RNA干扰技术抑制卵巢癌细胞系SKOV3.ip1细胞内Her-2/neu基因表达的研究

目的 探讨卵巢癌细胞株SKOV3．ip1细胞转染表达短发卡状RNA（shRNA）的质粒后，SKOV3．ip1细胞中Her-2／neu基因表达的变化，和对SKOV3．ip1细胞凋亡的影响。方法 将针对Her-2／neu基因的shRNA表达载体转染SKOV3．ip1细胞，从mRNA水平、蛋白表达水平和细胞凋亡的变化三个方面评价shRNA表达载体对Her-2／neu基因表达的抑制作用。结果shRNA表达载体可以有效的抑制SKOV3．ip1细胞Her-2／neu基因的表达，使Her-2／neu基因的mRNA和蛋白表达量明显下降，细胞凋亡增多。结论靶向Her-2／neu的shRNA表达载体可以有效抑制Her-2／neu基因的表达。     

慢病毒转染自杀基因后联合更音洛韦治疗卵巢上皮性癌的实验研究

目的探讨以抗Ⅰ型粘蛋白单链抗体(anti-MUC1)为靶向的慢病毒高效转染自杀基因后联合更昔洛韦(GCV)对Ⅰ型粘蛋白阳性(MUC1+)卵巢上皮性癌细胞的靶向性基因治疗.方法包装以anti-MUC1为靶向的慢病毒,介导单纯疱疹病毒胸苷激酶(HSV-tk)基因转染卵巢上皮性癌细胞株SKOV3(MUC1+)及正常成纤维细胞GF(MUC1-),荧光显微镜下观察转染效果.聚合酶链反应(PCR)、逆转录聚合酶链反应(RT-PCR)技术鉴定HSV-tk基因在靶细胞(SKOV3及GF)中的整合转录结果.光学显微镜观察GCV作用后细胞形态变化;用四甲基偶氮唑蓝(MTT)法测定GCV的体外杀伤效应.结果荧光显微镜下观察到以anti-MUC1为靶向的慢病毒可有效转染SKOV3细胞,PCR、RT-PCR均证明HSV-tk基因已在靶细胞SKOV3中整合且转录表达;而在GF细胞中未见HSV-tk基因的整合及转录表达.光学显微镜下观察到HSV-tk基因转录后的细胞在GCV作用后出现明显形态变化;MTT法显示GCV对转染HSV-tk基因后的SKOV3细胞有显著杀伤作用,其半数抑制浓度为0.10mg/L.结论以anti-MUC1为靶向的慢病毒可将HSV-tk基因高效、靶向性转染MUC1+卵巢上皮性癌细胞,联合应用GCV可高效、靶向性杀伤MUC1+卵巢上皮性癌细胞.     

~(131)I-Herceptin对卵巢癌细胞的体外作用

目的:研究~(131)I标记抗HER-2/neu单克隆抗体Herceptin(~(131)I-Herceptin)体外对高表达HER-2/neu卵巢癌细胞的生物学作用,探讨将其用于卵巢癌放射免疫治疗的可行性。方法:(1)流式细胞仪检测人卵巢癌SKOV3和HO8910细胞株HER-2/neu的表达。(2)Iodogen法~(131)I标记Herceptin,测定其标记率、放化纯度、稳定性及免疫活性。(3)MTT比色法检测~(131)I-Herceptin对人卵巢癌细胞株SKOV3和HO8910的抑制作用。(4)流式细胞法检测SKOV3在~(131)I-Herceptin作用下细胞周期改变和凋亡情况。结果:(1)SK-OV3细胞株HER-2/neu表达率为92.67%;HO8910表达率为9.59%。(2)~(131)I-Herceptin标记率为89.8%,放化纯度为98.4%,24h后仍大于80%,标记物免疫活性较好。(3)131I-Her-ceptin对SKOV3细胞株的抑制作用明显高于HO8910(P<0.001),且明显高于~(131)I组﹑Her-ceptin组以及~(131)I+Herceptin组(P<0.01)。(4)流式细胞检测~(131)I-Herceptin组SKOV3细胞的凋亡率明显高于~(131)I+Herceptin、Herceptin和~(131)I组(P<0.05),各干预组均检测到细胞周期阻滞,~(131)I-Herceptin组G0-G1期细胞比例明显高于其它三组(P<0.05)。结论:~(131)I-Herceptin对高表达HER-2/neu的人卵巢癌细胞SKOV3有明显的抑制和杀伤作用。     

HER-2小分子干扰RNA对卵巢上皮性癌细胞侵袭和趋化能力的影响

目的 探讨HER-2小分子干扰RNA（siRNA）对卵巢上皮性癌（卵巢癌）细胞侵袭和趋化能力的影响。方法 选用已知的干扰磷酸甘油醛脱氢酶（GAPDH）的特异性位点作为阳性对照,采用蛋白印迹法检测卵巢癌细胞株SKOV3细胞GAPDH蛋白和HER-2蛋白的表达水平,以吸光度（A）值表示。然后在HER-2基因的编码区内根据干涉位点的筛选原则,选择HER-2siRNAⅠ、HER-2siRNAⅡ、HER-2siRNAⅢ3段靶序列,体外转录合成HER-2siRNA。实验分为4组：空白对照组、脂质体组、非特异性转染组（非特异性siRNAⅢ）、特异性转染组（HER-2siRNAⅢ）,以B肌动蛋白（B-actin）作为内参照,用荧光实时定量PCR和蛋白印迹法检测转染前后SKOV3细胞HER-2mRNA和蛋白表达的变化;细胞侵袭实验和细胞趋化实验检测转染前后SKOV3细胞体外侵袭和趋化能力的变化。结果 GAPDHsiRNA能明显抑制SKOV3细胞内源性GAPDH蛋白的表达,空白对照及加入不同剂量（0．5、1．0、1．5、2．0μg）的GAPDHsiRNA转染SKOV3细胞后,其GAPDH蛋白的表达水平分别为0．6855±0．0259、0．5698±0．0275、0．4542±0．0296、0．3341±0．0178及0．1816±0．0180,各剂量间比较,差异有统计学意义（F=198．126,P〈0．01）。HER-2siRNAⅡ、HER-2siRNAⅢ均有抑制HER-2蛋白表达的作用,HER-2siRNAⅡ、HER-2siRNAⅢ转染后SKOV3细胞中HER-2蛋白的相对表达水平（分别为0．2162±0．1589、0．1562±0．0067）,分别与空白对照组、非特异性转染组及转染HER-2siRNAⅠ的SKOV3细胞（分别为0．3674±0．0350、0．3839±0．0188、0．3532±0．0197）比较,差异均有统计学意义（F=69．461,P〈0．01）。不同剂量（0．5、1．0、1．5、2．0μg）的HER-2siRNAⅢ转染后,SKOV3细胞中HER-2mRNA的相对表达水平比较,差异有统计学意义（F=174．53,P〈0．01）;HER-2siRNAⅢ转染后第1、3、6天,SKOV3细胞中HER-2mRNA的相对表达水平分别为0．0506±0．0017、0．0266±0．0011及0．0154±0．0020,不同时间点比较,HER-2mRNA的相对表达水平呈明显下降趋势（P〈0．01）;特异性转染组SKOV3细胞的侵袭和趋化能力均明显低于非特异性转染组及空白对照组,差异均有统计学意义（F=53．707,P〈0．01;F=11．361,P〈0．01）。结论 HER-2siRNA对卵巢癌细胞HER-2基因的抑制作用呈时间及剂量依赖性,HER-2siRNA能显著抑制细胞侵袭和趋化能力,这为卵巢癌的基因治疗提供了一种新策略。     

AD-hTERT-3E-EGFP-IRES-ELA构建及其对卵巢癌细胞体外抑制作用的研究

目的:构建溶瘤腺病毒载体系统AD-h TERT-3E-EGFP-IRES-ELA,观察ADh TERT-3E-EGFP-IRES-ELA在卵巢癌SKOV3细胞中的表达及对SKOV3细胞的体外杀伤、抑制迁移的作用。方法:利用基因重组技术构建腺病毒AD-h TERT-3E-EGFP-IRESELA。使用OLYMPUS共聚焦显微镜系统,观察EGFP在SKOV3细胞中的表达情况。通过MTS、流式细胞仪检测AD-h TERT-3E-EGFP-IRES-ELA对SKOV3细胞的杀伤效果及作用方式。通过划痕实验、Transwell检测重组病毒对SKOV3细胞体外迁移的作用。结果:成功构建AD-h TERT-3E-EGFP-IRES-ELA。AD-h TERT-3E-EGFP-IRES-ELA在卵巢癌细胞(SKOV3)中可表达EGFP,其荧光强度随时间增加而增强。AD-h TERT-3E-EGFP-IRESELA对卵巢癌细胞具有杀伤效果(P0.05)及抑制细胞迁移的特性(P0.05),主要作用是导致SKOV3细胞出现早期凋亡。结论:成功构建了一种新型的肿瘤特异的溶瘤腺病毒载体系统AD-h TERT-3E-EGFP-IRES-ELA,其在体外对卵巢癌细胞具有杀伤及抑制肿瘤迁移的作用。     

慢病毒转染自杀基因后联合更昔洛韦治疗卵巢上皮性癌的实验研究

目的：探讨以抗Ⅰ型粘蛋白单链抗体（anti-MUC1)为靶向的慢病毒高效转染自杀基因后联合更昔洛韦（GCV）对Ⅰ型粘蛋白阳性（MUC1^ ）卵 巢上皮性癌细胞的靶向性基因治疗。方法：包装以anti-MUC1为靶向的慢病毒,介导单纯疱疹病毒胸苷激酶（HSV－tk)基因转染卵巢上皮性癌细胞株SKOV3（MUC1^ ）及正常戊纤维细胞GF（MUC1^-）,荧光显微镜下观察转染效果,聚合酶链反应（PCR）、逆转录聚合酶链反应（RT－PCR）技术鉴定HSF－tk基因在靶细胞（SKOV3及GF）中的整合转录结果。学显微镜观察GCV作用后细胞形态变化,用四甲基偶氮唑蓝（MTT）法测定GCV的体外杀伤效应。结果：荧光显微镜下观察到以anti-MUC1为靶向的慢病毒可有效转染SKOV3细胞,PCR、RT－PCR均证明HSF－tk基因已有靶细胞SKOV3中整合且转录表达;而在GF细胞中未见HSF－tk基因的整合及转录表达,光学显微镜下观察到HSF－tk基因康后的细胞在GCV作用后出现明显形态变化;MTT法显示GCV对转染HSF－tk基因后的SKOV3细胞有显著杀伤的作用,其半数抑制浓度为0．10mg/L.结论：以anti-MUC1为靶向的慢病毒可将HSF－tk基因高效、靶向性转染MUC1^ 卵巢上皮性癌细胞,联合应用GVC可高效、靶向性杀伤MUC1^ 卵巢上皮性癌细胞。     

针对Her-2基因的小分子干扰RNA对卵巢上皮性癌细胞生物学行为影响的研究

目的通过小分子干扰RNA(siRNA)对Her-2基因表达的影响,探讨其对卵巢上皮性癌(卵巢癌)细胞生物学行为的影响。方法针对Her-2基因的siRNA质粒和阴性对照质粒在脂质体介导下转染到包装病毒细胞株PT67中,嘌呤霉素筛选细胞克隆,收集其上清液并感染SKOV3细胞,经过嘌呤霉素筛选得到稳定转染的细胞株SKOV3/siRNA、SKOV3/siRNA-negative,并通过RT-PCR和免疫组化方法鉴定Her-2基因表达的抑制效果。用四甲基偶氮唑蓝法检测细胞增殖并绘制生长曲线,通过流式细胞仪检测细胞周期、细胞凋亡,并将稳定转染的细胞株接种到裸鼠皮下检测成瘤情况。结果(1)SKOV3/siRNA细胞Her-2基因的表达明显减弱。(2)SKOV3/siRNA细胞的G0/G1期细胞占68·6%,S期细胞占15·1%,SKOV3/siRNA-negative细胞的G0/G1期占55·8%,S期占23·3%。(3)SKOV3/siRNA细胞的早期凋亡率为(10·500±0·250)%,而SKOV3/siRNA-negative细胞为(0·340±0·010)%,两者比较,差异有统计学意义(P<0·01)。(4)与SKOV3/siRNA-negative细胞比较,SKOV3/siRNA细胞的生长速度减慢(P<0·05),接种于裸鼠皮下的肿瘤生长速度明显减慢(P<0·01)。结论siRNA可以有效抑制Her-2基因的表达,抑制卵巢癌细胞的生物学行为。     

野生型p53基因对卵巢癌细胞株生长及凋亡的影响

目的 探讨野生型p53基因对卵巢癌细胞生长抑制及凋亡的作用，为卵巢癌的基因治疗提供实验依据。方法 构建野生型p53基因重组腺病毒载体、体外转染卵巢癌细胞株CaOV3；应用生化染色、免疫组化、聚合酶链技术，检测外源基因的转染率及表达效果；应用细胞计数、MTT法（四甲基偶氮唑盐微量酶反应比色法）、流式细胞术和TUNEL技术（TDL-mediated dUTP-hiotin end labeling)，检测细胞生长及凋亡的情况。结果 野生型p53基因重组腺病毒载体可以有效地转染卵巢癌细胞株CaOV3，转染后的CaOV3细胞内可以检测到p53基因的cDNA及p53蛋白的表达；转染后的CaOV3细胞生长受到明显抑制，63％的细胞生长停滞于G0／G1期，40％－50％的细胞TUNEL呈阳性。结论 野生型p53基因的导入，可抑制卵巢癌细胞的生长并诱导细胞凋亡。野生型p53基因导入可能成为今后临床基因治疗卵巢癌的可行性方法之一。     

RNA干扰技术抑制Her2基因表达对卵巢上皮性癌细胞生物学特性的影响

目的 应用RNA干扰技术,观察稳定转染Her2基因的短发夹状RNA(shRNA)对不同恶性程度的卵巢上皮性癌(卵巢癌)SKOV3和SKOV3.ipl细胞Her2基因表达及细胞生物学特性的影响.方法 将表达Her2的质粒pHer2 shRNA分别转染SKOV3和SKOV3.ipl细胞,经抗性筛选获得稳定转染的细胞系.RT-PCR技术、蛋白印迹法检测转染前后细胞Her2 mRNA和蛋白的表达,体外侵袭实验观察转染前后细胞侵袭能力的改变,裸鼠接种肿瘤细胞观察转染前后细胞成瘤能力的改变.结果 转染前后SKOV3.ipl、SKOV3细胞的Her2 mRNA表达量分别为(99.9±1.3)%和(42.4±2.5)%、(68.0±3.1)%和(40.8±2.0)%.Her2蛋白的表达量分别为(98.2±1.7)%和(40.7±2.1)%、(72.1±3.4)%和(36.4±1.5)%,稳定转染后,两种细胞的Her2基因表达均明显下降,分别比较,差异均有统计学意义(P<0.05).稳定转染pHer2 shRNA后,SKOV3.ipl细胞的穿膜细胞数由(36.2±9.7)个下降为(15.7±7.2)个,SKOV3的穿膜细胞数由(7.6±1.1)个下降为(1.8±0.8)个,细胞侵袭能力均显著下降,分别比较,差异均有统计学意义(P<0.05).稳定转染pHer2 shRNA后,SKOV3.ip1和SKOV3细胞的成瘤重量分别由(1.55±0.31)g下降为(0.69±0.20)g、(1.14±0.10)g下降为(0.38±0.03)g,两种细胞的成瘤能力均显著下降,分别比较,差异均有统计学意义(P<0.05).结论 (1)针对Her2基因的RNA干扰技术可以显著降低卵巢癌细胞Her2基因的表达水平.(2)经RNA干扰作用降低Her2基因表达后,卵巢癌细胞的生物学特性改变,恶性度降低.     

人端粒酶催化亚基基因反义寡核苷酸对卵巢癌细胞端粒酶活性及细胞生长的影响

目的：探讨人端粒酶催化亚基（hEST2)基因反义寡核苷酸（AODN）对卵巢 癌细胞系SKOV3、COC1细胞端粒酶活性及细胞生长的影响。方法：分别采用逆转录聚酶链反应技术、端粒重复扩增法、四甲基偶氮唑蓝法、 绘制细胞生长曲线的方法检测hEST2基因AODN转染前后SKOV3、COC1细胞hEST2 mRNA表达、端粒酶活性的变化,以及对细胞增殖能力及细胞生长的影响。结果：hEST2基因AODN能够下调SKOV3、COC1细胞hEST2 mRNA的表达,降低端粒酶活性,抑制细胞的生长,作用具有明显的时效性。以浓度为30μmol／L的AODN治疗48h的作用最显著,SKOVE、COC1细胞hEST2 mRNA的表达分别下降54．6％、44．6％,对两 株细胞的端粒酶活性抑制率分别达到47．9％、42．7％,与相同浓度的随机寡核苷酸（RODN）、正义寡核苷酸（SODN） 相比,差异有显著性（P＜0．05)。结论：hEST2基因的反义治疗能够抑制卵巢癌细胞的增殖能力,该基因有望成为卵巢治疗的新方向。     

卡培他滨对高表达HER-2/neu卵巢癌裸鼠的治疗作用

目的:探讨卡培他滨对高表达HER-2/neu卵巢癌裸鼠的治疗作用。方法:66只雌性健康裸鼠皮下接种人卵巢癌SKOV3细胞,建立试验模型。根据用药情况将裸鼠分成两组:对照组(塞来昔布灌胃治疗,33只)和观察组(塞来昔布联合卡培他滨灌胃治疗,33只)。比较两组SKOV3细胞的生长抑制率、肿瘤体积及肿瘤抑制率,同时观察两组裸鼠的p53标记指数、HER-2/neu标记指数。结果:观察组的细胞生长抑制率为(65.11±0.86)%,高于对照组(40.38±0.58)%,差异有统计学意义(P0.01)。观察组的肿瘤抑制率高于对照组,差异有统计学意义(P0.01)。观察组裸鼠的p53标记指数、HER-2/neu标记指数均低于对照组,差异有统计学意义(P0.01)。结论:卡培他滨在高表达HER-2/neu卵巢癌裸鼠的治疗中作用显著,能有效抑制肿瘤细胞的生长,作用机制可能是卡培他滨能调控p53、HER-2/neu基因表达。     

Synergistic interaction between an anti-p185HER-2 pseudomonas exotoxin fusion protein [scFv(FRP5)-ETA] and ionizing radiation for inhibiting growth of ovarian cancer cells that overexpress HER-2

OBJECTIVES: The HER-2 proto-oncogene is overexpressed in 15-30% of ovarian cancers, providing a target for therapy in a fraction of patients. We previously described the ability of a recombinant single-chain anti-p185HER-2-Pseudomonas exotoxin A fusion protein, scFv(FRP5)-ETA, to inhibit growth of cancer cells in culture and tumor xenografts in vivo. We have also described synergistic interaction between an anti-p185HER-2-ricin A chain immunotoxin and ionizing radiation for inhibiting growth of ovarian and breast cancer cells that overexpress HER-2. Our objective in this report was to evaluate the in vitro and in vivo effects of scFv(FRP5)-ETA against SKOv3 ovarian cancer cells that have been transfected to express >10(6) p185HER-2 receptors per cell (clone-9002-18). METHODS: Inhibition of clonogenic growth was measured in vitro by limiting dilution analysis. Inhibition of tumor growth was measured in vivo using heterografts established with the same ovarian cancer cell lines. RESULTS: Clone-9002-18 cells were substantially more sensitive than parental SKOv3 cells to the cytotoxic effects of scFv(FRP5)-ETA. Exotoxin fusion protein induced apoptosis in clone-9002-18 cells, but did not affect parental SKOv3 cells. Treatment of clone-9002-18 cells with 200- to 2000-cGy external beam irradiation in combination with scFv(FRP5)-ETA produced synergistic elimination of clonogenic tumor cells in culture. In vivo, subcutaneous or intraperitoneal growth of tumor xenografts in nu/nu mice was significantly inhibited (P = 0.004) by treatment for 10 days with scFv(FRP5)-ETA or with 131I-labeled-520C9 anti-p185HER-2 radionuclide conjugate, but additive effects were not observed with combined treatment. CONCLUSION: ScFv(FRP5)-ETA deserves further evaluation for intraperitoneal therapy of ovarian cancers that overexpress p185HER-2.     

Infectivity enhanced adenoviral-mediated mda-7/IL-24 gene therapy for ovarian carcinoma

OBJECTIVE: Melanoma differentiation associated gene-7 [mda-7/Interleukin (IL)-24] has been identified as a novel anti-cancer agent, which specifically induces apoptosis in cancer cells but not in normal epithelial, endothelial and fibroblast cells. The objective of this study was to evaluate the anti-tumor effect of adenovirus-mediated mda-7/IL-24 (Ad.mda-7) gene therapy in ovarian carcinoma and further improve anti-tumor effect by enhancing infectivity of Ad.mda-7. METHODS: A panel of human ovarian carcinoma cells, OV-4, HEY, SKOV3, SKOV3.ip1 and control normal human mesothelial cells, were infected by a replication deficient recombinant adenovirus encoding mda-7/IL-24 and control virus Ad.CMV.Luc. After 72 h, apoptosis was evaluated by TUNEL and Hoechst staining and further quantified by fluorescent activated cell sorter (FACS) analysis. Infectivity of Ad.mda-7 was enhanced by retargeting it to CD40 or EGF receptors overexpressed on ovarian cancer cells. Subsequently, enhancement in apoptosis of CD40- or epidermal growth factor receptor (EGFR)-retargeted Ad.mda-7 was evaluated. RESULTS: Adenoviral-mediated delivery of mda-7 induces apoptosis ranging from 10-23% in human ovarian cancer cells tested with the highest percentage of apoptosis noted in SKOV3 cells. Minimal apoptosis was noted in normal mesothelial cells. CD40- or EGFR-retargeted Ad.mda-7 increased apoptosis by 10-32% when compared to that achieved with untargeted Ad.mda-7. CONCLUSION: Ad.mda-7 exhibits ovarian cancer-specific apoptosis, but does not affect normal human mesothelial cells. Infectivity enhanced CD40- and EGFR-retargeted Ad.mda-7 augments apoptosis induction, thus increasing the therapeutic index and translational potential of Ad.mda-7 gene therapy.     

Ovarian cancer targeted adenoviral-mediated mda-7/IL-24 gene therapy

OBJECTIVE: We have previously shown that adenoviral-mediated melanoma differentiation-associated gene-7 (Ad.mda-7) therapy induces apoptosis in ovarian cancer cells. However, the apoptosis induction was low and directly correlated with infectivity of Ad.mda-7. The objective of this study was to derive ovarian cancer targeted infectivity-enhanced adenoviral vectors encoding mda-7 and evaluate their enhancement in therapeutic efficacy for ovarian carcinoma. METHODS: Infectivity-enhanced adenoviral vectors encoding mda-7 Ad.RGD.mda-7 and Ad.RGD.pK7.mda-7 were derived by incorporation of RGD and or RGD and Pk7 motifs in the fiber knobs by genetic modification. Viruses were validated by PCR for presence of mda-7 and by Western blot for expression of MDA-7 protein. To test the enhancement of therapeutic efficacy of these viruses, a panel of human ovarian carcinoma cells, OV-4, HEY, SKOV3, SKOV3.ip1, were infected by either Ad.mda-7 or Ad.RGD.mda-7 and Ad.RGD.pK7.mda-7 or their respective control viruses and the cell killing was evaluated by crystal violet staining in vitro. Further, therapeutic efficacy was evaluated in vivo using human ovarian cancer xenograft mouse models. RESULTS: Both Ad.RGD.pK7.mda-7 and Ad.RGD.mda-7 showed significant increase in cell killing in vitro compared to unmodified Ad.mda-7 with Ad.RGD.pK7.mda-7 showing highest cell killing. Further, Ad.RGD.pK7.mda-7 showed a significant increase in survival of mice bearing human ovarian cancer xenografts compared to Ad.mda-7 and other control groups. CONCLUSION: Infectivity-enhanced Ad.RGD.mda-7 and Ad.RGD.pK7.mda-7 viruses significantly enhanced ovarian cancer tumor cell killing in vitro. Significant prolongation of survival by Ad.RGD.pK7.mda-7 in murine ovarian cancer models demonstrates the high clinical translational potential of these viruses for ovarian cancer therapy.     

Aspirin-induced inhibition of ovarian tumor cell growth

OBJECTIVE: To determine if aspirin inhibits the growth of ovarian tumor cells in vitro and to investigate possible mechanisms involved in inhibition. METHODS: OVCAR-3 ovarian tumor cells were grown in monolayer cultures and then harvested for use in proliferation assays. The cells were then treated with vehicle (1% absolute ethanol), 1-5-mmol/L aspirin, 1-microg/mL of anti HER-2/neu monoclonal antibody, or 20-ng/mL heregulin, the ligand for the HER-2/neu receptor either alone or in combination. Cellular proliferation was determined spectrophotometrically by the reduction of tetrazolium dye. Expression of Her-2/neu was assessed by flow cytometry. RESULTS: Aspirin induced inhibition of OVCAR-3 tumor cell growth in a dose-dependent fashion ranging from little to no inhibitory response in cultures treated with 1-mmol/L aspirin to 68% in those treated with 5-mmol/L aspirin. Expression of HER-2/neu was likewise reduced in a dose-dependent manner from 87% expression in control cells to 16% in those treated with 5-mmol/L aspirin. Addition of heregulin alone resulted in 23% proliferation over the control. The combination of heregulin plus 2-mmol/L aspirin caused 66% inhibition of tumor cell growth, whereas the blocking of the HER-2/neu receptor with the monoclonal antibody resulted in an even greater inhibitory response of 82%. CONCLUSION: OVCAR-3 tumor cell growth is inhibited by aspirin, and suppression appears to be potentiated by blocking the HER-2/neu receptor.     

Growth suppression of human ovarian cancer cell lines by the introduction of a p16 gene via a recombinant adenovirus

OBJECTIVE: The cell cycle regulatory protein p16 (CDKN2/cyclin dependent kinase 4 inhibitor/multiple tumor suppressor-1) causes cell cycle arrest at the G1 checkpoint by inhibiting activity of cyclin D-CDK4 complexes. The purpose of this study is to assess the effect of introduction of the p16 gene into two ovarian cancer cell lines via a recombinant adenoviral vector (Ad5CMV-p16). METHODS: Cells lines used were SKOV3, which has a p16 deletion, and OVCA420, which has normal p16. Transduction efficiency was established by infecting cells with an adenovirus containing the Escherichia coli beta-galactosidase gene (Ad5CMV-beta-gal) at multiplicity of infection from 0 to 1000 and staining for X-gal. Cells were infected with Ad5CMV-p16 and cell growth was assessed by counting cells every other day for up to 7 days. Western blotting was done to assess for p16 expression after infection. Fluorescence-activated cell sorting after staining with propidium iodide was done to assess the effect of p16 on the cell cycle. RESULTS: The SKOV3 cell line was transduced with the adenovirus at a slightly lower MOI than the OVCA420 cell line. Growth of the Ad5CMV-p16-infected cells was suppressed 75-80% by cell count in both cell lines and caused morphologic changes of the cells consistent with apoptosis. The p16 protein expression was seen to increase within 24 h after introduction of the p16 gene. G1 arrest of cells occurred beginning 24 h after introduction of the p16 gene. CONCLUSIONS: These results suggest that Ad5CMV-p16 may be further studied as a potential therapeutic agent for ovarian cancer as introduction of the p16 gene into ovarian cancer cell lines causes a G1 arrest and attenuation of growth, regardless of the endogenous p16 status of the cells.     

尿激酶型纤溶酶原激活因子介导的卵巢上皮癌细胞浸润转移体外的实验研究

目地:探讨尿激酶型纤溶酶原激活因子在卵巢上皮癌浸润转移中的作用机制。方法:用RT-PCR技术从人卵巢上皮癌组织总RNA中逆转录uPA基因cDNA全长,克隆至pGEM-T Easy Vector,鉴定后与真核表达载体PCMV-HA连接,酶切及测序。用脂质体法将重组质粒DNA转染至SKOV3细胞。加压筛选并培养PCMV-HA-uPA及对照细胞。分别用RT-PCR和W estern blot方法检测转染前后SKOV3细胞uPA的表达。细胞增殖能力测定用四甲基偶氮唑蓝(MTT)法和集落形成实验,细胞周期测定用流式细胞仪法,细胞体外侵袭,迁移和黏附能力测定分别采用Matrigel Invasion,TranswellM igration和Adhersion Assay方法。结果:(1)uPA阳性表达能介导SKOV3细胞克隆形成,与对照组细胞的差异有统计学意义(P<0.05);(2)uPA阳性表达能介导SKOV3细胞的细胞周期中S期比例增加,与对照组细胞的差异有统计学意义(P<0.05);(3)uPA阳性表达介导SK-OV3细胞的体外侵袭,迁移和黏附能力均明显强于对照细胞株,差异有统计学意义(P=0.0002,<0.0001和0.0049)。结论:uPA通过促进肿瘤细胞侵袭,迁移和黏附能力在卵巢上皮癌浸润转移中起了重要作用。     

Selection of HER-2/neu-positive tumor cells in early stage cervical cancer: implications for Herceptin-mediated therapy

OBJECTIVE: The purpose of this study was to compare HER-2/neu expression on biopsies obtained from early stage cervical cancer, primary cell lines established therefrom, established cervical cancer cell lines, and metastatic or recurrent sites of disease; and to evaluate the sensitivity of primary cervical cancer cell lines to treatment with a humanized MAb against HER-2/neu (Herceptin). METHODS: Surface HER-2/neu expression on 18 cervical cancer cell lines was compared to HER-2/neu detection by immunohistochemistry on biopsies obtained from the original tumors (10 patients) and sites of recurrence (2 patients). Primary cell lines were tested for sensitivity to Herceptin-mediated antibody-dependent cellular cytotoxicity (ADCC) and sensitivity to Herceptin-mediated inhibition of proliferation. RESULTS: Nine out of 10 primary (90%) and 8 out of 8 (100%) established cervical cancer cell lines expressed HER-2/neu by flow cytometry. Surprisingly, all HER-2/neu-positive primary cell lines were derived from tumor biopsies that scored negative (i.e., 0 to 1+) for HER-2/neu expression by immunohistochemistry. Heavy staining for HER-2/neu (i.e., 3+) was found in the recurrent/metastatic lesions of the two relapsed patients. Importantly, all HER-2/neu-positive primary cell lines were highly sensitive to Herceptin-mediated ADCC, and their proliferation was also significantly inhibited by Herceptin. A significant enhancement of Herceptin-mediated ADCC was demonstrated when effector cells were exposed to low doses of IL-2 in vitro. CONCLUSIONS: Early stage cervical cancer may develop a population of HER-2/neu-positive cells with a selective growth advantage over HER-2/neu-negative cells. Therapy which targets HER-2/neu may be more effective in patients with cervical cancer than indicated by the commonly low expression of HER-2/neu in tumors removed at the time of primary treatment.     

脂质体转染白介素-12基因至卵巢癌SKOV3细胞检测VEGF表达及抗肿瘤作用探讨

目的:观察含mIL-12基因的质粒转染至SKOV3卵巢癌细胞,效应细胞存在情况下对卵巢癌细胞株血管内皮生长因子(VEGF)表达的影响。方法:用脂质体转染技术将基因质粒及空质粒转染至SKOV3卵巢癌细胞(SKOV3/IL-12)及SKOV3/neo;用ELISA法检测IL-12及INF-γ的表达;用逆转录聚合酶联反应(RT-PCR)半定量法测定SKOV3/IL-12+S(脾细胞)0,12,24,36,48,60hVEGFmRNA含量;用ELISA法测定细胞培养上清液中VEGF蛋白含量及其抑制率。结果:基因转染至SKOV3可检测到IL-12蛋白表达;分泌的IL-12蛋白具有活性,使脾细胞产生INF-γ,12h时迅速出现,作用后24h表达量最高;SKOV3/IL-12细胞与脾细胞作用12h后即出现VEGFmRNA表达抑制,24h达到高峰,与对照组有显著差异(P0.05);培养24h后上清中VEGF蛋白含量减少,与对照组有显著差异(P0.05)。结论:将IL-12基因转染至SKOV3卵巢癌细胞并表达IL-12;分泌的IL-12蛋白具有活性,使脾细胞产生INF-γ;SKOV3/IL-12与效应细胞脾细胞作用后产生INF-γ能在核酸水平下调VEGF并能抑制VEGF蛋白的表达。