盐酸拓扑替康、紫杉醇及顺铂对人卵巢癌细胞株OC_3体外作用的实验研究

目的 :研究国产盐酸拓扑替康 (TPTHCh)、紫杉醇 (PIX)与顺铂 (cDDP)对人卵巢癌细胞株OC3 的生长抑制作用。方法 :采用MTT法观察上述药物单药应用、双药联合及 3药联合使用对卵巢癌细胞株OC3 生长的影响 ,评价药物联合应用的效果。结果 :3种药物单药应用、双药联合及 3药联合应用均对卵巢癌细胞株OC3 有显著的抑制作用 ,以卵巢癌的标准化疗方案PIX加cDDP为对照组 ,3药联合应用的抑制效果显著增强 ,且三药全量及半量联合应用效果差异无显著性。结论 :PIX、TPTHCh及cDDP联合应用具有高效协同抑制卵巢癌细胞株OC3 的作用 ,可为临床难治性和复发性卵巢癌患者的化疗及小剂量化疗提供参考     

应用抑制性消减杂交筛选卵巢癌紫杉醇耐药差异表达基因

目的:应用抑制性消减杂交技术研究人卵巢癌紫杉醇耐药细胞株OC3/Tax300与其亲本细胞OC3(敏感株)之间基因表达的差异,筛选耐药相关基因,探讨基因表达差异与卵巢癌耐药的相关性。方法:以卵巢癌紫杉醇耐药细胞株OC3/Tax300 mRNA为检测子(tester),以其亲本细胞OC3(敏感株)mRNA为驱赶子(driver),构建cDNA消减文库。随机挑取文库克隆测序,所得结果在GenBank中做同源性比较分析。结果:成功构建了卵巢癌紫杉醇耐药细胞株的特异性cDNA消减文库。从文库中选取阳性克隆,其中76个测序成功,再随机选取36个序列与GenBank数据库进行初步比对,8个可能为新基因,1个为蛋白序列,另27个来源于16个已知基因,这些差异表达基因主要涉及细胞代谢、信号转导、细胞骨架、凋亡、蛋白翻译合成、发育等相关基因。结论:部分基因表达差异与卵巢癌紫杉醇耐药有关。     

CircASH2L调节miR-128-3p/MKNK2轴对卵巢癌细胞紫杉醇耐药性的影响

目的:研究环状RNA缺失-小同源异形-2样蛋白(CircASH2L)调节miR-128-3p/MAP激酶相互作用丝氨酸/苏氨酸激酶2(MKNK2)轴对卵巢癌细胞紫杉醇耐药性的影响。方法:实时荧光定量PCR检测人卵巢癌细胞株SKOV3和人卵巢癌紫杉醇耐药细胞株SKOV3-TR30中miR-128-3p与CircASH2L、MKNK2表达。体外培养SKOV3-TR30细胞，随机分为对照组、紫杉醇组、紫杉醇+阴性对照组、紫杉醇+CircASH2L敲低组、紫杉醇+CircASH2L敲低+miR-128-3p inhibitor组，分组处理后，检测各组细胞增殖率、凋亡率，miR-128-3p及CircASH2L、MKNK2、多重耐药1(MDR1)、多药耐药相关蛋白5(MRP5)mRNA表达，MKNK2及P-gp、MRP5蛋白表达。双荧光素酶报告基因实验检测SKOV3-TR30中CircASH2L对miR-128-3p的靶向调节及miR-128-3p对MKNK2的靶向调节。结果:与SKOV3细胞相比，SKOV3-TR30细胞中CircASH2L与MKNK2 mRNA表达升高，miR-128-3p表...     

Caspase-3活性改变与人卵巢癌细胞系COC1/DDP耐药的关系

目的 探讨人卵巢癌顺铂耐药细胞株COC1/DDP中抗凋亡蛋白Bcl-XL、Bcl-2、细胞色素C的表达及半胱天冬氨酸蛋白酶-3(caspase-3)活性与人卵巢癌顺铂耐药的关系。方法:采用RT-PCR和Western Blot分析人卵巢癌顺铂敏感细胞株COC1和顺铂耐药株COC1/DDP中Bcl-XL、Bcl-2、细胞色素C的表达和caspase-3的活性。并用流式细胞术测定顺铂作用后COC1和COC1/DDP细胞株的凋亡率。结果:在COC1/DDP细胞中,Bcl-XL和Bcl-2的表达明显高于COC1细胞;顺铂作用后,在COC1/DDP细胞株中细胞色素C的表达明显减少,caspase-3活性明显降低(P<0.05);其凋亡率也明显低于COC1细胞株(P<0.05)。结论:人卵巢癌细胞株COC1/DDP对顺铂产生耐药可能与细胞内Bcl-XL、Bcl-2过度表达抑制了线粒体细胞色素C的释放及caspase-3活性有关。     

紫杉醇耐药人卵巢癌细胞株中线粒体DNA基因突变的研究

目的:研究紫杉醇耐药的人卵巢癌细胞株SKOV3/Tax和OC3/Tax中线粒体DNA(mtDNA)突变。方法:提取细胞DNA,分析mtDNA突变,比较SKOV3/Tax和OC3/Tax与亲本敏感细胞SKOV3及OC3之间mtDNA突变数量和突变类型的差异。结果:紫杉醇耐药及敏感细胞mtDNA的突变热点依次是ND5基因、Cytb基因、ND4基因、CoⅡ基因及ND1基因;与敏感细胞株相比,耐药细胞株中mtDNA测序中发现更多的基因突变频率,主要表现为A→C,A→G,T→C,A→T,缺失A、C,插入T、C等,其中以缺失A最为明显。结论:人卵巢癌细胞株OC3及SKOV3中mtDNA编码区的ND5基因、Cytb基因、ND4基因和CoⅡ基因是具有高度突变性的区域,其与卵巢癌的发生、发展有重要关系。耐药细胞株中的突变明显高于敏感细胞株,推测突变的增加可能与卵巢癌耐药有关。     

MDR1及MDR3基因沉默对紫杉醇耐药细胞株A2780/Taxol凋亡能力的影响

目的:探讨采用RNA干扰技术沉默多药耐药基因MDR1及MDR3后对卵巢癌紫杉醇耐药细胞株A2780/Taxol凋亡能力的影响.方法:将MDR1和MDR3的siRNA重组质粒转染A2780/Taxol,通过MTT法、流式细胞术、免疫荧光法及Western blot方法分别检测细胞对紫杉醇的敏感性、细胞周期和凋亡率、细胞中caspase-3的活性及MDR编码的P-gp蛋白的表达的变化.结果:转染SiRNAs重组质粒后,A2780/Taxol对紫杉醇敏感性的相对逆转率分别为64.3%及53.9%,细胞凋亡率由1.59%分别增加到9.43%和8.66%,细胞中caspase-3活性增强,P-gp蛋白含量明显减少,与对照组比差异有统计学意义(P＜0.05).结论:MDR1和MDR3的siRNA质粒可通过诱导卵巢癌紫杉醇耐药细胞A2780/Taxol凋亡,恢复其对紫杉醇的敏感性.     

Beclin1基因对SKOV3/DDP细胞生长增殖与侵袭转移的影响

目的:观察自噬基因Beclin1对卵巢癌顺铂耐药细胞株SKOV3/DDP增殖与侵袭转移的影响,并探讨其可能作用机制。方法:脂质体包裹重组构建的pcDNA3.1-Beclin1和pSUPER-Beclin1质粒,分别体外转染SKOV3/DDP细胞并筛选稳定表达株。采用实时荧光定量RT-PCR检测不同细胞株中Beclin1 mRNA表达,Western blot法检测Beclin1、VEGF及MMP-9蛋白水平变化,MTT法测定细胞生长增殖情况,Transwell小室观察对细胞侵袭和转移能力的影响。结果:pcDNA3.1-Beclin1转染的SKOV3/DDP细胞株中,Beclin1表达上调、VEGF与MMP-9表达被抑制、细胞增殖受到抑制。Beclin1过度表达可显著降低SKOV3/DDP细胞侵袭与转移的能力,而干扰Beclin1表达可增强细胞的侵袭转移能力。结论:Beclin1能减弱卵巢癌SKOV3/DDP细胞的侵袭和转移能力,其机制可能与抑制肿瘤细胞增殖、降低VEGF和MMP-9的表达有关。     

抗凋亡基因NF-κB和Survivin对卵巢癌细胞株耐药性的诊断价值

目的:探讨凋亡抑制基因NF-κB、Survivin对卵巢癌细胞株耐药性的诊断价值。方法:采用卵巢上皮性癌细胞株A2780和由此细胞株诱导的耐顺铂细胞株AD4,分别加入顺铂(DDP)和紫杉醇(Taxol),并设空白对照,测定两组细胞中凋亡抑制基因核因子(NF-κB)、Survivin和多耐药基因(Mdr1)mRNA的表达;同时检测各组细胞的凋亡率。结果:经DDP处理后,Mdr1在两株细胞中均无明显表达,与G3PDH的比值均低于0.1;而Taxol作用后,Mdr1在两株细胞中表达均增加,与G3PDH的比值分别为1.98和1.86,但差异无显著性。NF-κB在各组细胞中均有表达,各组之间差异无显著性。Survivin有较强表达,与G3PDH的比值为4.5,耐药细胞株的比值为6.9。在两细胞株的不同实验中,Mdr1的表达与凋亡率无明显相关性,而Survivin则是负相关(P<0.05,r=-0.89)。结论:在卵巢癌及卵巢癌耐顺铂细胞株中,紫杉醇可诱导Mdr1高表达,而DDP对Mdr1表达无影响;NF-κB主要是通过活性增加,促进Mdr1表达来诱发卵巢癌细胞耐药;Survivin的表达与细胞凋亡率呈负相关,提示Survivin可作为肿瘤细胞耐药检测指标之一。     

mdr1启动子调控双自杀基因对卵巢癌SKOV3耐药细胞的靶向杀伤作用

目的观察多药耐药基因1(mdr1)启动子调控腺病毒介导的胞嘧啶脱氨酶∷尿嘧啶磷酸核糖转移酶(CD∷UPP)融合基因系统对人卵巢癌紫杉醇耐药细胞的靶向杀伤作用。方法2004年10月至2005年5月于华中科技大学附属协和医院,将重组腺病毒(Admdr1-CD∷UPP)转染卵巢癌紫杉醇耐药细胞(SKOV3/Taxol)及非耐药细胞(SKOV3),荧光显微镜下观察转染效率,用逆转录-聚合酶链反应(RT-PCR)检测目的基因CD∷UPP的表达,并给予不同浓度的5-氟胞嘧啶(5-FC),用四甲基偶氮唑蓝(MTT)法检测二组的细胞存活率及旁观者效应。结果重组腺病毒对SKOV3/Taxol的转染率随腺病毒滴度的递增而增加,感染复数(MOI)为50时,感染率约100%;CD∷UPP基因仅在SKOV3/Taxol中稳定表达;5-FC对耐药细胞转基因组的杀伤作用显著高于非耐药细胞转基因组,前者表现出明显的旁观者效应。结论mdr1启动子调控的CD∷UPP融合自杀基因系统对人卵巢癌紫杉醇耐药细胞有靶向杀伤作用。     

Wnt通路抑制因子甲基化与卵巢癌耐药的相关性

目的探讨Wnt通路抑制因子甲基化与卵巢癌耐药的相关性。方法2008年在四川大学华西第二医院采用甲基化特异性PCR(MSP)、RT-PCR检测卵巢癌细胞株SKOV3、OVCAR-8(紫杉醇耐药),A2780及A2780/D(顺铂耐药)中,Wnt通路抑制因子SFRP-1、2、4、5,WIF-1、DKK-3的甲基化状态、mRNA水平;四甲基偶氮唑蓝(MTT)法比较DNA甲基转移酶抑制剂5-氮-2-脱氧胞苷(5-Aza-CdR)处理后,OVCAR-8细胞耐药逆转的情况,并检测WIF-1基因甲基化状态、mRNA表达水平的改变。结果WIF-1基因在OVCAR-8细胞中为高甲基化表达抑制,5-Aza-CdR可使其去甲基化后重新表达,并部分逆转OVCAR-8细胞的紫杉醇耐药。结论WIF-1基因的高甲基化状态与卵巢癌耐药细胞株OVCAR-8的紫杉醇耐药相关,去甲基化后可部分逆转耐药,这为逆转卵巢癌耐药提供了新的研究方向。     

粒细胞集落刺激因子对人卵巢癌细胞株生长的影响

了解粒细胞集落刺激因子对人卵巢癌细胞株生长的影响,方法采用ＸＴＴ比色法,检测Ｇ－ＣＳＦ对卵巢癌细胞株ＳＫＯＶ３、３ＡＯ及ＧＡＯＶ３生长的影响;对有影响的细胞株进行流式细胞仪检测和Ｇ－ＣＳＦ受体的检测,观察外源性Ｇ－ＣＳＦ对该细胞株的裸鼠皮下瘤生长的影响。结论Ｇ－ＣＳＦ对所检测的卵巢癌细胞株生长无显著影响。     

A novel amplification at 17q21-23 in ovarian cancer cell lines detected by comparative genomic hybridization

OBJECTIVE: Little is known about the molecular mechanisms involved in the pathogenesis and/or progression of ovarian cancer (OC). To investigate the genomic imbalances and identify the cancer-related genes associated with this tumor, we applied comparative genomic hybridization (CGH) in OC cell lines. METHODS: Chromosomal aberrations among 17 OC cell lines were analyzed with CGH. Since novel chromosomal regions, including 17q21-23, were identified, we examined the involvement of two candidate genes, PS6K and ZNF147, mapped on this chromosomal region. We examined the status of amplification and expression by fluorescence in situ hybridization as well as by Southern blot analysis and by Northern blot analysis on two candidate genes, respectively. RESULTS: All lines displayed numerous chromosomal imbalances; the most frequent losses were observed on 18q22-23 (29.4%), 13q22-34 (23.5%), 9p (17.6%), 4p11-14 (17.6%), and 11p14-15 (17.6%). The most common gains were noted at 20q12-13 (47.1%), 8q23-24 (35.2%), 5p15 (23.5%), 7q32-36 (23.5%), and 20p (23.5%). High-level gains (HLGs) were detected at 20q12-13 (four cell lines), 8q24 (two cell lines), 12p11-12 (two cell lines), and 17q21-23 (two cell lines). PS6K and ZNF147 genes were amplified in two cell lines exhibiting HLGs at 17q21-23, but not overexpressed. CONCLUSIONS: Our CGH data indicate that OCs have various DNA copy number changes. Among these frequent changes, 17q21-23 may harbor another tumor-associated gene(s) responsible for OC carcinogenesis.     

Characterization of 10 vulvar carcinoma cell lines by karyotyping, comparative genomic hybridization and flow cytometry

OBJECTIVE AND METHODS: Ten vulvar squamous cell carcinoma cell lines established at the University of Michigan (UM-SCV-1A, -1B, -2, -3, -4, -6, -7) and at the University of Turku (UT-SCV-1, -2, -3) were characterized by G-banding karyotyping, comparative genomic hybridization (CGH), and deoxyribonucleic acid (DNA) flow cytometry. RESULTS: All cell lines had hyperdiploid DNA content as measured by flow cytometry. The DNA index (DI) remained relatively stable through different passages in 9 of 10 cases. DIs of UM-SCV-3 and UT-SCV-2 were near-diploid, as were the corresponding karyotypes. The 10 SCVs showed remarkable genetic similarities with respect to consistent chromosome rearrangements. Loss of 3p, noted in 8/10 SCVs, was narrowed to the smallest common region at 3p11-3p13. Loss of 8pter-p11 was observed in 10/10 cell lines. Loss of 11qter-q23 was present in UM-SCV-1 and -2, and in all four recently karyotyped SCVs. Other consistent losses include Xpter-p11 in 6/10, and 18qter-q11 in 7/10 cell lines. Common gains included gain of 8q in 8/10 and 3q in 6/10. Consistent copy number imbalances were confirmed by CGH; concerning loss of 3p, in 63%, to loss of 8p in 70%, to gain of 3q in 83%, and to gain of 8q in 75% of the cell lines. CONCLUSIONS: CGH and karyotyping showed concordance in defining copy number imbalances, thus supporting the accuracy of CGH to detect chromosome imbalances in tumors that cannot be karyotyped.     

Estrogen receptor-positive human epithelial ovarian carcinoma cells respond to the antitumor drug suramin with increased proliferation: possible insight into ER and epidermal growth factor signaling interactions in ovarian cancer

OBJECTIVES: Development of targeted therapeutics for ovarian cancer requires a basic understanding of ovarian epithelial carcinoma cell biology. The role of estrogen and epidermal growth factor (EGF) in control of cell growth was investigated in a panel of ovarian carcinoma lines. METHODS: EGF receptor (EGFR) was detected by flow cytometry and estrogen receptor (ER) by Northern blot. Western blotting and [(3)H]thymidine incorporation were used to determine receptor activation and the effects of ligand exposure and growth factor antagonists, including the antineoplastic agent, suramin, on cell growth. RESULTS: Only one cell line, OV266, expressed ER and responded to beta-estradiol with increases in DNA synthesis and cell proliferation that could be blocked by the pure antiestrogen ICI 182,780. All cell lines possessed functional EGFR as measured by flow cytometry and phosphorylation of the receptor and mitogen-activated protein kinase after EGF treatment, but only two cell lines (OV177 and OV266) proliferated in response to exogenous EGF. Suramin had limited effectiveness in inhibiting growth in four of five cell lines and had a striking dose-dependent stimulatory effect on OV266 cell growth. The proliferative response to suramin could be inhibited with EGFR antagonists. CONCLUSION: Cultured epithelial ovarian carcinoma cell lines express EGFR (5/5) and can express ER (1/5). Differential growth responses to EGF were observed despite uniform receptor and MAPK activation. Unexpectedly, the antineoplastic agent suramin increased growth of ER positive ovarian carcinoma cells in an EGFR-dependent manner. These studies provide insight into the complex interactions of these systems in control of ovarian cancer cell growth.     

Comparative study of primary and recurrent ovarian serous carcinomas: comparative genomic hybridization analysis with a potential application for prognosis

OBJECTIVES: The purpose of this study is to comparatively characterize genomic imbalances in primary and recurrent ovarian serous carcinomas and to identify genomic alterations that may be used as a marker for prognosis. METHODS: Twenty ovarian serous carcinomas were studied by comparative genomic hybridization (CGH). RESULTS: Genomic alterations were found in all of the tumors. The most common regions involving gain of DNA copy numbers are 1q41q44, 8q22q24, 19p12q13.1, 20q12q13, 3q26q29, 12p12p13, 2p22p25, 7p14p21, 5p15.2p15.3, and 17q22q25. The most common regions with loss of DNA copy numbers are Xp11.2q13, 4q31q35, Xp21p22.3, 18q22q23, 13q22q31, 9p22p24, and 16q22q24. High-level gains were detected at chromosomal regions of 1q41q44, 2p22p25, 3q26q29, and 19p12q13.1. Comparative analysis of primary and recurrent tumors showed that gains of 2p22p25, 19p12q13.1, and 20q12q13 and loss of 5q14q22 were more common in the recurrent high-grade tumors. About 85% of the tumors showed increases in DNA copy numbers in the regions (2p and 8q) harboring the myc family gene. Patients with tumor containing fewer than seven chromosomal aberrations showed longer survival time. CONCLUSION: The myc oncogene family may play a role in the pathogenesis of ovarian serous carcinomas. Our study suggests that tumors with gains of 2p22p25, 19p12q13.1, and 20q12q13 and loss of 5q14q22 may be at high risk for recurrence. Furthermore, the patients' survival time inversely correlates with the numbers of chromosomal alterations found in their tumors. CGH analysis may have a clinical application in predicting prognosis and risk of recurrence in patients with ovarian serous carcinomas.     

卵巢癌SKOV3细胞培养液对脐血树突细胞前体细胞亚群的影响

目的:研究卵巢癌细胞培养上清能否诱导树突细胞前体细胞亚群比例发生变化。方法:以脐血来源的CD34+干细胞通过细胞因子FLT3-L(100ng/ml)、SCF(50ng/ml)的诱导在体外扩增分化为DC前体细胞(pre-DC)。培养并收集卵巢癌细胞株SKOV3上清,经透析法浓缩。将上清培养的pre-DC,经不同浓度上清(0%、25%、50%、75%、100%、150%)作用后,用流式细胞仪(flowcytometry,FCM)检测pre-DC表面抗原CD123、CD11c、HLA-DR、CD80、CD86的表达。结果:上清促进CD11c和HLA-DR抗原的表达,不促进CD123、CD80、CD86抗原的表达。结论:卵巢癌细胞株上清影响两类DC前体细胞亚群的比例变化,可能通过此途径参与形成腹腔免疫缺陷。     

SV40 T基因诱导的人卵巢癌细胞永生化

目的 建立人卵巢癌永生化细胞系，为卵巢癌体外研究提供实验模型。方法 利用基因重组技术构建猴肾病毒40（SV40）T抗原基因的高效真核表达载体pcD2-SV40。将其转染10例原代培养的早期传代细胞，经G418筛选，抗性克隆扩大培养，建立永生化细胞系。免疫组化检测细胞的上皮起源，用PCR、RT－PCR和Southern Blot方法鉴定SV40 T基因在转染细胞中的表达。观察所建立的永生化细胞系的染色体核型。结果 10例转染细胞中，6例筛选出抗性克隆。角蛋白免疫组化证实3例为上皮起源。其中2例上皮起源细胞得以扩大培养成系，长期稳定传代，分别命名为BUPH：OVSC－2和BUPH：OVCA－3。经鉴定两种细胞系中存在SV40 T在基因水平及转录水平的表达。其染色体核型均符合人体恶性细胞特征。结论 利用SV40T抗原基因进行人卵巢癌原代细胞的 生化是一种可行的、可重复的建系方法，可提高卵巢癌细胞成系机率。     

Expression P53 protein and chromosome aberrations in benign tumors and ovarian carcinoma

OBJECTIVES: The epithelial ovarian tumors arise from the single layer of epithelial cells that line the ovarian surface or from underlying inclusion cysts. One from many theories of oncogenesis has been proposed that benign, borderline and invasive tumors represent sequential stages in the growth of an ovarian cancer, and p53 tumor suppressor gene mutation is the most common molecular genetic alteration. Because locus of p53 gene is located on 17 chromosome we performed the cytogenic analysis of tumor tissues. DESIGN: Analysis of mutated p53 protein expression and chromosomal aberrations in tissues of benign tumors and epithelial ovarian carcinomas. MATERIAL AND METHODS: Tissue samples from 19 women with benign and 17 women with invasive epithelial ovarian cancers were obtained for the study at the time of surgical procedures. From among benign tumors 12 were serous cystadenomas, 5 were endometrial cysts and 2 adult teratomas. All cases of invasive epithelial ovarian carcinomas were histologically recognized as serous adenocarcinomas, and were staged on I (9 cases), II (5 cases) and III (3 cases), according to the FIGO guidelines for ovarian cancers. Frozen tissue samples were stained immunohistochemically for mutated p53 protein using commercial monoclonal antibodies and standard detecting system. The fresh tumor samples were prepared for cytogenic analysis according to standard protocol. RESULTS: Among the all benign ovarian tumors overexpression of mutated form of p53 protein was not seen in any cases, but was noted in 6 cases in I stage and in all cases in II and III stages of advanced ovarian cancers. In 1 case of benign ovarian tumor deletion of X chromosome was observed. Most common numerical changes were observed in ovarian carcinomas e.g. loss of 8, 13, 17, and 22 chromosomes. Other chromosomes were involved at least once in structural rearrangements and several breakpoint cluster regions were identified: 19p13, 11p13-15, 1q21-23, 1p36, 19q13, and 6q21-23. CONCLUSIONS: The mutated form of p53 protein is often expressed in ovarian epithelial carcinoma tissues. This protein are unable to function effectively to inhibit proliferation and accumulate in the cells because is resistant to degradation. In tissues of ovarian carcinomas many chromosomal nondisjunctions (monosomics) and multiple structural rearrangements were observed, what means of genetically nonstable cell lines of neoplasms and probably it heterogenous origin.