原发上皮性卵巢癌组织中多药耐药基因表达及其临床意义

目的探讨多药耐药（ＭＤＲ１）基因表达与原发性上皮性卵巢癌（ＰＥＯＣ）耐药的关系及其临床意义。方法应用半定量逆转录—聚合酶链反应（ＲＴ－ＰＣＲ）技术,对３７例ＰＥＯＣ、１０例正常卵巢上皮组织和２０例正常女性外周血单核细胞（ＰＢＭＣ）新鲜标本ＭＤＲ１基因表达进行了检测;并用四氮唑蓝（ＭＴＴ）法,检测了这３７例ＰＥＯＣ患者的原代癌细胞对临床常用抗癌药物的敏感性。结果ＰＥＯＣ标本中ＭＤＲ１基因阳性表达率为３０％,而正常卵巢上皮组织和ＰＢＭＣ则无ＭＤＲ１基因表达;ＭＤＲ１基因表达与ＰＥＯＣ临床期别及组织学类型无关,而与组织学级别有关,且有随级别升高而升高的趋势;ＭＤＲ１基因阳性表达组与阴性组比较,ＤＡＣＴ、ＡＤＭ、Ｅ－ＡＤＭ、ＶＣＲ和ＶＰ１６对ＰＥＯＣ细胞抑制率差异有显著性,其余则无显著性;ＭＤＲ１基因表达与无进展生存期有关,而与总体生存期无关。结论ＭＤＲ１基因表达与ＰＥＯＣ耐药有关,是抗ＭＤＲ相关药物的主要原因;ＭＤＲ１基因表达的检测,对临床用药有指导意义,对临床疗效判断和患者预后预测有一定价值。     

RASSF1A基因在卵巢癌组织及腹腔冲洗液中的表达及意义

目的探讨RASSF1A(RAS 相关区域家族1A) 基因转录表达与卵巢癌发生、发展的关系.方法 2003年7月至2005年2月蚌埠医学院附属医院采用逆转录-聚合酶链反应(RT-PCR) 的方法检测42 例卵巢恶性肿瘤患者癌组织、腹腔冲洗液(或腹水)中RASSF1A mRNA 的表达水平,以及11例正常卵巢和19例卵巢良性肿瘤组织中RASSF1A mRNA的表达.结果 RASSF1A mRNA 在所有正常和卵巢良性肿瘤组织中均表达,而在卵巢恶性肿瘤组织中存在较高的表达缺失率(57.1%).RASSF1A mRNA的表达缺失率与卵巢恶性肿瘤的临床分期有关,Ⅲ～Ⅳ期卵巢恶性肿瘤组织中RASSF1A mRNA的表达缺失率显著高于Ⅰ～Ⅱ期,淋巴结转移阳性者RASSF1A mRNA 表达缺失率(73.9%) 明显高于淋巴结转移阴性者(36.8%) (P< 0.05) ;腹腔冲洗液(或腹水)细胞学阳性者,其RASSF1A mRNA的表达缺失率显著升高.结论 RASSF1A 转录表达缺失可能参与调控卵巢癌的发生、发展过程,并且提示RASSF1A表达可作为判断卵巢癌预后的一个指标.     

卵巢癌多药耐药相关药物的体外敏感性与MDR1基因表达

目的探讨卵巢癌多药耐药(MDR)相关药物的体外敏感性与MDR1基因表达的关系,为卵巢癌合理化疗提供理论依据.方法采用四甲基偶氮唑蓝(MTT)比色法体外药物敏感(药敏)试验及逆转录--聚合酶链反应(RT-PCR)技术,检测37例卵巢癌对MDR相关药物的敏感性,及其MDR1基因表达情况.结果卵巢癌MDR1基因表达阳性率为30%,与MDR相关药物耐药有关,与非MDR相关药物耐药无关.结论MDR1基因表达是卵巢癌抗MDR相关药物的主要原因.     

卵巢癌化疗耐药基因检测的研究进展

卵巢癌发生发展是细胞内外多种基因结构和功能异常改变的结果.化疗是目前卵巢癌重要的治疗手段之一,化疗耐药是卵巢癌患者治疗失败和死亡的主要原因,克服化疗耐药是提高远期疗效的关键.近年对卵巢癌化疗的耐药基因如多药耐药基因、DNA修复相关基因、紫杉醇耐药相关基因、p53基因、顺铂耐药性相关基因等进行了广泛研究,阐述有关卵巢肿瘤化疗耐药基因的最新研究动态.     

卵巢癌中微小RNA-210的表达及临床意义

目的：检测上皮性卵巢癌组织中微小RNA-210（miR-210）的表达水平,初步探讨miR-210在卵巢癌发病中的作用。方法：采用多聚腺苷酸加尾实时荧光定量反转录聚合酶链反应［ｐｏｌｙ（Ａ）－RT-qPCR]检测上皮性卵巢癌、良性上皮性卵巢肿瘤及正常卵巢组织标本中miR-210的表达。结果：miR-210在卵巢癌组中表达低于良性卵巢肿瘤组和正常卵巢组,3组间差异有统计学意义（F=893.213,P=0.000）;且随着卵巢癌组织临床分期及组织分化的进展,miR-210表达有下降的趋势。结论：miR-210在上皮性卵巢癌组织中低表达,在卵巢癌发生发展中发挥重要的抑癌基因作用,且与卵巢癌分期、组织分级及淋巴结转移密切相关。     

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。     

卵巢癌中PARP抑制剂耐药机制的研究进展

卵巢癌在妇科恶性肿瘤中预后最差、病死率最高.在全球范围内,女性恶性肿瘤中,卵巢癌发病率是第7位,死亡率是第8位,5年生存率低于45％[.虽然早期患者预后较好,但大多数患者在诊断时已属晚期.其中上皮性卵巢癌约占80％,手术减瘤、含铂化疗(如卡铂、紫杉醇等)是公认的一线治疗方案.但这些治疗长期效果并不满意,超过70％的晚期...     

应用组织芯片技术分析卵巢癌组织中Bit1基因的表达

目的:研究B it1基因在正常卵巢组织和不同病理分级的卵巢癌组织中的表达。方法:应用组织芯片技术,通过免疫组织化学方法检测82例卵巢癌标本和78例正常卵巢组织标本中B it1基因的表达。结果:B it1基因在卵巢癌和正常卵巢组织中的弱阳性及阳性表达率分别为100%和33.3%,差异有统计学意义(P<0.01);B it1基因在不同病理分级的卵巢癌组织中的阳性表达率分别为:Ⅰ级,53.1%;Ⅱ级,28.1%;Ⅲ级,7.1%。统计学分析表明,B it1基因在Ⅰ级与Ⅱ级或Ⅲ级的卵巢癌组织中的表达存在显著的统计学差异(P=0.042,0.003),而在Ⅱ级和Ⅲ级中的表达则无统计学差异(P=0.143)。结论:B it1基因在正常卵巢组织中的表达明显低于卵巢癌组织,而且卵巢癌组织中B it1基因的表达与病理分级密切相关。     

原发上皮性卵巢癌组织中多药耐药基因表达及其临床 …

目的 探讨多药耐药（ＭＤＲ）基因表达与原妇性上皮性卵巢癌（ＰＥＯＣ）耐药的关系及其临床意义。方法 应用半定量逆转录－聚全酶链反应（ＲＴ－ＰＣＲ）技术,对３７例ＰＥＯＣ、１０例正常卵巢上皮组织和２０例正常女性外因单核细胞（ＰＢＭＣ）新鲜标本ＭＤＲ１基因表达进行了检测;并用四氮唑蓝（ＭＴＴ）法,检测了这３７例ＰＥＯＣ患者的原代癌细胞对临床常用抗癌药物的敏感性。结果 ＰＥＯＣ标本中ＭＯＲ１基因阳怀表达率     

miRNA表达谱改变与卵巢癌转移潜力相关性的研究

目的:探讨卵巢癌转移的相关机制,检测有不同侵袭力卵巢癌细胞的miR-NA差异表达谱。方法:应用包含924条成熟miRNA探针的哺乳动物miRNA芯片V3.0杂交检测具有不同侵袭力的人卵巢乳头状腺癌SKOV3.ip1细胞与其母系SKOV3细胞miRNA表达谱的差异,并与cDNA芯片基因检测结果进行比对分析。结果:miRNA芯片检测到39个2倍以上差异表达miRNA,对比SKOV3细胞,SKOV3.ip1细胞中有11个miRNA表达上调和28个miRNA表达下调,其中包括了hsa-miR-200a/b、-10b、-34a、-224、-148a等已知与侵袭转移相关的重要miRNA。比对cDNA芯片检测结果,提供了IG-FBP1、VEGFB、NME1等重要侵袭相关基因可能调控miRNA的机制。结论:卵巢癌侵袭转移过程中,多个miRNA参与其中,担当重要的调控作用。miRNA有望成为卵巢癌转移的标记物和治疗的靶点。     

KDM5B基因在卵巢癌中的表达及其对卵巢癌耐药能力的影响

目的:检测组蛋白去甲基化转移酶KDM5B(JARID1B/PLU-1)在正常卵巢、卵巢癌组织及细胞株中的表达,以及其对卵巢癌细胞株耐药能力的影响。方法:RT-q PCR法检测KDM5B在36例卵巢癌、15例正常卵巢、6例术后化疗耐药复发患者组织及卵巢上皮细胞株IOSE和5株卵巢癌细胞中的表达情况;构建p MSCVpuro-KDM5B过表达质粒和p GIPZ-sh1、p GIPZ-sh2干扰质粒,并筛选得到稳定过表达的SKOV3和稳定干扰的Caov3细胞株,RT-q PCR和Western blot法鉴定调控效果;用浓度梯度递增的顺铂处理SKOV3过表达和Caov3干扰细胞株,CCK-8法检测顺铂半抑制浓度(IC50)变化。结果:RTq PCR结果示,正常卵巢组织中KDM5B相对表达量(1.950±0.3003)低于卵巢癌组织(4.924±0.2989),差异有统计学意义(t=5.909,P0.0001)。KDM5B表达量与卵巢癌患者的FIGO分期、病理分级、转移和耐药发生明显相关,与患者年龄、CA125水平不相关。6例复发患者中,5例KDM5B表达明显升高(P0.001)。KDM5B的mRNA和蛋白水平在卵巢上皮性细胞株IOSE中最低,在卵巢癌细胞株SKOV3、HO-8910、ES-2、A2780、Caov3中表达逐渐增加。RT-q PCR结果示,SKOV3过表达组的KDM5B表达量为对照组的27.4倍,Caov3干扰sh1和sh2组分别为对照组的0.38和0.41倍,Western blot也显示过表达和干扰有效。用浓度梯度递增的顺铂处理Caov3细胞株,KDM5B表达量随顺铂浓度增加而逐渐升高;检测SKOV3细胞株KDM5B过表达组IC50为3.666(95%CI为3.067~4.382)μg/ml高于对照组[1.676(95%CI为1.553~1.810)μg/ml],Caov3细胞株干扰sh1组和sh2组分别为3.359(95%CI为2.875~3.925)μg/ml、2.881(95%CI为2.49~3.324)μg/ml,均低于对照组[6.972(95%CI为6.182~7.864)]。结论:组蛋白去甲基化转移酶KDM5B在卵巢癌中表达升高,并且具有促进卵巢癌细胞株耐药的作用。     

间隙连接蛋白43的磷酸化调节在卵巢上皮性癌化疗耐药中的作用

目的 通过检测卵巢上皮性癌(卵巢癌)间隙连接蛋白43(Cx43)、非磷酸化Cx43及蛋白激酶C(PKC)的表达,探讨Cx43的磷酸化调节在卵巢癌化疗耐药中的作用.方法 采用免疫组化法检测29例化疗敏感(化疗敏感组)和28例化疗耐药(化疗耐药组)卵巢癌患者癌组织中以及PKC抑制剂--星孢菌素处理后的卵巢癌顺铂耐药细胞株SKOV3/DDP细胞中Cx43、非磷酸化Cx43及PKC蛋白的表达,并采用三磷酸腺苷-生物荧光肿瘤药敏实验(ATP-TCA)检测SKOV3/DDP细胞对化疗药物的敏感性.结果 (1)免疫组化法检测显示,化疗耐药组Cx43和非磷酸化Cx43蛋白的阳性表达率(分别为54%和14%)明显低于化疗敏感组(分别为83%和59%,P<0.05);化疗耐药组PKC蛋白的阳性表达率明显高于化疗敏感组(分别为64%和31%,P<0.05).卵巢癌组织中,PKC蛋白的阳性表达率与Cx43、非磷酸化Cx43蛋白的阳性表达率呈明显负相关关系(r=-0.626和-0.714,P<0.05).(2)免疫组化法检测显示,星孢菌素处理后SKOV3/DDP细胞中PKC蛋白的表达强度减弱,Cx43及非磷酸化Cx43蛋白的表达强度增强,随着星孢菌素处理时间的延长,Cx43蛋白的表达强度进一步增强.(3)ATP-TCA检测显示,体外培养的SKOV3/DDP细胞对紫杉醇和顺铂均耐药;紫杉醇和顺铂分别联合星孢菌素处理后细胞敏感度增加,其中联合低浓度(1×10-8 mol/L)星孢菌素者为中度敏感.联合高浓度(1×10-7 moL/L)星孢菌素者为高度敏感.结论 PKC通过对Cx43的磷酸化作用导致Cx43蛋白的表达下降,降低卵巢癌对化疗药物的敏感性,这种效应可以被星孢菌素逆转.     

Identification of genetic alterations related to chemoresistance in epithelial ovarian cancer

OBJECTIVE: After the completion of primary chemotherapy, the majority of advanced ovarian cancer patients have persistent, chemoresistant disease. Comparative genomic hybridization (CGH) has been used to study genetic alterations that may be responsible for chemoresistance in ovarian cancer. CGH is a useful, genomewide screen but resolution is limited to 5-10 Mb. Recently, quantitative microsatellite analysis (QuMA), a TaqMan-based quantitative PCR technology, has been used for higher resolution of DNA copy number abnormalities. Our goal is to identify specific chromosomal aberrations correlated with platinum resistance. METHODS: Snap-frozen ovarian tissue samples taken from 22 patients with ovarian cancer between 1994 and 1998 were analyzed. Patients whose ovarian cancer actually demonstrated growth during platinum-combination treatment or no objective evidence of regression following four to six cycles of therapy were considered to have clinically defined platinum-resistant disease. QuMA was carried out at the following loci using the ABI Prism 7700 (TaqMan) instrument with a microsatellite repeat probe: D3S1553, D3S1617, D5S464, D5S630, D6S1581, D6S446, D8S557, D19S208, D20S196, DXS1068. Fisher's exact test, exact logistic regression, and the Cochran-Armitage trend test were used. Because of multiple hypothesis testing, the P values were adjusted with the Bonferroni procedure to limit the familywise error rate to at most 5%. RESULTS: Of the 22 patients, 12 (54.5%) were platinum-sensitive and 10 (45.5%) were platinum-resistant. When comparing sensitive and resistant patients, no statistically significant difference was noted among stage, grade, histology, and age (P = 0.1292, P = 1.0000, P = 1.0000, P = 1.0000, respectively). In the QuMA analysis, 10 of the 14 (71.4%) patients who had a low copy number of D6S1581 were platinum-resistant, while none of the patients with a normal or high copy number of D6S1581 were platinum-resistant. This was statistically significant when the marker data were treated as either a continuous or a categorical variable (P = 0.0410 and P = 0.0170, respectively). No other loci correlated significantly with platinum resistance. CONCLUSIONS: D6S1581 was the only genetic marker, of those examined, significantly related to chemoresistance. Patients with a loss of D6S1581 are more likely to be platinum-resistant. Identification of genetic alterations associated with platinum resistance detected by QuMA may contribute to a better understanding of clinical behavior and chemotherapy treatment options for patients.     

应用组织芯片技术研究卵巢癌组织中缺氧诱导因子1α与血管生成的关系

目的　探讨缺氧诱导因子 1α(hypoxiainduciblefactor 1α ,HIF 1α)在卵巢癌组织中的表达及其与血管生成的关系。方法　联合应用组织芯片和原位杂交、免疫组化技术 ,检测 2 95份卵巢上皮性肿瘤 (其中卵巢癌 2 38份、交界性卵巢肿瘤 19份、良性卵巢肿瘤 38份 )及 13份正常卵巢组织中HIF 1αmRNA和血管内皮生长因子 (VEGF)的表达情况 ,以CD3 4 单克隆抗体标记血管内皮细胞来检测微血管密度 (MVD)。结果　卵巢癌、交界性卵巢肿瘤和良性卵巢肿瘤、正常卵巢组织中HIF 1αmRNA的阳性表达率分别为 81 9%、4 2 1%、13 2 %和 0。交界性卵巢肿瘤和卵巢癌组织中HIF 1αmRNA的表达高于正常卵巢和良性卵巢肿瘤 ,卵巢癌高于交界性肿瘤 ,差异均有统计学意义 (P <0 0 1)。卵巢癌组织中HIF 1αmRNA的表达与手术病理分期和病理类型无关 (P >0 0 5 ) ,而与病理分级呈正相关 (r=0 2 4 6 ,P <0 0 1)。卵巢癌组织中HIF 1αmRNA表达与VEGF表达 (r =0 2 0 6 ,P =0 0 1)和MVD计数 (r =0 4 5 1,P <0 0 1)均呈显著正相关。结论　卵巢癌组织过度表达HIF 1αmRNA ,并通过诱导VEGF促进肿瘤的新生血管形成。     

Screening and detection of ovarian cancer

According to the National Cancer Institute, ovarian cancer is the sixth most common cancer in women and the leading cause of death from gynecologic malignancies. Most often the disease is advanced before symptoms are evident. It is estimated that only 15% to 30% of women in advanced stages will survive 5 years, whereas, of women in stage I at the time of diagnosis, 95% are likely to be alive in 5 years, and most are cured following surgery. Current screening techniques recommended for women with known strong risk factors include combination transvaginal sonography with cancer antigen (CA-125). Transvaginal sonography and serum CA-125 have limited diagnostic predictability. A new early detection method that uses proteomic technology will soon be available. The OvaCheck test, as researchers purport, is a highly specific and sensitive early detection method for ovarian cancer in women with strong risk factors. The Food and Drug Administration has yet to approve nationwide marketing of OvaCheck for early detection, because trials are not yet complete. Anticipated commercial availability is scheduled for early 2005.     

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。     

Differences of chemoresistance assay between invasive micropapillary/low-grade serous ovarian carcinoma and high-grade serous ovarian carcinoma

The objective of this study was to evaluate the pattern of chemoresistance in invasive micropapillary/low-grade serous ovarian carcinoma (invasive MPSC/LGSC) and high-grade serous ovarian carcinoma (HGSC) according to extreme drug resistance (EDR) assay testing. Surgical specimens of 44 recurrent ovarian cancer patients harvested at the time of cytoreductive surgery between August 1999 and February 2004 were identified retrospectively from the tumor registry database. Thirteen patients (29.5%) had recurrent invasive MPSC/LGSC and 31 (70.5%) patients had recurrent HGSC. Eight drugs were evaluated; EDR assay results were compared between LGSC and HGSC groups using Fisher exact tests and exact logistic regression models. Compared to HGSC, invasive MPSC/LGSC were more likely to manifest EDR to the drugs paclitaxel (69% vs 14%, P < 0.001), carboplatin (50% vs 17%, P= 0.05), cyclophosphamide (40% vs 23%, P= 0.41), gemcitabine (36% vs 19%, P= 0.40), and cisplatin (33% vs 28%, P= 0.72) and less likely to be resistant to etoposide (0% vs 44%, P= 0.007), doxorubicin (8% vs 45%, P= 0.03), and topotecan (8% vs 21%, P= 0.65). Exact logistic regression estimates revealed that invasive MPSC/LGSC patients had significantly increased probabilities of paclitaxel resistance odds ratio (OR) = 12.5 (95% CI: 2.3-100.0), P= 0.001 and carboplatin resistance OR = 4.8 (95% CI: 0.9-25.0), P= 0.07, while the HGSC cases were more likely to be resistant to etoposide OR = 12.1 (95% CI: 1.7-infinity), P=0.009 and doxorubicin OR = 8.6 (95% CI: 1.0-413.7), P= 0.05. In this retrospective analysis, patients with recurrent invasive MPSC/LGSC were more likely to manifest EDR to standard chemotherapy agents (platinum and paclitaxel). These observations may help to guide chemotherapeutic decision making in these patients if confirmed in a large-scale study.