人胃癌顺铂耐药细胞系的建立过程

目的:建立人胃癌顺铂耐药细胞系方法:采用逐步递增顺铂浓度,间歇作用体外诱导法建立人胃癌顺铂耐药细胞系SGC7901/ DDP,每月作相关检测;MTT法测定药物敏感性:光镜、电镜、MTT法计数活细胞、流式细胞仪等方法观察其生物学特征的改变.结果:历时4 mo建成人胃癌顺铂耐药细胞系SGC7901/DD P,1,2,3,4mo的耐药指数分别为2.13,5.32,12.60,12.93,并且与5-氟尿嘧啶、丝裂霉素等多种抗癌药有不同程度的交叉耐药性:SGC7901/DDP的细胞形态逐渐发生改变;体外群体倍增时间较亲代细胞逐渐延长:细胞周期分析发现其S期与G_2/M期细胞逐渐减少,G_0/G_1期细胞逐渐增多.结论:SGC7901/DDP细胞具有耐药表型特征,耐药性能稳定,增殖能力逐渐下降.     

Participation of poly(ADP-robose) polymerase in the drug sensitivity in human lung cancer cell lines

Summary Poly(ADP-ribose) polymerase has been generally assumed to be involved in DNA repair. The level of the enzyme in various lung cancer cell lines was examined to determine if it is involved in drug resistance. Among nine cell lines of lung cancer tested, small-cell lung cancer lines, which showed higher sensitivity to cisplatin and etoposide, were unexpectedly found to contain significantly higher poly(ADP-ribose) polymerase activity than five non-small-cell lung cancer cell lines. This activity inversely correlated with IC₅₀ values of lung cancer cell lines to etoposide, an inhibitor of topoisomerase II. The polymerase activity was also examined in several cisplatin-resistant variants of the cell lines. However, no difference was observed between parental and cisplatin-resistant cells. There was no significant relation between poly(ADP-ribose) polymerase activity and IC₅₀ values for cisplatin and carboplatin. Although this enzyme was considered to play some role in the resistance to specific drugs, it might not be a critical factor in cisplatin-induced cytotoxicity.     

胃癌细胞系SGC7901耐阿霉素和顺铂细胞系的建立及其耐药可持续性的质控研究

目的建立胃癌耐药细胞模型，并进行耐药细胞耐药可持续性研究。方法以脉冲式诱导方法诱导胃癌耐阿霉素细胞SGC7901／ADR和胃癌耐顺铂细胞SGC7901／DDP。检测细胞的生长曲线和群体倍增时间，电镜观察细胞的超微结构，并进行了体外药物敏感性实验和细胞药物转运实验，Western blot检测耐药细胞内P—gP和MRP的表达，尤其是进行了耐药细胞的耐药可持续性的质控研究。结果耐药细胞的增殖能力比药敏细胞强（P〈0．05）；电镜下耐药细胞较药敏细胞表现为更多的核大核畸形及细胞膜间突触样联系和细胞间小管结构；体外药物敏感性实验和细胞药物转运实验显示耐药细胞的耐药性明显增强；而且耐药细胞中MRP和P—gP的表达均比药敏细胞SGC7901高；耐药可持续性的质控研究表明在不给与抗肿瘤药维持状况下细胞自第ll代耐药性明显下降。结论成功构建了胃癌耐阿霉素细胞系SGC7901／ADR和耐顺铂细胞系SGC7901／DDP；对实验用耐药细胞系建议质控在9代以内以保证耐药细胞的耐药稳定性。     

铜离子转运蛋白家族与肺癌顺铂耐药的研究进展

铂类药作为化疗一种关键药之一,被广泛用于治疗各种恶性肿瘤,如卵巢、膀胱、头颈部肿瘤及肺癌.但铂类耐药的发生限制了化疗反应,影响了患者的预后.目前在铂类耐药的机制方面已经有一些重要的发展,其中之一是肿瘤铂类耐药与细胞内浓度的蓄积之间的相关性,摄入的减少和泵出过多均可减少药物在细胞内的聚积,导致耐药.但是具体耐药机制尚不清楚.铜离子动态平衡是由铜离子转运蛋白及其分子伴侣来维持.铜离子转运蛋白家族包括铜离子转运蛋白和铜离子转运磷酸化ATP酶.本文将就铜离子转运蛋白家族与肺癌顺铂耐药作一综述.     

非小细胞肺癌肿瘤药敏与耐药基因MDR1、MRP、GST-π表达的相关性研究

目的研究非小细胞肺癌(NSCLC)多药耐药(MDR1)、谷胱甘肽转移酶(GST-π)、多药耐药相关蛋白(MRP)基因表达和肿瘤药物敏感试验之间的相关性及临床指导意义.方法 48例NSCLC(可手术)进入研究,药敏方法采用磷脂结合蛋白V(Annexin V)联合碘化丙啶(PI)双参数法,耐药基因MDR1、GST-π、MRP采用逆转录聚合酶链反应(RT-PCR)检测.结果抗癌药物检测显示紫杉醇(商品名泰素)、顺铂、去甲长春花碱、丝裂霉素、鬼臼乙叉甙、双氟胞苷、长春碱酰胺、长春新碱的平均抑瘤率分别为(15.7±21.8)%, (20.7±22.2)%,(7.9±16.2)%,(10.3±17.1)%,(9.7±20.1)%,(11.2±13.8)%,(5.6±14.9)%,(4.7 ±8.7)%.耐药基因MDR1、MRP、GST-π阳性率分别为67%(32/48),42%(20/48),48%(23/48).耐药基因MRP、GST-π阳性及阴性表达与病理类型之间未见明显相关;而在MDR1组中鳞癌及腺癌的MDR1阳性表达明显高于阴性表达组(P<0.05).NSCLC各期别中MDR1、MRP、GST-π阳性表达统计学上未见明显差异.在所检测的所有抗癌药物与MDR1耐药基因阳性与阴性表达未见相关性.MRP表达阳性者对去甲长春花碱、长春碱酰胺、长春新碱和丝裂霉素的肿瘤抑制率显著低于阴性表达组(P<0.05).而MRP表达与顺铂、鬼臼乙叉甙、紫杉醇肿瘤药敏未见明显相关.耐药基因GST-π阳性表达者对顺铂、去甲长春花碱、丝裂霉素的抑瘤率显著低于阴性表达组(P<0.05).结论肺癌耐药基因GST-π、MRP表达与部分化疗药物肿瘤药敏存在相关性,耐药基因检测对指导临床化疗药物的选择具有一定的帮助.     

耐药相关蛋白在肺癌组织中的表达及意义

目的　探讨耐药相关蛋白在肺癌组织中的表达及意义。方法　应用免疫组化法检测治疗前 90例肺癌组织标本中 P-糖蛋白 (P- gp)、多药耐药相关蛋白 (MRP)、肺耐药蛋白 (L RP)、谷胱苷肽 S转移酶 (GST- π)、DNA拓扑异构酶 (Topo- )蛋白的表达 ;流式细胞术检测肺癌组织增值指数 (PI)、DNA指数 (DI)及细胞周期各时相分布。结果　非小细胞癌 (NSCL C组 ) P- gp、L RP表达明显高于小细胞癌 (SCL C)组 (P <0 .0 5 ) ,MRP+L RP共表达者高于 SCL C组 (P <0 .0 5 ) ;中、高分化癌组 P- gp、L RP表达高于 SCL C组 (P <0 .0 5 ) ,MRP表达高于低分化癌组 (P <0 .0 5 ) ;腺癌组 MRP、L RP、MRP+L RP、MRP+L RP+GST- π、MRP+L RP+P- gp+GST-π表达高于鳞癌组、SCL C组、低分化癌组 (P <0 .0 5 ) ,MRP+GST- π共表达者高于鳞癌组、低分化癌组 (P <0 .0 1)。P- gp与 Topo 蛋白表达呈正相关 (P <0 .0 5 )。MRP与肺癌组织增殖指数 (PI)、G2 / M期细胞比例呈正相关 (P <0 .0 5 ) ,与 G0 / G1 期细胞比例呈负相关 (P <0 .0 5 )。结论　联检肺癌组织中耐药相关基因蛋白的表达有助于判断化疗疗效及预后。 P- gp、L RP、MRP、GST-π可作为判断肺癌细胞原发性耐药的指标。     

肺癌药敏与耐药基因MRP1、P-gp、GST-π和TopoⅡ表达的相关性研究

目的 研究多药耐药相关蛋白1 （MRP1）、P-糖蛋白（P-gp）、谷胱甘肽S转移酶-π（GST-π）、拓扑异构酶Ⅱ（TopoⅡ）基因表达和肺癌药物敏感试验相关性.方法 药物敏感试验采用MTT比色法,MRP1、P-gp、GST-π及TopoⅡ采用免疫组化检测.结果 MRP1、P-gp、GST-c及TopoⅡ在肺癌中总的阳性率分别为72.5％、67.5％、57.5％、50.0％.在鳞癌和腺癌中,这四种因子其表达无显著性差异（P＞0.05）,但P-gp、GST-π在鳞癌或腺癌中与在小细胞肺癌中表达有明显差异（P＜0.05）.P-gp和GST-π表达与顺铂、吉西他滨、长春瑞滨、紫杉醇耐药性呈正相关（P＜0.05）,与异环磷酰胺的耐药性无相关性（P ＞0.05）;MRP1的表达与顺铂、吉西他滨、长春瑞滨的耐药性均呈正相关（P＜0.05）,其表达与紫杉醇、异环磷酰胺的耐药性无相关性（P＞0.05）;TopoⅡ的表达与顺铂、吉西他滨、紫杉醇、异环磷酰胺的耐药性均呈正相关（P＜0.05）,其表达与长春瑞滨的耐药性无相关性（P＞0.05）.结论 MRP1、P-gp和GST-π的高表达及TopoⅡ的低表达共同介导参与了肺癌耐药的机制,且与检测的化疗药物耐药性有不同程度的相关性.     

耐药相关蛋白在肺癌组织的表达与药物敏感性的关系

目的探讨耐药相关蛋白在肺癌组织中的表达与药物敏感性的关系。方法应用免疫组化法、四唑蓝快速比色法(MTT法)分别检测P-糖蛋白(P-gp)、多药耐药相关蛋白(MRP)、肺耐药蛋白(LRP)、谷胱苷肽s转移酶(GST-π)蛋白在87例肺癌组织中的表达和10种抗癌药的体外药敏试验。结果P-gp、MRP、GST-π阳性表达及高表达者中,腺癌组药物敏感数明显低于鳞癌组(P<0.05),LRP阳性表达者中NSCLC组药物敏感数明显低于LRP阴性表达者(P<0.05)。LRP与MMC、5-FU、VP-16、VCR均呈负相关(P<0.05或0.01)。P-gp与顺铂(CDDP)呈负相关(P<0.05)。GST-π与MTX、HCPT呈正相关(P<0.05行)。结论P-gp、MRP、LRP、GST-π的蛋白表达及共表达与体外药敏试验存在较好相关性,可作为监测肺癌细胞原发性耐药的指标。     

Studies on tumor-cell-induced platelet aggregation in human lung cancer cell lines

We investigated the ability of human lung cancer cells of different histological subtypes to cause platelet aggregation. Tumor-cell-induced platelet aggregation (TCIPA) was studied in vitro in 13 human lung cancer cell lines [small-cell lung cancer (SCLC), squamous-cell lung cancer, large-cell lung cancer, adenocarcinoma and alveolar-cell lung cancer]. Three tumor cell lines failed to aggregate platelets in plateletrich plasma, whereas platelet aggregation was induced by 12 cell lines when added to washed platelets and minimal amounts of platelet-poor plasma (0.5% v/v). The thrombin antagonist hirudin inhibited TCIPA in non-small-cell lung cancer cell lines (NSCLC). In SCLC, TCIPA was fully abolished only when the ADP scavenger apyrase was added to hirudin. Thus ADP and thrombin generation by these tumor cell lines are responsible for platelet aggregation. The ability to activate platelets independently of coagulation factors VII and X was demonstrated for 8 cell lines. Electronmiicroscopically, direct tumor-cell/platelet contact was found to be the initiating mechanism of TCIPA in SCLC, whereas tumor-cell/platelet contacts in NSCLC could only be observed at the peak of the aggregation curve. Lung cancer cells activate platelets in vitro by generation of thrombin and/or ADP.Abbreviations TCIPA tumor-cell-induced platelet aggregation - SCLC small-cell lung cancer - NSCLC non-small-cell lung cancer     

顺铂诱导的人NCI-H460细胞获得性耐药与p53基因突变的关系

目的　探讨p53基因突变与肺癌化疗后获得性耐药的关系。方法　采用顺铂(DDP)大剂量(50μmol/L)间歇诱导法,体外诱导具有野生型p53基因的人大细胞肺癌NCIH460细胞株,建立多药耐药细胞系H460/DDP;用免疫细胞化学法检测P糖蛋白(Pgp)、多药耐药相关蛋白(MRP1)、肺相关蛋白(LRP)、拓扑异构酶Ⅱ(TOPOⅡ)、谷胱甘肽转移酶π(GSTπ)及P53蛋白的表达状况;用荧光免疫法测定P53蛋白磷酸化状态;对p53cDNA全长进行扩增及测序,用含野生型p53基因的质粒pShuttleCMVwtp53cDNA转染耐药细胞,测定转染细胞的药物敏感性。结果　新建立的人大细胞肺癌多药耐药株H460/DDP,对DDP及卡铂的耐药指数分别为1021和998,对氟尿嘧啶、多柔比星、表柔比星、甲氨蝶呤、依托泊苷及异长春新碱等有不同程度的交叉耐药。多药耐药株H460/DDP细胞的P53蛋白滞留于细胞浆,在DDP刺激下不能发生磷酸化;LRP表达明显增加(P<005);但其他耐药相关蛋白水平与DDP诱导前相比差异无统计学意义(P>005);耐药株细胞p53基因第277位点后插入一个“t”;耐药株转染pShuttleCMVwtp53cDNA后,其耐药性可发生部分(532%)逆转。结论　铂类化疗药物诱导的NCIH460细胞p53基因突变与其对化疗药物耐药性的产生有密切关系,p53基因替代疗法可能是克服这类获得性耐药的一个有效方     

胃癌耐药细胞株OCUM-2M/VP16的建立及其耐药机制

目的:建立耐VP16的胃癌耐药细胞株, 并初步探讨细胞发生VP16耐药的可能机制.方法:采用药物浓度梯度递增法诱导建立OCUM-2M/VP16耐药株. 细胞计数法绘制细胞生长曲线, 计算细胞倍增时间. MTT法检测细胞对6种化疗药物的IC50. 流式细胞仪分析细胞周期分布. 逆转录多聚酶链反应检测caspase-3, P53, DAPK-1, DAPK-2, DAPK-3,Bcl-2, ERCC-1, MDR-1及MRP基因mRNA的表达.结果:OCUM-2M/VP16成功实现了对VP16的耐药, 耐药指数为40.53; 其增殖速率及细胞倍增时间明显低于OCUM-2M(细胞倍增时间:22.96±0.96 h vs 30.29±2.55 h, P <0.01).OCUM-2M/VP16对Sn38、奥沙利铂及吉西他滨发生了交叉耐药, 但对5-FU及紫杉醇敏感性与亲代类似. OCUM-2M/VP16凋亡相关基因DAPK-2, DAPK-3和Bcl-2表达下调(OCUM-2M表达值为:0.61、0.79及0.81, OCUM-2M/VP16为, 0.24、0.45、0.44, P <0.01), 耐药相关基因 ERCC-1和MDR-1表达上调(OCUM-2M为0.53及0.20, OCUM-2M/VP16则为0.84及0.41, P <0.01). caspase-3, P53, DAPK-1及MRP表达无变化.结论:成功构建胃癌耐药细胞株OCUM-2M/VP16, 该细胞株具有交叉耐药特性, 耐药机制涉及凋亡、耐药等多个基因表达变化.     

托泊替康联合顺铂治疗广泛期小细胞肺癌

目的：观察托泊替康联合顺铂治疗广泛期小细胞肺癌临床疗效及毒性反应。方法回顾性分析我科2010年1月至2012年6月收治的68例经病理证实的广泛期小细胞肺癌患者,其中 TC 组(托泊替康/顺铂)36例,21例为初治患者,15例为复治患者,EP 组(依托泊苷/顺铂)32例,均为初治患者。结果68例患者均可评价疗效,TC 组一线治疗有效率明显高于二线治疗组(χ2=0.461,P =0.006),与 EP 组差异无统计学意义(χ2=0.874,P =0.553)。TC 组一线治疗患者与 EP 组患者生存期差异无统计学意义(P =0.847),显著长于二线治疗患者(P =0.021)。不良反应主要为骨髓抑制、消化道反应及脱发。结论托泊替康联合方案治疗广泛期小细胞肺癌安全有效。     

Influence of priming with 5-hydroxytryptophan on APUD characteristics in human small cell lung cancer cell lines

Two established human small cell cancer lines (SCCL) were exposed to 5-hydroxytryptophan (5-HTP) for various time intervals. The levels ofl-dopa decarboxylase (DDC), formaldehyde-induced fluorescence (FIF), number and volume density of dense-cored granules, and levels of bombesin-like immunoreactivity (BLI) and of immunoreactive calcitonin (ICT) were examined in controls and after 5-HTP treatment. The levels of DDC and BLI were not changed by 5-HTP treatment, while FIF was significantly increased in all 5-HTP treated groups. Immunoreactive calcitonin was undetectable in all groups including the controls. A quantitative increase in number and volume density of dense-cored granules was measured in one cell line after 18 h of 5-HTP and was accompanied by exocytosis of granules. The data demonstrate that while 5-HTP increased cellular activity in one of the cell lines (increase in number of densecored granules and evidence of secretion), not all of the measured parameters of APUD cells correlate under these experimental conditions.     

体外香菇多糖对顺铂抑制胃癌细胞增殖的促进作用

目的:研究体外香菇多糖(Lentinan)对多药耐药基因表达的影响和促进顺铂抑制胃癌细胞增殖的作用.方法:分别用顺铂(Cisplatin)、香菇多糖和顺铂联合香菇多糖处理SGC-7901胃癌细胞,将其分为4组:对照组(Con组),香菇多糖组(Len组),顺铂组(Cis组)和顺铂联合香菇多糖组(L+C)组.应用RT-PCR检测多药耐药基因MDR1、MRP1和LRP基因mRNA表达;应用CCK-8试剂盒检测Con组和药物处理组前后的胃癌细胞增殖状态.结果:正常SGC-7901胃癌细胞中多药耐药基因MDR1、MRP1和LRP均表达mRNA,香菇多糖能显著降低多药耐药基因表达而对细胞增殖无明显影响;顺铂明显增加多药耐药基因表达,同时抑制细胞增殖(P<0.05);香菇多糖联合顺铂作用后,MDR1和MRP1基因表达完全受到抑制,LRP表达显著降低,细胞增殖速度明显低于Con组、Len组和Cis组,差异具有统计学意义(10d:0.54vs1.90,1.88,0.92,均P<0.05).结论:香菇多糖联合顺铂后因抑制多药耐药基因表达而显著增强顺铂的抑制细胞增殖作用.     

小细胞肺癌多药耐药机制研究进展

小细胞肺癌对多种化疗药物产生的多药耐药特性是造成化疗失败的主要原因.小细胞肺癌产生多药耐药的机制涉及膜糖蛋白介导的药物外排机制、细胞内pH、ROS、HIF-1、GSH的改变,以及DNA损伤修复机制异常等.系统性分析小细胞肺癌多药耐药产生的机制,将有助于指导临床用药及为耐药逆转的新药研发提供理论依据.     

Preliminary study on mechanism of drug resistance in human colon cancer cell line HCTV2000

AIM: To investigate the mechanisms of multidrug resistance in human colon cancer cell line HCT_V2000. METHODS: P-glycoprotein (P-GP) was detected by immunocytochemical staining. Expression and amplification of mdr1 gene were detected by nucleic acid hybridization. Cytotoxicity was measured by MTT method. RESULTS: HCT_V2000 cells showed a positive response to monoclonal antibodies against P-GP encoded by the mdr1 gene. Overexpression of mdr1 mRNA was found, but no evidence of mdr1 gene amplification or rearrangement was suggested. Verapamil increased the accumulation of ³H-VCR inside the cells and partially reversed drug resistance to VCR. CONCLUSION: Overexpression of mdr1 mRNA is one of the important mechanisms of drug resistance in HCT_V2000 cell line; however, there may be some other mechanisms which are also involved in this.     

In vitro investigations on induction and reversal of cisplatin resistance in a rat ovarian tumor cell line

Summary Resistance of a rat ovarian tumor cell line (O-342/DDP) to cisplatin was induced in vitro by stepwise increase of cisplatin concentrations. Both chemosensitive parental cells (O-342) and resistant O-342/DDP cells grow in a monolayer and enter log-phase growth about 24 h after seeding (cell population doubling time in log-phase growth is about 24 h). O-342/DDP cells show a 33-fold resistance to cisplatin as compared to 0-342 cells (ID₅₀=33 M in O-342/DDP vs 1 M in O-342 cells). The intracellular total glutathione (GSH+GSSG) of O-342/DDP cells was twice as high as that of O-342 cells (3.04 vs 1.37 nmol/10⁶ cells), while the intracellular GSSG was increased by 26% in O-342/DDP cells compared to O-342 cells. DNA interstrand crosslinks were found to be 8.5 times higher in O-342 cells than in O-342/DDP cells (204 vs 24 rad equiv.), following cisplatin treatment. DNA single-strand breaks were approximately doubled in the sensitive line as compared to the resistant line following exposure to cisplatin. Chromosome analysis uncovered a change in the karyotype of O-342/DDP cell as compared to O-342 cells. In the sensitive line hyperploid (3n) clones, in the resistant line near-diploid clones predominated. Both DL-buthionine-(S,R)-sulfoximine and 3-aminobenzamide were able to sensitize the resistant line towards cisplatin. Thus, the present results suggest that mechanisms for cisplatin resistance in this tumor line apparently are multifactorial, and include a higher intracellular GSH content and an increased repair activity.Abbreviations GSH glutathione - GSSG glutathione disulfide - BSO DL-buthionine-(S,R)-sulfoximine Dedicated to Professor Dr. D. Schmähl on the occasion of his 65th birthday     

EGFR-TKIs在非小细胞肺癌治疗中耐药机制及应对策略的研究进展

肺癌是全球范围内肿瘤性疾病死亡的主要原因,且治疗方法有限.表皮生长因子受体-酪氨酸激酶抑制剂在非小细胞肺癌的靶向治疗中具有重要意义,但患者使用后容易出现耐药,影响治疗效果.本文就非小细胞肺癌靶向治疗的耐药机制的研究现状及应对策略作一综述.     

Inhibition of protein-kinase-C — dependent cell proliferation of human lung cancer cell lines by the dihydropyridine dexniguldipine

The dihydropyridine, dexniguldipine hydrochloride (B859-35), has shown therapeutic activity in experimentally induced neuroendocrine hamster lung tumors and demonstrated antiproliferative effects in a mammary cancer cell line via inhibition of Ca²⁺ calmodulin. Studies in NIH 3T3 fibroblasts have provided evidence that dexniguldipine may also inhibit protein kinase C (PCK). In this study, we have tested the hypothesis that dexniguldipine may inhibit the proliferation of lung cancer cells in response to autocrine or exogenous activation of PKC. Using a panel of human lung cancer cell lines, we show that dexniguldipine is a potent inhibitor of mitogenic signal transduction pathways dependent on PKC activation in several small-cell and non-small-cell lung cancer cell lines while it failed to inhibit cyclic-AMP-dependent cell proliferation.Supported in part by grant R55 CA 51211-01A1 with the National Cancer Institute