抗肿瘤多药耐药的研究进展

肿瘤多药耐药是化疗失败的主要原因,其机制很多,如转运P糖蛋白与mdr- 1基因,谷胱甘肽(GSH)解毒酶系统,DNA拓扑异构酶含量或性质的改变,多药耐药相关蛋白(MRP) ,蛋白激酶(PK)等。有关药物有,针对P -gp/MDR1的逆转剂,针对蛋白激酶C的逆转剂,DNA修复相关酶的活性抑制剂,针对MPR的逆转剂,抑制GSH合成酶。现将MDR的耐药机制及目前可供临床治疗的MDR肿瘤的药物:MDR逆转剂和抗肿瘤MDR药物以及对MDR的基因治疗研究作一综述。     

siRNA特异抑制树突状细胞BAK基因表达的研究

本文探讨了小干扰RNA(siRNA)对人树突状细胞(DC)中促凋亡基因BAK表达的影响。针对BAK基因区设计了2条高效特异的siRNA,合成相应的oligoDNA;构建重组质粒psi-BAK-1与psi-BAK-2,并对这些质粒进行了限制性酶切、测序等鉴定工作;在脂质体的介导下转染树突状细胞,转染后72h收集细胞,用蛋白印迹法检测Bak蛋白的表达量;并通过流式细胞仪检测转染后72h树突状细胞的存活率。结果表明,经过酶切鉴定和测序证实两个重组质粒psi-BAK-1、psi-BAK-2分别构建成功;WesternBlot结果显示转染后72h,si-BAK-1对Bak蛋白表达的抑制率为(51.5±3.54)%,si-BAK-2对Bak蛋白表达的抑制率为(26.5±6.36)%;转染重组质粒后72h树突状细胞存活率分别为93.95%、89.66%,均高过对照组的88.00%。si-BAK-1、si-BAK-2都能有效降低树突状细胞中BAK基因的表达,为延长树突状细胞的寿命,增强抗原递呈能力提供了新的思路和方法。     

鉴定活性维生素D细胞模型的构建

本文构建了维生素D受体(Vitamin D receptor,VDR)融合蛋白的真核表达载体,并获得稳定表达VDR融合蛋白的细胞体系,为含有活性维生素D食品及药物的鉴定建立成熟的细胞模型。设计并合成特异性引物扩增VDR基因,将扩增产物克隆至pcDNA3.1/His真核表达载体上,将其转染HEK293细胞。采用免疫沉淀(IP)技术鉴定VDR融合蛋白的表达效率。在构建pcDNA 3.1/VDR-His成功的基础上,用活性1,25(OH)_2D_3处理转染的细胞,收集总蛋白和m RNA,分别利用免疫印迹(Western blot,WB)和实时定量PCR(q RT-PCR)技术,检测VDR融合蛋白以及其下游基因CYP24A1基因的表达。IP结果证实VDR融合蛋白具有转录活性。WB和q RT-PCR结果显示,1 nmol/L浓度的活性1,25(OH)_2D_3处理转染细胞能有效激活VDR蛋白的表达,以及显著增强CYP24A1的m RNA表达(p0.01)。利用该细胞模型检测维生素D类药物的活性,结果显示不同药物中维生素D的活性与药物种类有关。pcDNA3.1/VDR-His融合表达载体构建成功,并以此构建了鉴定活性维生素D的细胞模型。     

HA-His/PEI-His/DNA纳米复合载体的制备及基因转染研究

为了克服PEI介导的基因转染毒性高、无肿瘤细胞选择性的缺点,采用组氨酸(His)分别对聚乙烯亚胺(PEI)和透明质酸(HA)进行修饰,得到PEI-His(PH)和HA-His(HH)。利用静电相互作用制得HH、PH与DNA的复合物HH/PH/DNA(HPD)并用于基因转染。动态光散射测定HPD复合物的平均粒径为109 nm,ζ-电位为-15 mV。HPD复合物可有效包载DNA并保护其不被酶降解,表面的HA还可避免血清粘附。细胞实验结果表明,经His修饰可有效降低PEI的细胞毒性,HPD复合物介导的基因转染对肿瘤细胞具有选择性,在肿瘤细胞中的转染效率为正常细胞中的2倍。     

Rspo1-EGFP重组腺相关病毒载体的构建

本文通过构建携带Rspo1-EGFP融合基因的重组腺相关病毒载体,包装出含有目标基因的重组腺相关病毒.首先利用PCR从cDNA文库中把Rspo1基因扩增出来,插入到pcDNA6-EGFP中EGFP的上游形成融合基因Rspo1-EGFP.采用AAV Helper-free包装系统,将融合基因用PCR方法从质粒pcDNA6-Rspo1-EGFP上扩增出来,亚克隆到AAV表达质粒pAAV-MCS多克隆位点中,构建出重组质粒pAAV-Rspo1-EGFP.重组质粒与包装质粒pAAV-RC和辅助质粒pHelper磷酸钙法共转染AAV-293细胞制备重组病毒rAAV-Rspo1-EGFP.重组病毒感染AAV-HT1080细胞,荧光显微镜检测病毒介导的目标基因表达,流式细胞技术测定病毒滴度.结果:酶切鉴定和测序确定重组病毒载体pAAV-Rspo1-EGFP构建成功.病毒感染的细胞中检测到绿色荧光,表明重组病毒包装成功并介导融合基因在宿主细胞里表达,病毒滴度达107VP/mL.本研究成功包装出具有侵染性的重组朦相关病毒agV-Rspo1-EGFP,为今后利用腺相关病毒载体进行Rspo1相关的体内体外研究提供了实验基础.     

重组人凝血酶真核表达载体的构建及其在CHO细胞中的稳定表达

目的 构建含重组人凝血酶基因的重组真核表达载体,并转染哺乳动物细胞CHO,建立稳定表达重组人凝血酶的细胞株.方法 利用限制性内切酶EcoR I和XbaI将凝血酶基因插入真核表达载体pcDNA3.1（＋）中,构建含人凝血酶基因的重组质粒pcDNA3.1 （＋）/thrombin,利用双酶切和测序法鉴定.采用阳离子脂质体介导方法,将构建的表达载体转染到CHO细胞中,通过G418加压筛选出阳性细胞克隆,建立稳定表达人凝血酶的细胞株,并扩大培养.采用RT-PCR和SDS-PAGE法检测其mRNA和蛋白质的表达,并对其生物活性进行鉴定.结果 重组质粒pcDNA3.1 （＋）/thrombin经双酶切和测序鉴定证实插入序列准确无误;经RT-PCR和SDS-PAGE法鉴定,转染的CHO细胞可表达、分泌人凝血酶,得到的重组人凝血酶表观相对分子质量（Mr）为43000左右,与预测一致,且具有降解并凝固牛纤维蛋白原的活性,其比活为234.1 U/mg.结论 成功构建了真核表达载体pcDNA3.1 （＋）/thrombin,并成功地在CHO细胞中表达了具有生物活性的重组人凝血酶,该体系的建立为进一步规模化生产重组人凝血酶奠定了基础.     

蓖麻毒素A链基因的克隆表达、纯化及其活性

为了实现蓖麻毒素A链基因(rta)的克隆表达,制备有高生物活性的重组蓖麻毒素A链蛋白(RTA),借助重组腺病毒介导表达的RTB进入细胞,发挥RTA的细胞毒作用,检测其活性。重组质粒pET32a-His-RTA能正确表达RTA融合蛋白,相对分子质量约47 000,每升细菌培养物回收约50 mg的纯化蛋白质,在有RTB表达的系列中,细胞死亡率明显上升,最高可达50%~60%。说明利用pET32a表达系统可以快速获得大量有高生物活性的可溶性RTA融合蛋白质。以腺病毒为载体表达RTB可以帮助RTA进入细胞,对细胞发挥毒性作用。     

药食同源类中药在P-糖蛋白介导的多药耐药中的应用

目前, 肿瘤细胞产生多药耐药(multidrug resistance, MDR)严重制约了化疗疗效, 由多药耐药基因编码的P-糖蛋白(P-glycoprotein, P-gp)的过表达是多药耐药产生的重要原因之一。我国中药资源丰富, “药食同源”历史悠久, 药食两用类中药, 安全性高, 毒副作用小, 可通过多成份, 多靶点、多途径来预防和治疗肿瘤。本文根据P-gp介导MDR机制, 介绍了部分可以抑制P-gp实现逆转MDR的“药食同源”类中药, 为肿瘤的预防和辅助治疗提供新思路。     

RNA干扰技术抑制乳腺癌细胞系MDA-MB-231中SATB1的表达

构建含有靶向satb1基因的siRNA质粒,体外观察对乳腺癌细胞系MDA-MB-231的SATB1mRNA以及蛋白质表达的影响。设计合成三对靶向人源satb1基因的siRNA,并分别克隆在质粒载体上,在脂质体的介导下转染高表达SATB1的乳腺癌细胞系MDA-MB-231。运用RealTime-PCR方法分析SATB1mRNA的表达情况,Western-Blot方法检测蛋白的表达量。结果表明:经过酶切鉴定和测序证实三个重组质粒psiS823、psiS2567、psiS3373分别构建成功,转染乳腺癌细胞系MDA-MB-231能明显抑制SATB1mRNA及蛋白的表达。说明本试验成功构建的重组质粒psiS823、psiS2567、psiS3373都能有效降低的人乳腺癌细胞MDA-MB-231中SATB1的表达,为SATB1高表达的乳腺癌基因治疗提供了新方法。     

青稞对60Co-γ射线照射小鼠造血功能的保护作用

目的:探讨青稞对辐射小鼠造血功能的保护作用。方法:以含20%青稞饲料饲养C57BL/6J小鼠12周后,采用4 Gy剂量60Co-γ射线照射全身。照射后继续干预2周,监测其体重、摄食量、外周血象、骨髓与脾脏组织形态、骨髓有核细胞数、脏器指数及血生化水平,以评价其造血功能恢复情况。结果:与辐射对照组相比,青稞干预后小鼠在辐照第4天体重明显恢复,第1周摄食量显著上升,总胆固醇(TC)水平明显恢复,小鼠的骨髓有核细胞数、红细胞、血红蛋白、白细胞及血淋巴细胞水平明显恢复,且小鼠骨髓结构损伤较小。同时,青稞干预可有效恢复辐照导致的脏器指数的异常并保护脾脏结构。本研究结果表明,青稞的预防性干预通过减轻小鼠辐照后脾脏组织形态的损伤、白细胞、淋巴细胞、脏器指数及血生化水平的异常来防止对免疫功能的损伤。通过恢复小鼠辐照后骨髓结构的损伤,促进骨髓有核细胞数及外周血红细胞的恢复来保护造血功能。结论:青稞预防性干预是通过保护机体免疫功能,有效恢复辐射引起的造血功能损伤。     

An isoquinoline alkaloid from the Chinese herbal plant Corydalis yanhusuo W.T. Wang inhibits P-glycoprotein and multidrug resistance-associate protein 1

Overexpression of P-glycoprotein (P-gp) and multidrug resistance-associate protein 1 (MRP1) is a major mechanism leading to multidrug resistance (MDR) of cancer cells. These transporters expel anti-cancer drugs and greatly impair therapeutic efficacy of chemotherapy. A Chinese herbal plant Yanhusuo (Corydalis yanhusuo W.T. Wang, YHS) is frequently used in functional food and traditional Chinese medicine to improve the efficacy of chemotherapy. The objective of this work was to study effects of glaucine, an alkaloid component of YHS, on P-gp and MRP1 in resistant cancer cells.The resistant cancer cell line, MCF-7/ADR and corresponding parental sensitive cells were employed to determine reversal properties of glaucine. Glaucine inhibits P-gp and MRP1-mediated efflux and activates ATPase activities of the transporters, indicating that it is a substrate and inhibits P-gp and MRP1 competitively. Furthermore, glaucine suppresses expression of ABC transporter genes. It reverses the resistance of MCF-7/ADR to adriamycin and mitoxantrone effectively.     

2016年中国26个省市食源性沙门菌耐药性特征分析

目的 了解2016年中国26个省、直辖市和自治区食源性沙门菌的耐药状况。方法 采用微量肉汤稀释法测定755株食源性沙门菌对10类16种抗生素的药物敏感性,采用实时荧光定量聚合酶链式反应方法检测mcr-1基因的存在情况。结果 72.7%(549/755)的沙门菌对受试的16种抗生素呈现不同程度的耐药性,其中萘啶酸(NAL)、四环素(TET)、氨苄西林(AMP)、氨苄西林/舒巴坦(SAM)4种抗生素的耐药率较高,均在34%以上,未见碳青霉烯类[亚胺培南(IPM)、美罗培南(MEM)]耐药菌株,44.4%(335/755)的沙门菌同时耐受3类或3类以上抗生素,表现为多重耐药,同时耐受抗生素种类最高为8类。共存在134种耐药谱,优势耐药谱型为NAL、TET和AMP-SAM-NAL。全部菌株中检出2株携带mcr-1基因的菌株,分别为八重耐药的德尔卑沙门菌(Salmonella Derby)和七重耐药的鼠伤寒沙门菌(Salmonella Typhimurium)。部分省份沙门菌耐药率较高。结论 2016年中国26个省、直辖市和自治区食源性沙门菌整体耐药水平较高,多重耐药情况严重,我国食源性沙门菌中存在携带mcr-1基因的多重耐药菌株,应引起关注。     

Enhancement of drug efflux activity via MDR1 protein by spheroid culture of human hepatic cancer cells

Human hepatic cancer cells, HepG2, formed spheroids on a poly-L-glutamic acid-coated dish. Doxorubicin (DOX) efflux activity of the cells in spheroid culture was higher than that in monolayer culture due to the higher expression of MDR1 protein of the cells in spheroids compared with those in monolayer. The amount of MDR1 per cell in spheroids was similar to that of hepatic tumor tissue in vivo. Consequently, it was suggested that the drug efflux activity of cells in spheroid culture reflected the activity of hepatic cancer cells. Furthermore, the IC(50) of DOX and epirubicin (EPI) in HepG2 cells, both of which are known to be exported by MDR1, were higher in spheroid compared with monolayer cells, while IC(50) of 5-fluorouracil (5-FU), which is not exported by MDR1 protein, was almost the same in both types of culture. The higher IC(50) of DOX and EPI in HepG2 cells in spheroid culture was associated with a higher efflux activity of the drugs in the spheroid-cultured cells, which appeared to reflect the IC(50) of DOX and EPI in cancer cells in vivo. Therefore, a spheroid culture of hepatic cancer cells seems to provide a promising cell-based in vitro assay system for examining the proper IC(50) values of anticancer agents that would reflect the drug resistance of cancer cells in vivo. In addition, the system would be useful in screening for inhibitors of MDR1 activity, which will help to overcome the multidrug resistance of cancer cells.     

The SCR1 gene from Schwanniomyces occidentalis encodes a highly hydrophobic polypeptide,which confers ribosomal resistance to cycloheximide

In Saccharomyces cerevisiae, the SCR1 gene from Schwanniomyces occidentalis is known to induce ribosomal resistance to cycloheximide (cyh). A 2.8 kb DNA fragment encoding this gene was sequenced. Its EMBL Accession No. is AJ419770. It disclosed a putative tRNA(Asn) (GUU) sequence located downstream of an open reading frame (ORF) of 1641 nucleotides. This ORF was shown to correspond to SCR1. It would encode a highly hydrophobic polypeptide (SCR1) with 12 transmembrane domains. SCR1 is highly similar to a variety of yeast proteins of the multidrug-resistance (MDR) family. However, SCR1 only conferred resistance to cyh but not to benomyl or methotrexate. The cyh-resistance phenotype induced by SCR1 was confirmed in several S. cerevisiae strains that expressed this gene to reside at the ribosomal level. In contrast, a beta-galacosidase-tagged SCR1 was found to be integrated in the endoplasmic reticulum (ER). It is proposed that the ribosomes of yeast cells expressing SCR1 undergo a conformational change during their interaction with the ER, which lowers their affinity for cyh-binding. If so, these findings would disclose a novel ribosomal resistance mechanism.     

一株食源性多重耐药单核细胞增生李斯特菌的耐药性研究

本研究对食品样本中分离的一株多重耐药单核细胞增生李斯特菌进行耐药机制的探讨,以期对食源性单核细胞增生李斯特菌多重耐药现象的控制提供理论依据。本文通过聚合酶链式反应筛选耐药决定因子,质粒消除及自然转化实验对耐药决定因子进行定位及传播能力的探讨,最后通过传代实验验证该菌株多重耐药性传播的稳定性。结果表明,对检测到的多重耐药菌株LM78(耐受氯霉素、红霉素、链霉素、四环素、复方新诺明)进行相关耐药基因检测,检测到cat、erm B、tet S 3个耐药基因。质粒消除后MIC值下降到敏感范围,且该质粒可通过自然转化在不同菌属间传递,说明这些耐药基因存在于质粒上。该质粒在无抗生素选择压力下连续传代,仍具有较高稳定性。食源性致病菌多重耐药性有可能通过不同细菌种属间转移,进而由食物链向人类传播,对人类健康造成潜在的威胁。     

白血病的多药耐药性及其逆转策略

多药耐药是化疗失败的主要原因,现对白血病的多药耐药机制的研究进展及其可逆转的策略进行了论述。     

CaALK8, an alkane assimilating cytochrome P450, confers multidrug resistance when expressed in a hypersensitive strain of Candida albicans

We report the isolation of a novel C. albicans gene designated CaALK8, by its ability to complement drug hypersensitivity of a pdr5 (ABC: ATP-binding cassette drug extrusion pump) null mutant of S. cerevisiae (JG436). CaALK8 in JG436 conferred resistance to drugs such as cycloheximide (CYH), fluconazole (FCZ), O-phenanthroline (PHE) and 4-nitroquinoline oxide (NQO). The gene was so designated because its sequence was identical to a partial sequence entry named as ALK8 in the Candida database (http://alces.med.umn.edu/candida.html). CaALK8 encodes for a putative 515 amino acid protein highly homologous to alkane-inducible cytochromes P450 (CYP52 gene family) of C. maltosa and C. tropicalis. The ability of CaALK8 to confer drug resistance was also established by its expression in another drug-hypersensitive strain of S. cerevisiae (AD 1234568), which was deleted in seven ABC efflux pumps. The homozygous disruption of CaALK8 in a wild-type C. albicans strain (CAI4) did not result in altered drug susceptibilities. The overexpression of CaALK8 in CAI4 resulted in only FCZ resistance. However, a distinct MDR phenotype was evident when CaALK8 was overexpressed in a drug-hypersensitive C. albicans strain disrupted in both CDR1 and CDR2 (ABC drug extrusion pumps of C. albicans). Alk8p, similar to other Alk proteins from C. maltosa and C. tropicalis, could hydroxylate alkanes and fatty acids. In this study we demonstrate that several drugs could compete with the hydroxylation activity by directly interacting with CaAlk8p. Taken together, our results suggest that a member of the CYP52 gene family could mediate MDR in C. albicans, although it does not seem to be involved in the development of azole resistance in clinical isolates. The nucleotide sequence reported in this paper has been submitted to GenBank under Accession No. Y14766.