紫杉醇耐药卵巢癌细胞SKOV3／TAX基因表达谱的变化

目的比较紫杉醇耐药卵巢癌细胞SKOV3／TAX及敏感细胞SKOV3基因表达谱的差异,筛查获得性紫杉醇卵巢癌耐药的相关基因。方法取对数生长期SKOV3、SKOV3／TAX细胞,抽提细胞总RNA,合成双链eDNA,生物素标记,用高通量Affymetfix人类基因组U133A基因芯片检测其基因谱。结果与SKOV3相比,SKOV3／TAX有111条基因表达发生变化（45条基因表达上调,66条基因表达下调）。结论SKOV3／TAX的部分基因表达发生变化,与卵巢癌化疗耐药有关。     

沉默DCR3表达增加卵巢癌细胞对紫杉醇敏感性的实验研究

目的研究沉默诱骗受体(DCR)3对卵巢癌细胞紫杉醇敏感性的影响。方法以靶向DCR3慢病毒干扰载体感染卵巢癌细胞ES-2,用Realtime PCR和Western印迹检测干扰效果。用不同浓度的紫杉醇处理ES-2细胞,细胞计数试剂盒(CCK-8)测定细胞存活情况,用紫杉醇处理沉默DCR3的ES-2细胞,CCK-8检测细胞增殖,细胞克隆实验检测细胞克隆能力,流式细胞术检测细胞凋亡,Western印迹检测细胞中活化的含半胱氨酸的天冬氨酸蛋白水解酶(C-Caspase)-12、C-Caspase-3、CCAAT/增强子结合蛋白同源蛋白(CHOP)水平。结果靶向DCR3慢病毒干扰载体下调ES-2细胞中DCR3 mRNA和蛋白水平。不同浓度的紫杉醇抑制ES-2细胞增殖,10~(-6) mol/L的紫杉醇处理后的卵巢癌细胞存活率为(53.26±4.15)%,选用10~(-6)mol/L的紫杉醇处理沉默DCR3的ES-2细胞。沉默DCR3后的ES-2细胞增殖能力显著降低,细胞克隆形成能力显著降低,细胞凋亡率显著升高,细胞中C-Caspase-12、C-Caspase-3、CHOP蛋白水平显著升高。紫杉醇处理沉默DCR3后的ES-2细胞增殖和克隆形成能力显著降低,细胞凋亡率显著升高,C-Caspase-12、C-Caspase-3、CHOP蛋白水平显著升高,与未沉默DCR3的ES-2细胞比较,差异有统计学意义(P0.05)。结论沉默DCR3协同紫杉醇抑制卵巢癌细胞增殖,诱导卵巢癌细胞凋亡,增加卵巢癌细胞紫杉醇敏感性。     

HABP1在人卵巢癌细胞中的表达及其对紫杉醇敏感性的影响

目的探讨透明质酸结合蛋白1（HABP1）在5种人卵巢癌细胞系中的表达及其对紫杉醇敏感性的影响。方法采用荧光定量RT-PCR法检测各细胞系中HABP1 mRNA的表达量,激光共聚焦显微镜观察HABP1蛋白的表达及定位,MTT法检测各细胞系对紫杉醇的敏感性。结果 HABP1 mRNA在人卵巢癌细胞系SKOV3、OVCA429和HO-8910PM中的表达水平分别是OVCA420的3.7、2.8和2.1倍（P均〈0.05）,HO-8910LM和OVCA420中HABP1 mRNA表达量差异无统计学意义（P均〉0.05）,HABP1蛋白在五种卵巢癌细胞系中的胞质中均有表达,在SKOV3、OVCA429和HO-8910PM细胞中可见到明显的核周聚集,在HO-8910LM和OVCA420中其核周聚集量明显减少,SKOV3、OVCA429、OVCA420、HO-8910PM和HO-8910LM细胞对紫杉醇的中效浓度分别为0.047、0.221、0.7230、.092、0.672μM,相关分析显示HABP1 mRNA表达水平与卵巢癌细胞对紫杉醇的中效浓度之间的相关系数为-0.888,P〈0.05。结论 HABP1高表达能增加人卵巢癌细胞对紫杉醇的敏感性,HABP1可作为筛选卵巢癌化疗药物的新指标。     

基因芯片技术筛选卵巢癌紫杉醇耐药差异表达基因的研究

目的应用基因芯片技术研究人卵巢癌紫杉醇耐药细胞株OC3/Tax300与其敏感细胞系OC3之间的基因表达谱差异,筛选耐药相关基因.方法分别提取OC3/Tax300与OC3细胞的总RNA并纯化mRNA;将等量的mRNA逆转录,以Cy5和Cy3标记的cDNA做探针,在BiostarH140S基因表达谱芯片上进行杂交.扫描芯片荧光信号图像,用基因图像分析软件对扫描图像进行数字化处理和分析.结果共筛选出显著表达差异基因234种,其中217种基因表达水平下调,17种基因表达上调;下调基因主要为EB病毒编码核蛋白（EBNA-3）、信号蛋白（COP9）,上调的基因主要是酪氨酸激酶（JAK2）、热休克蛋白（HSPs）、还原型辅酶烟胺腺嘌呤二核苷酸（NADH）等.结论本研究筛选出的基因可能为进一步探讨卵巢癌肿瘤细胞耐药机制提供新的途径.     

miR-21转染对卵巢癌细胞株A2780/Taxol紫杉醇耐药性的影响及机制探讨

目的探讨microRNA-21(miR-21)在卵巢癌化疗耐药中的作用及机制。方法荧光定量RT-PCR法检测miR-21在人卵巢癌细胞株A2780及A2780/Taxol的表达水平,合成miR-21的干扰序列,以脂质体为载体转染A2780/Taxol细胞;MTT法检测细胞转染前后对紫杉醇的耐药性;流式细胞仪检测细胞凋亡情况;蛋白质印迹法检测磷酸酶基因(PTEN)、BAX及Bcl-2蛋白。结果 miR-21表达下调后,A2780/Taxol对紫杉醇的IC50值明显降低(P<0.05),细胞凋亡率明显增加(P<0.05),细胞内PTEN、BAX蛋白表达水平增加,Bcl-2表达降低。结论 miR-21在人卵巢癌耐药株中呈高表达,其可能是介导人卵巢癌耐药的主要因素之一,具体机制可能是降低PTEN、BAX及升高Bcl-2来介导肿瘤细胞耐药。     

67kD层粘连蛋白受体在三种卵巢癌细胞株中表达的研究

目的 检测三种不同来源卵巢癌细胞株中67kD层粘连蛋白受体（67k DLN-R）的表达,探讨其在卵巢癌侵袭转移中的作用。方法 体外培养腹水来源的人卵巢癌细胞株HRA、SKOV3及性实体瘤来源的人卵巢腺癌细胞株3AO。以免疫组化和逆转录-聚合酶链反应技术（RT-PCR）检测各细胞株中67kD LN-R蛋白表达及其mRNA水平。结果 三种细胞的胞膜及胞浆中67kD LN-R蛋白均呈阳性表达,但HRA、SKOV3明显强于3AO细胞,差异有显著性（P均〈0．05）;HRA、SKOV3均可见明显的474bp的67kD LN-R特异性条带,其mRNA分别为1．156、1．081;3AO特异性条带极弱,其mRNA为0．358;前两者与后者相比,差异有显著性（P均〈0．05）。结论 67kD LN-R在卵巢癌侵袭转移中发挥重要作用。     

白蛋白结合型紫杉醇治疗晚期耐药复发卵巢癌的疗效及安全性

目的探讨白蛋白结合型紫杉醇(nab-p)治疗晚期耐药复发卵巢癌的疗效及耐受性。方法对2012年1月至2015年12月在吉林大学第一医院接受以nab-p为基础化疗方案的21例复发/难治性晚期卵巢癌患者进行回顾性临床分析。均为经多线药物治疗后复发的患者,给予nab-p 120 mg/m2,第1、8天,联合或不联合卡铂[卡铂曲线下面积(AUC)=4~5,第2天]静脉滴注,21 d为1个疗程。结果 21例患者均可评估疗效,无完全缓解病例,部分缓解13例,疾病稳定5例,疾病进展3例。21例患者接受nab-p为基础化疗方案的有效率为61.9%(13/21),CA125完全缓解率为28.6%(6/21);铂类敏感者的有效率为88.9%(8/9),铂类耐药者的有效率为41.6%(5/12),两者差异无统计学意义(P>0.05)。治疗过程中1例出现了肝功能1级异常,2例出现了2级粒细胞减少不伴发热,2例出现Ⅱ度外周神经病变。未记录到剂量减少或治疗中断。结论 nab-p在先前接受过多线药物治疗的复发/难治性卵巢癌患者中表现出较好的疗效性及耐受性。     

雷公藤内酯醇联合紫杉醇对人卵巢癌耐顺铂细胞株体外活性的影响及机制

目的探讨雷公藤内酯醇（TP）联合紫杉醇对人卵巢癌耐顺铂细胞株（CoC1/DDP）体外活性的影响及其可能机制。方法将对数生长期CoC1/DDP细胞随机分为4组,TP组采用10 ng/ml TP作用于细胞,紫杉醇组采用3.13μg/ml紫杉醇,联合组采用10 ng/ml TP和3.13μg/ml紫杉醇,空白对照组不作任何处理（调零组）,采用MTT法检测各组细胞生长抑制率,透射电子显微镜下观察细胞超微结构变化,流式细胞仪检测细胞周期及细胞凋亡情况。结果联合组生长抑制率明显高于单药组,且呈时间依赖性（P〈0.05）;透射电子显微镜示TP组、紫杉醇组、联合组细胞均呈凋亡改变,联合组细胞凋亡率明显高于单药组,紫杉醇组与联合组细胞周期中G2/M期的比例明显增高（P均〈0.05）。结论 TP联合紫杉醇可协同抑制CoC1/DDP细胞生长,促进其凋亡,其机制可能为使细胞阻滞于G2/M期。     

老年晚期铂类耐药复发性卵巢癌患者白蛋白结合型紫杉醇治疗方案的临床疗效

目的探讨白蛋白结合型紫杉醇对老年晚期铂类耐药复发性卵巢癌患者的临床疗效。方法选取老年晚期铂类耐药复发性卵巢癌患者172例。按照治疗方案分为对照组(n=84)和观察组(n=88),对照组给予普通溶剂型紫杉醇治疗,观察组给予白蛋白结合型紫杉醇治疗,两组均给予6个月化疗。比较两组治疗前后血液肿瘤标志物CA125、CA199及CA724水平,分析近期治疗疗效、不良反应发生率、功能状态KPS、生活质量(QOL)评分及远期疗效患者生存率。结果治疗后,两组CA125、CA199及CA724水平明显下降,观察组明显低于对照组(P<0.05)。观察组近期疗效明显好于对照组,不良事件发生率明显低于对照组,1年整体生存率明显高于对照组(均P<0.05)。观察组功能状态KPS评分及QOL评分明显高于对照组(P<0.05)。结论白蛋白结合型紫杉醇对老年晚期铂类耐药复发性卵巢癌患者近远期临床疗效明显优于紫杉醇治疗,安全性高,患者生存率高。     

卵巢癌紫杉醇耐药细胞株的建立及其耐药机制探讨

目的 为探讨卵巢癌细胞多药耐药的发生机制,寻找克服卵巢癌耐药的方法而建立人卵巢癌紫杉醇(TAX)耐药细胞株SKOV3/TAX.方法 选用卵巢癌细胞株SKOV3,以浓度为300ug/ml的TAX反复大剂量冲击用药,培养8个月后建立TAX耐药细胞株,命名为SKOV3/TAX.该细胞已反复传代3个月以上.结果 SKOV3/TAX细胞耐药性稳定,耐药指数(RI)为10.57.除对紫杉醇耐药外,对环磷酰胺(CTX)、5-氟尿嘧啶(5-Fu)、足叶乙甙(VP-16)有明显的交叉耐药,与亲本细胞相比,细胞倍增时间明显缓慢(P<0.01),G2/M期明显高于亲本细胞,S期比例明显降低(P<0.05),细胞内P糖蛋白(P-gp)表达增加.产生耐药的机制可能与细胞周期改变、细胞凋亡及P-gp的表达增加,导致细胞内TAX聚集量减少有关.结论 SKOV3/TAX细胞耐药性稳定,具有典型的多药耐药表型.     

Enhanced expression of DNA polymerase eta contributes to cisplatin resistance of ovarian cancer stem cells

Cancer stem cells (CSCs) with enhanced tumorigenicity and chemoresistance are believed to be responsible for treatment failure and tumor relapse in ovarian cancer patients. However, it is still unclear how CSCs survive DNA-damaging agent treatment. Here, we report an elevated expression of DNA polymerase η (Pol η) in ovarian CSCs isolated from both ovarian cancer cell lines and primary tumors, indicating that CSCs may have intrinsically enhanced translesion DNA synthesis (TLS). Down-regulation of Pol η blocked cisplatin-induced CSC enrichment both in vitro and in vivo through the enhancement of cisplatin-induced apoptosis in CSCs, indicating that Pol η-mediated TLS contributes to the survival of CSCs upon cisplatin treatment. Furthermore, our data demonstrated a depletion of miR-93 in ovarian CSCs. Enforced expression of miR-93 in ovarian CSCs reduced Pol η expression and increased their sensitivity to cisplatin. Taken together, our data suggest that ovarian CSCs have intrinsically enhanced Pol η-mediated TLS, allowing CSCs to survive cisplatin treatment, leading to tumor relapse. Targeting Pol η, probably through enhancement of miR-93 expression, might be exploited as a strategy to increase the efficacy of cisplatin treatment.Ovarian cancer is the most lethal malignancy of the female reproductive tract with a 5-y survival rate of only 27% in advanced stages (1). The American Cancer Society estimates that in 2014, about 21,980 new cases of ovarian cancer will be diagnosed and 14,270 women will die of ovarian cancer in the United States (1). The mainline treatment of ovarian cancer is cytoreductive surgery followed by platinum (Pt)-based chemotherapy (2). Chemotherapy with Pt is initially effective for most patients. However, the majority eventually becomes refractory to Pt treatment, and around 70% of patients have tumor relapses (3). Poor understanding of the underlying mechanisms of this acquired drug resistance and tumor relapse poses a critical cancer research challenge.cis-diamminedichloroplatinum(II) (cisplatin), the first member of Pt-based chemotherapeutic agents, has been widely used to treat various malignant tumors, including ovarian cancer (4). Mechanistically, cisplatin reacts with DNA bases to cross-link adjacent purines. These cross-links block DNA replication and invoke apoptosis in rapidly dividing cells (5). Thus, the preferential activation of the DNA damage responses, especially the efficient removal of these DNA lesions, or prompt rescue of the replication, will prevent replication fork collapse and promote survival of the cells upon cisplatin treatment, eventually leading to cisplatin resistance. The cisplatin-induced DNA cross-links are primarily removed by the nucleotide excision repair (NER) pathway (6) or bypassed during replication through translesion DNA synthesis (TLS) (7–10). TLS is mediated by specialized DNA polymerases (Pols), which are characterized by low fidelity and an ability to replicate across certain types of damaged sites in template DNA with the assistance of monoubiquitylated proliferating cell nuclear antigen (ub-PCNA) (11). TLS rescues cells from the collapse of the replication fork and thus is believed to contribute to the development of cisplatin resistance (8, 12–17).It has been increasingly evident that heterogeneous ovarian cancers contain a subpopulation of cancer stem cells (CSCs) with enhanced tumorigenicity and chemoresistance. These CSCs are believed to be responsible for treatment failure and tumor relapse. Ovarian CSCs have been successfully isolated, based on the expression of distinctive cell surface markers CD44 and CD117 (18, 19), their ability to efflux the Hoechst 33342 fluorescent dye (Side population, SP) (20), the activity of ALDH (21), and their ability to grow as floating spheres in serum-free medium (19). The CD44⁺CD117⁺ cells, SP cells, ALDH⁺ cells, and spheroid cells isolated from both ovarian cancer cell lines and primary human ovarian tumors fulfill all currently accepted criteria for the existence of a subpopulation of tumor-initiating cells (19, 22, 23). Most importantly, these CSCs also demonstrate increased cisplatin resistance. However, it is still unclear how CSCs survive cisplatin treatment. In this study, we demonstrated that the expression level of TLS Pol η is higher in ovarian CSCs isolated from both cancer cell lines and primary tumors than the bulk cancer cells. Down-regulation of Pol η expression blocked cisplatin-induced enrichment of the CSC population, through facilitating the killing of CSCs by cisplatin. Mechanistic investigation demonstrated that decreased expression of miR-93 in ovarian CSCs contributes, at least partially, to the enhanced expression of Pol η. Taken together, our study suggests that Pol η-mediated TLS could be a target to facilitate the eradication of ovarian CSCs by cisplatin.     

Changes of TIZ expression in epithelial ovarian cancer cells

Objective:To study the change of TIZ expression in epithelial ovarian cancer cells.Methods:HO8910 cells were transinfected with siRNA to inhibit the expression of TIZ.pcDNA3.1-TIZ vectors were combined to increase the TIZ expression level.The cell viabilily,colony forming efficiency and cycle distribution of HO8910,HO8910/NC,HO8910/pcDNA3.1-NC,HO8910/ TIZ-573 and HO8910/pcDNA3.l-TIZ were compared,and the invasion rate,migration rate and adhesion rate between 5 groups of cells were compared.Results:Compared with those of HO8910,HO8910/NC and HO8910/pcDNA3.1-NC,the cell viability,colony forming efficiency and cell cycle distribution of HO8910/ TIZ-573 were increased,while the indexes of HO8910/pcDNA3.1-NC were decreased with statistical significant difference(P0.05).There was no statistical significant difference in the invasion rate,migration rate and adhesion rate between 5 groups of cells(P0.05).Conclusions:The expression of TIZ can inhibit the proliferation of epithelial ovarian cancer cells.     

p53 expression and resistance against paclitaxel in patients with metastatic breast cancer

PURPOSE: Paclitaxel is an important agent in the pharmacological treatment of metastatic breast cancer. Despite its efficacy in selected patients, the majority of patients have a resistance against paclitaxel. The aim of this study was to identify the responding patients and hence prevent the other patients from ineffective treatment. Identifying these patients could spare them an ineffective treatment and could in turn characterize a subgroup of patients with a higher response rate. MATERIAL AND METHODS: Thirty-three patients with metastatic breast cancer received paclitaxel 175 mg/m(2 )either as first- (15 patients) or as second-line (18 patients) treatment. Immunohistochemistry was performed on the blocks of the primary tumors with monoclonal antibodies against p53, HER-2/ neu, P-glycoprotein, Glutathione-S-Transferase-pi, and beta-tubulin II. The expression of those factors was then correlated with the objective response to paclitaxel. RESULTS: Ten of 33 patients had an objective response to treatment. A significant correlation with the objective response was found for the expression of p53. None of the tumors with p53 expression ( n=11) responded to paclitaxel. In contrast, 10 of the 22 patients without p53 expression showed an objective response ( P=0.013). Expression of HER-2/ neu, P-glycoprotein, Glutathione-S-Transferase-pi, and beta-tubulin II did not show a correlation with the response to paclitaxel. CONCLUSION: The immunohistochemical detection of p53 characterizes patients with metastatic breast cancer unlikely to respond to paclitaxel.     

MACC1在卵巢癌组织中表达及对卵巢癌细胞生物学特性的影响

目的探讨结肠癌转移相关基因(MACC)1在卵巢癌组织中的表达及对卵巢癌细胞生物学特性的影响。方法收集卵巢癌组织及对应的癌旁组织,Western印迹检测组织中MACC1表达水平。以卵巢癌细胞A2780为研究对象,细胞转染MACC1小干扰RNA(MACC1 siRNA)、siRNA对照(siRNA control),细胞分为未转染组(只加入转染试剂)、siRNA control组(转染siRNA control)、MACC1 siRNA组(转染MACC1 siRNA)。培养24 h后,Western印迹检测细胞中MACC1、基质金属蛋白酶(MMP)-2、MMP-9表达水平,噻唑蓝(MTT)检测细胞增殖,细胞划痕实验检测细胞迁移能力,Transwell小室检测细胞侵袭能力。结果卵巢癌组织中MACC1表达水平高于癌旁组织。siRNA control组细胞中MACC1、MMP-2、MMP-9表达水平及细胞存活率、迁移率、侵袭细胞数目与未转染组相比均没有差异,MACC1 siRNA组细胞中MACC1、MMP-2、MMP-9表达水平及细胞存活率、迁移率、侵袭细胞数目均明显低于未转染组。结论 MACC1在卵巢癌组织中过度表达。干扰MACC1能够抑制卵巢癌细胞增殖、迁移、侵袭能力,作用机制可能与抑制MMP-2、MMP-9表达有关。     

Chk1基因沉默对人卵巢癌细胞 A2780放疗敏感性的影响

目的：探讨Chk1基因沉默对人卵巢癌细胞A2780放疗敏感性的影响。方法构建Chk1基因的短发夹RNA（shRNA）质粒转染人卵巢癌细胞A2780,同时以转染空载体和未转染组作为对照组,运用RT-PCR、蛋白免疫印迹方法观察转染前后Chk1基因表达的差异;四甲基偶氮唑蓝试验绘制细胞生长曲线;克隆形成试验观察细胞的放射敏感性变化。结果与对照组相比,构建shRNA表达载体可抑制人卵巢癌细胞A2780中Chk1 mRNA转录和蛋白的表达;Chk1基因沉默后细胞生长明显慢于对照组;并通过解除G2/M期阻滞,显著提高肿瘤细胞对放射线的敏感性。结论靶向Chk1的shRNA能有效抑制人卵巢癌细胞A2780的Chk1基因表达,促进细胞凋亡,增强肿瘤对放疗的敏感性。     

全反式维甲酸对卵巢癌SKOV3细胞增殖、分化的影响

目的 探讨全反式维甲酸（ATRA）对卵巢癌SKOV3细胞增殖、分化的影响.方法 以1×10^-6 mol/L的ATRA处理人卵巢癌SKOV3细胞为实验组,细胞不加ATRA处理为对照组.应用MTT法测定细胞增殖抑制率,流式细胞仪测定细胞周期分布,免疫组化法检测鼠抗人分化相关基因1（NDRG1）表达,显微镜观察细胞形态变化.结果 与对照组比较,实验组细胞增殖抑制率、NDRG1表达升高（P均＜0.05）;随着作用时间延长,实验组G/G0期细胞增多、S期细胞减少,显微镜下可见细胞生长差,增殖缓慢,分裂像少见.结论 ATRA有较强的抑制卵巢癌SKOV3细胞增殖和诱导其分化作用.     

Diffuse scleroderma occurring after the use of paclitaxel for ovarian cancer

 A case of diffuse scleroderma in a 56-year-old woman who received paclitaxel for the treatment of a metastatic ovarian cancer is presented. The clinical cutaneous alterations, as well as the capillaroscopic and histological findings, were indistinguishable from those encountered in definite systemic sclerosis (SSc). In contrast to SSc, Raynaud's phenomenon and cutaneous calcinosis were absent and antinuclear antibodies were negative. The temporal relationship between the onset of skin involvement and administration of the drug may indicate an effect of paclitaxel. Received: 11 January 2002 / Accepted: 10 June 2002