine123 （Rh123） was used to test the function of Pgp. The Rz- transfected HepG2 cells became doxorubicin-sensitive, which was concomitant with the decreased MDR1 expression. The study showed that the retrovirus vector encoding the anti-MDR1 ribozyme may be applicable to the treatment of MDR cells.     

Effect of Ursolic Acid on Breast Cancer Resistance Protein-mediated Transport of Rosuvastatin In Vivo and Vitro

Objective To evaluate whether ursolic acid can inhibit breast cancer resistance protein (BCRP)-mediated transport of rosuvastatinin vivo andinvitro. Methods Firstly, we explored the pharmacokinetics of 5-fluorouracil (5-FU, a substrate of BCRP) in rats in the presence or absence of ursolic acid. Secondly, we studied the pharmacokinetics of rosuvastatin in rats in the presence or absence of ursolic acid or Ko143 (inhibitor of BCRP). Finially, the concentration-dependent transport of rosuvastatin and the inhibitory effects of ursolic acid and Ko143 were examined in Madin-Darby Canine Kidney (MDCK)Ⅱ-BCRP421CC (wild type) cells and MDCKⅡ-BCRP421AA (mutant type) cells. Results As a result, significant changes in pharmacokinetics parameters of 5-FU were observed in rats following pretreatment with ursolic acid. Both ursolic acid and Ko143 could significantly affect the pharmacokinetics of rosuvastatin. The rosuvastatin transport in the BCRP overexpressing system was increased in a concentration-dependent manner. However, there was no statistical difference in BCRP-mediated transport of rosuvastatin betweent the wild type cells and mutant cells. The same as Ko143, ursolic acid inhibited BCRP-mediated transport of rosuvastatinin vitro. Conclusion Ursolic acid appears to be a potent modulator of BCRP that affects the pharmacokinetic of rosuvastatinin vivo and inhibits the transport of rosuvastatinin vitro.     

siRNAs介导的基因沉默技术和相关应用的研究进展

RNA干扰（RNAi）是由双链RNA（double-slranded RNA,dsRNA）介导的、序列特异的转录后基因沉默机制,是一古老的细胞反应通路,其在抵抗病毒感染、抑制转座子活动、调控内源性基因表达等方面发挥重要作用。近年来,短链RNA干扰（siRNAs）已成功地应用于哺乳动物中,该文就其介导的基因沉默技术和相关应用的研究进展进行综述。     

质粒表达型siRNA对SARS-CoV复制与感染干扰的初步研究

目的构建针对SARS-CoV的特异性siRNA质粒,利用RNA干扰技术抑制该病毒复制与感染.方法根据SARS-CoV的全长基因序列,以4个不同的部位为靶点,分别设计、合成含有19 nt干扰序列的一段寡核苷酸,将两两互补的寡核苷酸经退火后所形成的序列克隆到pSilencer 3.1-H1载体中,构建表达siRNA的质粒.采用脂质体法将质粒转染Vero E6细胞,经潮霉素抗性筛选后,再对稳定表达的细胞进行细胞病变、病毒空斑形成和MTT实验,以观察干扰效应.结果对所构建的质粒分别测序,确定其含有siRNA的序列与预期的特异性干扰序列一致.用不同浓度的SARS-CoV感染转染质粒后的细胞,与阴性对照相比,其细胞病变减轻、活细胞显著增加,病毒空斑明显减少.结论利用RNA干扰能有效的抑制SARS-CoV在细胞中的复制,并对细胞有保护作用,为预防、治疗SARS提供了新的思路和方法.     

应用RNA干扰抑制目的基因在小鼠体内表达

目的研究RNA干扰(RNAi)对体外和体内基因表达的沉默作用。方法以PCR方法从基因组DNA中克隆出依赖于RNA聚合酶Ⅲ的H1启动子,并用于驱动RNAi片段的合成;构建特异性针对目的基因(绿色荧光蛋白,GFP)的RNAi载体(Pi)。将RNAi干扰载体和GFP载体转染NIH3T3细胞,应用荧光显微镜观察、RT-PCR、流式细胞技术(FACS)分析上述细胞中RNAi对目的基因GFP表达的抑制效果,进一步在个体水平将RNAi载体注入小鼠体内,研究RNAi对GFP表达的抑制情况。结果RNAi能有效地使NIH3T3细胞中GFP表达量降低约60%以上,并能有效地抑制GFP在转基因小鼠体内的表达。结论RNAi技术能抑制目的基因在体内外的表达,是一种有效的基因治疗工具。     

Induction of apoptosis of human colon cancer cells by siRNA recombinant expression vector targeting survivin gene

This study constructed siRNA recombinant expression vector targeting survivin gene and observe the apoptosis induction effect of it in human colon cancer cells, siRNA recombinant expression vector targeting survivin gene was constructed and transfected into human colon cancer cells. The effect of siRNA recombinant expression vector was detected by RT-PCR, Western blot, MTT reduction assay and flow cytometry. It was confirmed by restriction endonuclease and sequence analysis that siRNA recombinant expression vector targeting survivin gene was constructed successfully. Inhibition rate of survivin siRNA at mRNA and protein levels was 36.33% and 44.65% respectively. Growth of cancer cells was inhibited and the apoptosis rate was （17.24±2.13）%. The siRNA recombinant expression vector targeting survivin gene has been constructed successfully. It not only can inhibit the expression of survivin gene, but also can induce apoptosis in human colon cancer cells remarkably.     

RNAi逆转肾癌细胞MDR1基因表达导致的替尼泊苷耐药

目的：研究RNA干扰（RNAinterference,RNAi）对肾癌细胞多药耐药基因（MDRl）表达的抑制作用,并分析干扰前后肾癌细胞对替尼泊苷（VM一26）敏感性的变化。方法：根据MDRI基因设计3条小干扰RNA（smalJinterferingRNA,siRNA）序列,在质粒的介导下转染肾癌A498细胞,RT—PCR法分析转染前后MDRlmRNA表达水平,筛选出干扰效率最高的siRNA序列;进而利用慢病毒包装siRNA重组质粒,感染A498细胞,RT—PCR法筛选沉默效果最好的细胞株进行克隆,Westernbolt法检测MDRl蛋白表达水平,MTT法对比干扰前后替尼泊苷对细胞半抑制浓度（ICSO）的变化。结果：3条siRNA序列均能不同程度地抑制细胞MDRl基因的表达,其中siRNA一1序列能更有效地封闭MDRl基因,使MDRlmRNA表达水平下降67％;筛选出的稳转细胞株与未干扰细胞株相比,MDRl蛋白表达量明显下降,并使替尼泊苷对细胞的IC50降低约79．8％,差异具有统计学意义（P〈O．01）。结论：RNAi可抑制肾癌A498细胞MDRl基因的表达从而逆转其对替尼泊苷的耐药,使肾癌细胞化疗耐药逆转成为可能。     

苦参碱逆转人结肠癌细胞株（HT-29）奥沙利铂耐药性的作用及机制研究

目的：研究苦参碱对结肠癌耐药细胞HT-29/奥沙利铂(Oxaliplatin, OXA)耐药性的逆转作用并探讨其可能的作用机制。方法采用逐步增加药物浓度的方法建立奥沙利铂耐药结肠癌细胞株HT-29/OXA;CCK-8法测定苦参碱对HT-29/OXA细胞的耐药性逆转作用,流式细胞术检测细胞凋亡、周期变化;实时定量PCR 检测各组细胞肺耐药蛋白（lung resis-tance protein LRP)和 P-glycoprotein (P-gp) mRNA表达水平; Western blot 检测各组细胞LRP和P-gp蛋白的表达水平。结果苦参碱使HT-29/OXA细胞对奥沙利铂的敏感性增加,耐药性得到部分逆转。奥沙利铂(0.5μg/mL)和苦参碱(3.0μg/mL)单独用药对结肠癌耐药细胞株HT-29/OXA细胞周期以及细胞凋亡没有影响;联合用药对HT-29/OXA生长增殖具有明显抑制作用并且能够通过改变细胞周期引起凋亡（P<0.05）。奥沙利铂(0.5μg/mL)和苦参碱(3.0μg/mL)单独用药对HT-29/OX-ALRP和P-gp mRNA以及蛋白的表达没有影响;联合用药作用后LRP mRNA和P-gp mRNA表达水平降低（P<0.01）,同时下调了LRP和P-gp蛋白表达（P<0.01）。结论苦参碱部分逆转HT-29/OXA细胞对奥沙利铂的耐药性,其机制可能与细胞内LRP和P-gp表达降低有关。     

汗防已甲素联合塞来昔布逆转白血病细胞耐药的体外研究

目的：通过汗防已甲素联合塞来昔布逆转白血病细胞耐药的实验研究,探讨有关多药联合逆转肿瘤细胞的耐药机制.方法： 噻唑蓝（MTT）比色法检测汗防已甲素联合塞来昔布对K562细胞的细胞毒试验;用流式细胞技术（FCM）检测各组K562细胞内的药物浓度;用FCM检测各组的早期凋亡（Annexin Ⅴ）;用RT-PCR检测各组COX-2/COX-1的表达.结果： 汗防已甲素联合塞来昔布对K562细胞的抑制作用与其他组相比有显著性差异（P〈0.05）;细胞内的药物浓度高于其他实验组;诱导凋亡率（17.67%）高于其他实验组;COX-2/COX-1的表达明显低于其他实验组.结论： 汗防已甲素联合塞来昔布逆转K562细胞的效果明显,能够增加K562细胞内药物浓度,诱导细胞凋亡,降低 COX-2/COX-1的表达.     

Enhanced Chemotherapy Sensitivity of Human Colon Cancer Cells to 5-Fluorouracil by siRNA Recombinant Expression Vector Targeting Survivin Gene

Objective To investigate the effects of small interfering RNA （siRNA） recombinant expression vector targeting survivin gene on chemotherapy sensitivity of human colon cancer cells to 5-fluorouracil. Methods siRNA recombinant expression vector targeting survivin gene was constructed and transfected into human colon cancer cell lines LOVO. After 48 hours of transfection, cells were harvested for analysis of survivin mRNA and protein expressions using RT-PCR and Western blot. In addition, after human colon cancer cell lines were treated with Survivin siRNA and/or 5-fluorouracil, MTT assay and flow cytometry were used to analyze cell proliferation and apoptosis. Results Restriction endonuclease analysis confirmed that siRNA recombinant expression vector targeting survivin gene was successfully constructed. Inhibitory ratios of survivin mRNA and protein expressions by Survivin siRNA were 36.33% and 44.65%, respectively. Survivin siRNA combined with 5-fluorouracil significantly increased the cell proliferation inhibitory ratio and apoptosis ratio compared with 5-fluorouracil treatin~ alone （P〈0.05）. Conclusion The siRNA recombinant expression vector targeting survivin gene can inhibit the expression of survivin gene, and enhance chemotherapy sensitivity of human colon cancer cells to 5- fluorouracil.