腹腔热化疗中卵巢癌细胞对顺铂敏感性影响的体外研究

目的:探讨腹腔热疗对卵巢癌细胞顺铂敏感株的化疗增敏作用及对顺铂耐药株的逆转耐药作用,并分析其作用机制,为腹腔热疗增加顺铂化疗敏感性及逆转卵巢癌顺铂耐药提供实验依据。方法:MTT 法研究常温及41℃不同作用时间下温热联合顺铂对卵巢癌细胞敏感株SKOV 3、卵巢癌细胞顺铂耐药株SKOV 3/DDP 的生长抑制作用。Western bloting法检测常温及41℃不同作用时间下ERCC1 基因表达水平。结果:41℃下作用60、90min时对SKOV 3 细胞生长的抑制作用大,差异有显著性,P<0.001。热处理对SKOV 3/DDP 细胞生长有抑制作用,不同时间差异有统计学意义。热疗联合顺铂在常温或41℃下,SKOV 3 细胞对顺铂的敏感性不同,差异有统计学意义（P<0.001）。 且41℃作用90min时敏感性最大（P=0.002）,对顺铂的敏感性提高79.885% 。热处理不同时间,SKOV 3/DDP 细胞对顺铂的敏感性增加,差异有显著性,P<0.001。41℃90min SKOV3/DDP 细胞对顺铂的敏感性提高37.129% 。在SKOV 3 细胞中,ERCC1 表达水平较SKOV 3/DDP 细胞降低,F=32.175,P<0.001。热疗联合顺铂与常温下相比,SKOV 3、SKOV 3/DDP 细胞中ERCC1 基因表达水平差异无统计学意义,F=0.962,P=0.443。结论:热疗对卵巢癌细胞SKOV 3 及其顺铂耐药亚株SKOV 3/DDP 细胞有生长抑制作用;热疗联合顺铂可以增加SKOV 3 细胞对顺铂的敏感性,可以部分逆转SKOV 3/DDP细胞对顺铂的耐药性,且最佳作用时间为41℃90min;ERCC1 表达增加与卵巢癌顺铂耐药有关,但热疗联合顺铂对ERCC1 基因表达水平无明显影响,热疗可能通过其他途径增加肿瘤细胞对顺铂的敏感性。      

RNAi抑制HIF-1α基因逆转卵巢癌多药耐药的实验研究

目的 探讨靶向缺氧诱导因子1α(hypoxia inducible factor-1α,HIF-1α)载体表达的小干扰RNA(small interfering RNA,siRNA)逆转卵巢癌细胞多药耐药的可行性.方法 构建靶向HIF-1α短发夹状siRNA 基因表达载体,脂质体介导转染人卵巢癌COC1/DDP细胞.Western blot法检测HIF-1α蛋白和P-糖蛋白(P-glycoprotein,P-gp)的表达;实时定量PCR(real time PCR)检测HIF-1α和多耐药基因1(mdr-1)mRNA的表达;MTT法检测COC1/DDP细胞对化疗药物顺铂的抗性.结果 转染HIF-1α短发夹状siRNA真核表达载体的COC1/DDP细胞,低氧条件下HIF-1α蛋白及mRNA表达水平下降,同时mdr-1基因的mRNA及其编码的P-gp蛋白水平也明显下降,对顺铂的药物敏感性增加.结论 HIF-1α短发夹状siRNA真核表达载体可有效地抑制卵巢癌COC1/DDP细胞HIF-1α和mdr-1基因的表达,逆转卵巢癌细胞的多药耐药.     

RNA干扰ERCC1基因表达对肺腺癌细胞A549/DDP顺铂耐药的影响

背景与目的核苷酸切除修复机制可修复顺铂(DDP)造成的DNA损伤,作为其中的关键酶之一,切除修复交叉互补基因1(excision repair cross-complementing gene 1,ERCC1)的表达可能与DDP耐药有关.本研究旨在探讨小干扰RNA沉默ERCC1基因表达对肺腺癌耐药细胞A549/DDP药物敏感性的影响.方法将体外设计合成针对ERCC1的小分子RNA(siRNA)转染A549/DDP细胞,RT-PCR检测ERCC1 mRNA水平;Western blot检测ERCC1蛋白表达的变化;MTT检测RNA干扰后细胞药物敏感性改变.结果 ERCC1 RNAi干扰后ERCC1 mRNA表达指数降低,明显低于对照组,干扰后ERCC1 mRNA表达抑制率为90.4%.转染ERCC1 siRNA降低TERCC1蛋白的表达水平.MTT显示分别用2μg/mL、4μg/mL、8μg/mL、16μg/mL、32μg/mL顺铂处理细胞48 h,转染ERCC1 siRNA细胞组较对照组敏感性明显增高,干扰前A549/DDP细胞IC50为12.49μg/mL,干扰后A549/DDP细胞IC50下降为9.27 μg/mL.结论 ERCC1siRNA干扰可以降低ERCC1的表达水平,并可提高A549/DDP细胞对顺铂的敏感性.     

LncRNA ITGA9-AS1在卵巢癌中的表达及与顺铂化疗敏感性的关系

目的:观察长链非编码RNA（LncRNA） ITGA9-AS1在卵巢癌中的表达水平,探究其与顺铂化疗敏感性的关系。方法:选取2017年01月至2019年01月本院收治的64例卵巢癌患者,卵巢摘除手术中采集卵巢癌组织,另收集因卵巢囊肿需作手术切除但已证实无瘤细胞的正常卵巢组织标本,采用实时定量PCR（RT-qPCR）检测卵巢癌及正常卵巢组织、顺铂（DDP）耐药卵巢癌细胞SKOV3/DDP、卵巢癌细胞（OVCAR5、OVCAR8、SKOV3）及永生化卵巢上皮细胞（IOSE29、IOSE80）中LncRNA ITGA9-AS1表达水平。采用慢病毒介导LncRNA ITGA9-AS1转染并筛选稳定细胞株,MTT法检测过表达LncRNA ITGA9-AS1对SKOV3、SKOV3/DDP增殖的影响及对不同浓度（0、1、2、4、8、16 mg/L）DDP的敏感性。结果:与正常卵巢组织比较,卵巢癌组织中LncRNA ITGA9-AS1表达水平降低（P＜0.05）。与IOSE29、IOSE80细胞比较,OVCAR5、OVCAR8、SKOV3及SKOV3/DDP细胞中LncRNA ITGA9-AS1表达水平显著降低（P＜0.05）;与SKOV3细胞比较,SKOV3/DDP细胞中LncRNA ITGA9-AS1表达水平显著降低（P＜0.05）。与空白对照组及慢病毒对照组比较,慢病毒过表达组SKOV3、SKOV3/DDP细胞中LncRNA ITGA9-AS1表达水平升高（P＜0.05）,增殖率降低（P＜0.05）,细胞存活率呈DDP浓度依赖降低（P＜0.05）。结论:LncRNA ITGA9-AS1在卵巢癌组织及癌细胞中低表达,过表达LncRNA ITGA9-AS1可增加卵巢癌SKOV3及卵巢癌顺铂耐药细胞SKOV3/DDP的顺铂化疗敏感性。     

Neu RNAi对卵巢癌细胞生长、凋亡及药物敏感性的作用

目的探讨nenRNAi对卵巢癌耐药细胞株SKOV3／DDP细胞生长、凋亡及药物敏感性的作用,寻找下调基因表达并逆转化疗耐药的方法,为卵巢癌的基因治疗探索新途径。方法应用含有u6启动子的pSilencer 1．0-U6表达载体构建neu基因的小干扰RNA（siRNA）表达载体（pSilencer—neu）,用脂质体方法将pSilencer—neu转染卵巢癌顺铂耐药细胞株SKOV3／DDP,另设未转染组及转染pSilencer—control组为对照。显微镜下观察转染前后细胞的变化;用RT—PCR及Western Blot检测neu基因mRNA及蛋白的表达;MTT法检测细胞生长情况;流式细胞仪检测细胞凋亡及周期的变化;加入顺铂后检测RNAi对SKOV3／DDP顺铂药物敏感性的影响。结果neuRNAi可明显下调卵巢癌耐药细胞株SKOV3／DDP中neu的基因表达;使其细胞生长受到抑制,生长速度减慢,生长曲线低平;细胞凋亡增加且细胞周期发生变化,G1／G0期细胞增多,S期细胞则减少;顺铂耐药实验显示,转染RNAi的SKOV3／DDP细胞IC50明显下调,细胞凋亡率显著增加。结论应用RNAi可以部分阻抑neu基因在卵巢癌顺铂耐药细胞株SKOV3／DDP中的表达,使SKOV3／DDP细胞生长减慢、凋亡增加,而且对顺铂的敏感性增强。     

p38MAPK对卵巢癌顺铂耐药作用及机制的探讨

目的:研究p38MAPK在卵巢上皮癌顺铂化疗耐药中的作用,并探讨其作用机制.方法:蛋白质印迹法检测顺铂对卵巢癌中p38MAPK的激活情况 ;MTT法检测p38MAPK抑制剂SB203580处理后及p38 shRNA干扰后细胞顺铂耐药指数的变化 ;实时定量PCR技术检测SB203580处理后及p38 shRNA干扰后,耐药蛋白ERCC1、MDR、LRP、GST-π及凋亡蛋白Caspase-3、Survivin等mRNA的表达变化.结果:在一定时间浓度梯度下,顺铂可诱导卵巢癌顺铂敏感株COC1及耐药株COC1/DDP细胞内p38MAPK的激活,但耐药株的激活程度弱于敏感株 ;p38MAPK抑制剂SB203580及p38 shRNA干扰可增加卵巢癌细胞株的耐药指数(t=4.610,P=0.041 ;t=11.621,P=0.000) ;SB203580及p38 shRNA干扰后Survivin mRNA(t=5.152,P=0.007 ;t=6.008,P=0.004)、ERCC1 mRNA(t=13.742,P=0.005;t=11.621,P=0.000)及LRPmRNA(t=8.173,P=0.001 ;t=16.815,P=0.000)的表达明显上调.结论:p38MAPK受抑制可能导致卵巢上皮性癌顺铂耐药的产生,其机制可能是通过上调Survivin、ERCC1及LRP的mRNA表达来实现.     

细胞色素c 介导的半胱天冬氨酸蛋白酶-3活性改变与人卵巢癌顺铂耐药的关系

目的：探讨人卵巢癌顺铂耐药细胞株A2780／DDP、COC1／DDP中抗凋亡基因bcl-XL、细胞色素c的表达和半胱天冬氨酰蛋白酶－3（caspase-3)活性对人卵巢癌顺铂耐药的影响。方法：采用逆转录聚合酶链反应（RT－PCR）和Western blot检测人卵巢癌顺铂敏感细胞株A2780、COC1和顺铂耐药株A2780／DDP、COC1／DDP中bcl-XL的表达,以及顺铂作用后细胞色素c的含量和caspase-3活性的变化,并应用流式细胞仪测定顺铂作用后A2780、COC1、A2780／DDP、COC1／DDP细胞的凋亡率。结果：bcl-XL在A2780/DDP、COC1／DDP细胞中的表达明显高于A2780、COC1细胞;顺铂作用后,细胞色素c在A2780／DDP、COC1／DDP细胞中的表达明显减少,caspase-3活性和凋亡率也较A2780和COC1细胞明显降低（P＜0．05）。结论：人卵巢部细胞对顺铂产生耐药可能与细胞内bcl-XL过度表达、细胞色素c释放受抑制和caspase-3活性下降有关。     

凋亡相关蛋白的表达及Caspase-3活性改变与人卵巢癌细胞顺铂耐药的关系

背景与目的:以顺铂为基础的化疗是卵巢癌治疗的重要组成部分,对顺铂的耐药是卵巢癌治疗失败的原因之一。本研究探讨人卵巢癌顺铂耐药细胞株中凋亡相关蛋白表达及caspase-3活性与人卵巢癌细胞顺铂耐药的关系。方法:采用Westernblot法分析人卵巢癌顺铂敏感细胞株COC1和顺铂耐药株COC1/DDP中凋亡相关蛋白bcl-2、bcl-xL、bax、bcl-xS的表达,caspase-3活性和其底物多聚ADP核糖聚合酶(PARP)的变化,并应用流式细胞仪检查不同浓度顺铂作用COC1和COC1/DDP细胞后的细胞凋亡率。结果:在COC1/DDP细胞中,bcl-2和bcl-xL的表达明显高于COC1细胞,bax的表达无明显改变,bcl-xS在COC1和COC1/DDP细胞中均无表达。用顺铂处理后,COC1/DDP细胞中caspase-3活性、PARP裂解片段和凋亡率较COC1细胞均明显降低(P<0.05),且呈浓度依赖性。结论:人卵巢癌细胞对顺铂产生耐药可能与肿瘤细胞内凋亡抑制蛋白过度表达、caspase-3活性下降有关,而与凋亡诱导蛋白的表达无关。     

RNA干扰靶向沉默EGFR基因对卵巢癌耐药细胞凋亡影响的实验观察

目的:探讨RNA干扰表皮生长因子受体(EGFR)基因对卵巢癌顺铂耐药细胞株SKOV3/DDP凋亡的影响。方法:利用基因重组技术构建携带EGFR小发夹干扰RNA(pEGFR-shRNA)的重组质粒表达载体,脂质体法转染SKOV3/DDP细胞。实验分组:对照组(不进行干扰)、S1组(转染非特异性质粒)、S2组(转染特异性质粒)。用逆转录聚合酶链反应(RT-PCR)和免疫细胞化学法(ICC)检测转染后细胞内EGFRmRNA和蛋白的表达,流式细胞仪(FCM)分析细胞周期和凋亡率。结果:与对照组相比,S2组细胞EGFRmRNA的表达明显受抑制,抑制率达57%(P<0.01)。蛋白质的表达水平显著降低(P<0.01)。流式细胞仪检测结果显示,顺铂作用24h后,特异性转染组细胞周期分布发生明显改变,G0/G1期细胞比例增多,而S期细胞比例减少,凋亡率显著升高(P<0.01)。结论:RNA干扰EGFR基因通过抑制SKOV3/DDP细胞中EGFR基因的表达,恢复细胞对顺铂的敏感性。     

鼻咽癌组织ERCC1的表达及其临床意义

目的:探讨核苷酸切除修复交叉互补基因(ERCC1)在鼻咽癌(NPC)组织中表达及其与顺铂(DDP)化疗疗效的关系。方法:82例经鼻咽部肿物活检确诊为鼻咽低分化鳞癌患者接受DDP为基础化疗,应用免疫组化法检测鼻咽部癌组织ERCC1基因蛋白表达,并设34例NPC癌旁上皮组织为对照组。结果:在NPC癌旁上皮组织中ERCC1的阳性率为88.2%,明显高于NPC组织中ER-CC1阳性率(63.4%),P<0.05;ERCC1基因蛋白表达阳性患者化疗有效率为36.5%,ERCC1阴性患者有效率为63.3%,其差异有统计学意义,P<0.05。ERCC1表达与NPC患者年龄有关(P<0.05),与性别、T分期、N分期及M分期无关,P>0.05。结论:ERCC1异常表达是NPC DDP化疗耐药的一个重要因素,检测ERCC1基因有助于预测NPC患者对顺铂化疗敏感性。     

Aberrant methylation of breast and ovarian cancer susceptibility gene 1 in chemosensitive human ovarian cancer cells does not involve the phosphatidylinositol 3′‐kinase–Akt pathway

Methylation is an important silencing mechanism of breast and ovarian cancer susceptibility gene 1 (BRCA1) expression in sporadic ovarian cancer. However, the role of BRCA1 methylation in chemotherapy in sporadic ovarian cancer and the related pathways have not been understood completely. This study has determined the roles of BRCA1 hypermethylation in chemotherapy of sporadic ovarian cancer and its related signaling pathways. We used bisulfite sequencing, real‐time polymerase chain reaction, and western blotting to check the methylation state and expression levels of BRCA1 of the following cell lines: platinum‐sensitive human ovarian cancer cell line COC1, platinum‐resistant cell line COC1/DDP, SKOV‐3, and 5‐Aza‐dC treated COC1. The cisplatin sensitivity of ovarian cancer cells was examined by MTS (methyl‐thiazol tetrazolium) assay. Tumorigenicity in vivo and DDP‐based chemosensitivity were compared among the above cells. Phosphatidylinositol 3′‐kinase (PI3K)–Akt pathway activation in ovarian cancer cells was studied by western blotting. The frequency of BRCA1 methylation in the COC1 cell line was higher than in COC1/DDP and SKOV‐3 cell lines, whereas the mRNA and protein expression of BRCA1 were lower than in the COC1/DDP and SKOV‐3 cell lines. DNA demethylation decreased the chemosensitivity of COC1 cells and partially increased the expression levels of BRCA1. The activation of the PI3K‐Akt pathway was low in ovarian cancer cells. Our results indicate that hypermethylation of BRCA1 might play an important role in the chemosensitivity of ovarian cancer, and that the PI3K–Akt pathway is not involved in this response. (Cancer Sci 2010)     

lncRNA FAL1通过调控MAPK通路对卵巢癌细胞化疗耐药性的影响及其机制

目的 观察lncRNA FAL1在卵巢癌细胞及其耐药细胞株中的表达差异,探索下调lncRNA FAL1对细胞化疗耐药性的影响及机制.方法 qRT-PCR法检测SKOV3和COC1细胞及其耐药细胞株FAL1基因表达水平,转染FAL1-siRNA下调FAL1基因,MTT检测细胞增殖能力,Transwell法检测细胞侵袭能力...     

Effect of lung resistance-related protein on the resistance to cisplatin in human ovarian cancer cell lines

The mechanisms of drug-resistance in human ovarian cancer cells have not been entirely clarified. The purpose of this study was to investigate whether LRP is involved in the resistance of ovarian cancer cell lines to cisplatin and its molecular mechanism. Human ovarian cisplatin-resistant cancer cell lines (A2780/DDP and COC1/DDP) and their parental cisplatin-sensitive cell lines (A2780 and COC1), alone or transfected with antisense LRP-specific oligonucleotides (ODN) or sense ODN, were treated with cisplatin to induce differentiation. Expression of LRP was examined by RT-PCR and Western blot analysis. The sensitivities of cells to cisplatin were assessed using sulforhodamine B (SRB) assay and flow cytometry, and the accumulation and efflux of cisplatin in the cells and isolated nuclei were examined by high performance liquid chromatographic (HPLC) assay. The expressions of LRP in A2780/DDP and COC1/DDP cells were higher than those in A2780 and COC1 cells and conferred resistance to cisplatin. Transfection of LRP AsODN into A2780/DDP and COC1/DDP cells down-regulated LRP expression and reversed the resistance phenotype. Levels of cisplatin accumulating in cells were increased by LRP-specific AsODN and anti-LRP monoclonal antibody. Isolated nuclei from A2780 and COC1 cells or A2780/DDP and COC1/DDP cells incubated with anti-LRP antibody contained more cisplatin than the nuclei of A2780/DDP and COC1/DDP cells not treated with anti-LRP antibody. Efflux of cisplatin was greater from the nuclei of A2780/DDP and COC1/DDP cells than those of A2780 and COC1 cells, and was inhibited by anti-LRP monoclonal antibody. Thus, LRP was involved in the resistance of ovarian cancer cells to cisplatin and has an important role in the transport of cisplatin both in exocytotic vesicles and between the nucleus and cytoplasm.     

Tanshinone IIA acts via p38 MAPK to induce apoptosis and the down-regulation of ERCC1 and lung-resistance protein in cisplatin-resistant ovarian cancer cells

Tanshinone IIA is known to induce apoptosis in several types of cancer cells. However, little is known about its activity in chemoresistant cells. The aim of this study was to investigate the anticancer properties of tanshinone IIA in cisplatin-resistant human ovarian cancer COC1/DDP cells in vitro. We used a variety of methods to measure cell viability, the resistance index (RI) of cisplatin, cellular apoptosis, p38 mitogen-activated protein kinase (MAPK) expression and phosphorylation, and the mRNA expression of several genes implicated in drug resistance including survivin, Caspase-3, excision repair cross-complementing gene 1 (ERCC1), multidrug resistance (MDR), lung resistance protein (LRP) and glutathione-S-transferase-π (GST-π). We found that tanshinone IIA time- and dose-dependently inhibited the proliferation of COC1/DDP cells and caused significant apoptosis. Western blotting revealed that tanshinone IIA also increased phospho-p38 MAPK in a time- and dose-dependent manner. After treatment by tanshinone IIA for 48 h, the RI of cisplatin and the mRNA expression of survivin, ERCC1 and LRP were all significantly decreased. Furthermore, blockade of p38 signal transduction decreased apoptotic cell rates and dramatically elevated the mRNA expression of the survivin, ERCC1 and LRP genes. We therefore conclude that tanshinone IIA induces apoptosis and reduces cisplatin resistance in COC1/DDP cells and thus causes significant growth inhibitory effects. This mechanism appears to involve p38-mediated downregulation of survivin, ERCC1 and LRP mRNA expression.