抑制Her-2基因的表达对NK-92细胞杀伤SKOV3细胞活性影响的研究

目的:探讨抑制Her-2基因的表达对NK-92细胞杀伤SKOV3细胞活性的影响。方法:Her-2siRNA质粒在脂质体介导下转染到包装病毒细胞株PT67中,将病毒上清转染SKOV3,经嘌呤霉素筛选得到稳定抑制Her-2基因表达的SKOV3/siRNA-1、SKOV3/siRNA-2细胞株,并分别通过RT-PCR和免疫组化法鉴定抑制Her-2表达的效果。应用LDH法检测NK-92细胞对SKOV3、SKOV3/siRNA-1、SKOV3/siRNA-2、SKOV3/siRNA-neg-ative control的杀伤活性。结果:Her-2/siRNA-1、Her-2/siRNA-2均可有效沉默Her-2mR-NA和蛋白的表达。NK-92细胞对SKOV3、SKOV3/siRNA-negative control的杀伤率分别为21%、20%,而对SKOV3/siRNA-1、SKOV3/siRNA-2的杀伤率分别为33%、45%(P<0·05)。结论:抑制Her-2基因的表达可增强NK-92细胞对SKOV3细胞的杀伤活性。RNA干扰技术联合NK-92细胞为高表达Her-2基因卵巢癌的生物治疗提供了一种新的策略。     

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。     

RNA干扰技术沉默survivin基因对卵巢上皮性癌细胞株SKOV3生物学行为的影响

survivin基因是凋亡抑制蛋白家族（IAP）成员之一,具有肿瘤组织表达特异性,与肿瘤化疗耐药相关,已有研究证明,survivin基因在卵巢肿瘤组织高表达,与肿瘤的恶性程度及预后密切相关.RNA干扰（RNA interference,RNAi）技术具有很强的转录后基因沉默作用,已广泛用于抗肿瘤的研究中.     

短发夹状RNA抑制AKT2基因表达增强卵巢上皮性癌细胞对紫杉醇敏感性的研究

RNA干扰（RNAi）技术是运用双链RNA引发的转录后水平基因沉默机制,特异性抑制靶基因转录,进而下调相应蛋白表达水平及功能.新近的RNAi技术,是利用DNA载体直接在体内表达短发夹状RNA（shRNA）,特异性封闭相应的靶基因mRNA,抑制mRNA的翻译.与早期的RNA干扰技术相比,该方法的特异性和干扰效率均有较大提高,已广泛应用于多种研究领域.利用RANi技术特异地抑制癌基因、癌相关基因或突变基因的过度表达,使这些基因保持在沉默或休眠状态,从而使肿瘤细胞增殖速度减慢,凋亡加快,显示了RNAi技术用于肿瘤基因治疗的可行性和有效性.     

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.     

RNA干扰技术逆转卵巢上皮性癌细胞多药耐药的研究

目的探讨应用RNA干扰(RNAi)技术逆转卵巢上皮性癌(卵巢癌)细胞多药耐药的可行性。方法将设计合成的针对多药耐药基因MDR1的特异性小分子干扰RNA(siRNA),用脂质体转染具有MDR1基因高表达的卵巢癌紫杉醇耐药细胞株OVCAR8/TR。用荧光定量RT-PCR技术和流式细胞仪分别测定转染前后细胞MDR1mRNA及糖蛋白P-gp表达的变化;三磷酸腺苷(ATP)生物荧光法检测转染前后细胞对顺铂、氟尿嘧啶、阿霉素和紫杉醇药物敏感性(以ATP抑制率判断)的变化情况。结果转染后24、48、72、96和120h,OVCAR8/TR细胞MDR1mRNA的抑制率分别为26·42%、84·00%、78·43%、45·85%和0;转染后48h MDR1mRNA的抑制率达到最高峰。转染后24、48、72、96和120h,OVCAR8/TR细胞P-gp的抑制率分别为16·71%、49·64%、85·23%、65·98%和9·44%;转染后72h P-gp的抑制率达到最高。转染MDR1siRNA后,能够明显提高OVCAR8/TR细胞对紫杉醇和阿霉素的敏感性,在紫杉醇的作用下,转染前后OVCAR8/TR细胞的ATP抑制率分别为(25·8±3·1)%和(78·0±9·8)%,转染前后比较,差异有统计学意义(P<0·05)。结论在OVCAR8/TR细胞中,针对MDR1合成的siRNA能够有效地抑制MDR1mRNA和P-gp的表达,并能恢复其对紫杉醇和阿霉素的敏感性。应用RNAi技术,能够逆转卵巢癌细胞对化疗药物的多药耐药。     

RNA干扰技术抑制切除修复交叉互补基因1对卵巢上皮性癌细胞顺铂敏感性的影响

目的探讨RNA干扰技术抑制切除修复交叉互补基因(ERCC)1对卵巢上皮性癌(卵巢癌)细胞顺铂敏感性的影响。方法体外设计、合成针对ERCC1的小分子干扰RNA(siRNA),并转染卵巢癌细胞ES2,应用RT PCR、蛋白印迹法(westernblot)和免疫组化链霉菌抗生物素蛋白过氧化物酶(SP)连接法检测转染siRNA后ES2细胞ERCC1基因和蛋白的表达变化,应用四甲基偶氮唑蓝比色法检测干扰ERCC1后ES2细胞对顺铂敏感性的变化。结果转染针对ERCC1的siRNA后24、48、72h,ES2细胞ERCC1基因和蛋白的表达水平下降;转染ERCC1siRNA后,ES2细胞对顺铂的半数抑制量从(10.475±1.713)μg/ml提高到(0.194±0.021)μg/ml,ES2细胞对顺铂的敏感性增加了53.88倍。结论RNA干扰技术抑制ERCC1能增加卵巢癌细胞ES2对顺铂的敏感性。     

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。     

小分子干扰RNA诱导卵巢上皮性癌细胞凋亡的实验研究

表皮生长因子受体2（HER-2）是HER-2原癌基因编码的一种酪氨酸蛋白激酶，研究表明，过度表达的HER-2基因通过激活瑚等信号分子，导致细胞过度增殖和恶性变，抑制细胞凋亡，与肿瘤的恶性程度和转移能力密切相关。已有研究证明，30％的卵巢上皮性癌（卵巢癌）存在HER．2基因的高表达。RNA干扰（RNA interference，RNAi）是生物进化过程中保留的一种防御机制，可以介导互补基因的靶向性降解。本研究拟应用RNAi技术，合成针对HER-2基因的特异性小分子干扰RNA（small interference RNA，siRNA），转染卵巢癌细胞株SKOV3细胞，探索siRNA诱导卵巢癌细胞凋亡的机制。     

AKT2基因沉默抑制卵巢癌细胞侵袭和粘附能力的实验研究

目的:探讨AKT2基因对人卵巢癌细胞A2780侵袭和黏附活性的影响。方法:构建AKT2基因的短发卡状RNA质粒转染人卵巢癌细胞A2780,运用RT-PCR、蛋白质免疫印迹法比较转染前后AKT2表达的差异;用transwell小室检测细胞侵袭人工基底膜的能力;MTT法检测卵巢癌细胞与细胞外基质黏附能力。结果:与对照相比,构建shR-NA表达载体可抑制AKT2 mRNA转录和蛋白的表达;AKT2表达下降后,穿透重组基底膜的细胞数明显下降(P<0.05),且可显著抑制细胞与细胞外基质的黏附(P<0.05)。结论:靶向AKT2 shRNA能有效地抑制卵巢癌细胞中AKT2基因的表达,并抑制卵巢癌细胞体外侵袭和黏附能力。AKT2基因有可能成为治疗卵巢癌的新途径。     

The antiobesity drug Orlistat induces cytotoxic effects, suppresses Her-2/neu (erbB-2) oncogene overexpression, and synergistically interacts with trastuzumab (Herceptin™) in chemoresistant ovarian cancer cells

Abstract.   Menendez JA, Vellon L, Lupu R. The antiobesity drug Orlistat induces cytotoxic effects, suppresses Her-2/neu (erbB-2) oncogene overexpression, and synergistically interacts with trastuzumab (Herceptin^™) in chemoresistant ovarian cancer cells. Int J Gynecol Cancer 2006; 16: 219–221.     

Her-2/neu expression and amplification in early stage ovarian surface epithelial neoplasms

OBJECTIVE: The aim of this study is to examine Her-2/neu gene amplification and protein overexpression in a spectrum of ovarian neoplasms using both immunohistochemical (IHC) and fluorescence in situ hybridization (FISH) techniques that are FDA approved. This study is focused on early stage tumors including both carcinomas and borderline tumors. METHODS: FDA-approved IHC and FISH for Her-2/neu were performed on formalin-fixed, paraffin-embedded tissue from 79 ovarian neoplasms representing a broad spectrum of tumor types as well as four normal ovaries. All tumors were either stage I or stage II. Tumor and normal tissue were studied collectively using a tissue microarray (TMA). HercepTest (DAKO) and PathVysion Her-2/neu probe kit (Vysis Inc.) were used for IHC and FISH analysis. RESULTS: FISH analysis of serous carcinomas demonstrated Her-2/neu gene amplification in 3 (18%) of 17 cases. Two of three cases showing Her-2/neu gene amplification were scored 1+ using IHC, while the remaining case was scored as 0. Analysis of endometrioid carcinomas demonstrated Her-2/neu amplification using FISH in 1 of 10 (10%) cases. IHC in this case was scored 2+ (positive). None of the remaining 44 tumors, including clear cell carcinoma (n = 12), transitional cell carcinoma (n = 1), mixed epithelial carcinoma (n = 7), carcinoma not otherwise specified (n = 1), and 31 borderline tumors (mucinous, n = 17; endometrioid, n = 7; serous, n = 7), showed Her-2/neu gene amplification or protein overexpression. Normal ovaries were negative as well. CONCLUSIONS: Amplification of Her-2/neu in early stage ovarian neoplasms is infrequent, 6.7% overall. Due to the limited number of informative cases, we were unable to determine the clinical significance of Her-2/neu amplification in this study. Her-2/neu amplification was restricted to carcinomas and was not encountered in ovarian borderline tumors.     

Assessment of Her-2/neu expression in hydatidiform moles for prediction of subsequent gestational trophoblastic neoplasia

OBJECTIVE: The aim of the present study was to asses the ability of Her-2/neu immunohistochemical staining of the molar tissue to predict the risk of developing gestational trophoblastic neoplasia (GTN). METHODS: Sections prepared from 33 consecutive formalin-fixed paraffin-embedded archival reconfirmed hydatidiform mole tissue blocks were immunohistochemically stained for Her-2/neu. The staining was scored according to the subjectively evaluated intensity of staining and the proportion of stained villous cytotrophoblastic cells. Clinical data were abstracted from medical files. RESULTS: 23 patients had a complete and 10 a partial mole. Nine patients (27.3%) were diagnosed with GTN [7 of 23 patients with a complete mole (30.4%) and 2 of the 10 (20.0%) with a partial mole]. A positive immunohistochemical Her-2/neu stain was found in 6 (18.2%) of the patients with hydatidiform mole (3 with a complete mole). The rate of Her-2/neu expression was somewhat higher in moles with subsequent GTN than in moles with an uneventful course (22.2% vs. 16.6%, respectively). The difference did not reach significance (Fisher's Exact Test, P=0.55) possibly due to the small number of cases (power of <5%). The sensitivity and specificity of Her-2/neu expression for prediction of GTN was 22.2% and 83.3%, respectively, and the positive and negative predictive value 33.3% and 74.1%, respectively. CONCLUSION: While the specificity of Her-2/neu immunohistochemical staining for prediction of GTN is relatively high, the low sensitivity and low positive and negative predictive value precludes its practical clinical use for prediction of post-molar GTN. The quest for a precise predictor of post-molar GTN should continue.     

Amplification of HER-2/neu oncogene in human ovarian cancer

Amplification and/or increased expression of the HER-2/neu oncogene has been reported to occur in ovarian tumors and possibly to correlate with biologic behavior and prognosis. The frequency with which amplification is reported to occur is quite variable ranging from 0–30% in different series and this variability is probably accounted for by technical and methodologic factors. The variability and lack of reproducibility has raised questions about the usefulness of assessing amplification of the HER-2/neu oncogene and in particular its clinical relevance. In this study by using strict criteria for amplification and using multiple controls we could demonstrate unequivocal amplification of the HER-2/neu oncogene by Southern blot analysis in only 11% of malignant ovarian tumors. The potential pitfalls with the techniques used to detect HER-2/neu oncogene amplification and overexpression are reviewed and possible ways to overcome some of the problems are suggested.     

Expression of HER-2/neu oncoprotein, DNA-ploidy and S-phase fraction in advanced ovarian cancer

The immunohistochemical expression of HER-2/neu and cytofluorimetric data were retrospectively analyzed in a group of primary advanced ovarian cancers. Thirty-three out of 94 (35%) cases showed a specific p185/neu immunoreaction. No correlation between p185/neu expression and any of the clinico-pathologic parameters examined was observed. As far as cytofluorimetric data are concerned, 38 out of 69 (55%) of the tumors were diploid (DNA index = 1) while 31 (45%) were aneuploid (DNA index from 1.10 to 2.50 with a median value of 1.50). Ovarian tumors were defined as of low and high S-phase fraction in 68% and 32% of the cases, respectively. Tumor ploidy and S-phase fraction did not correlate with the clinico-pathologic characteristics or p185/neu oncoprotein expression. Aneuploid tumors had a higher S-phase fraction (mean: 15.81 ± 13.44) than diploid tumors (mean: 8.89 ± 7.98) ( P < 0.01). p185/neu expression failed to affect significantly both overall and progression free survival. On the other hand tumor ploidy was found to be related to the prognosis of advanced ovarian cancer patients although the difference was not statistically significant. As far as progression free survival is concerned, the median time to recurrence was not reached for diploid cases whereas it was 21 months for aneuploid cases ( P < 0.05). The 5-year survival for patients with a low S-phase fraction (58%) was significantly higher than for patients with high S-phase fraction tumors (28%) ( P < 0.01). Median time to recurrence was 48 and 17 months for low and high S-phase fraction tumor patients, respectively ( P < 0.05). However, in a multivariate analysis both tumor ploidy and S-phase fraction did not retain their prognostic value. The assessment of the role of the parameters examined in improving the prognostic characterization of ovarian cancer patients should be investigated in large multicenter clinical trials.     

WWOX基因干扰对卵巢上皮性癌细胞顺铂耐药的影响

目的 研究应用RNA干扰(RNAi)技术逆转卵巢上皮性癌(卵巢癌)耐药细胞中WWOX基因的表达,并探讨WWOX基因与卵巢癌细胞顺铂耐药的关系.方法 将设计合成的针对WWOX基因的特异性小分子干扰RNA(siRNA),用脂质体瞬时转染法将其转染进WWOX基因高表达的卵巢癌顺铂耐药细胞株SKOV3/SB细胞中,用RT-PCR技术和蛋白印迹法分别测定转染前后SKOV3/SB细胞中WWOX Mrna及蛋白表达的变化;采用四甲基偶氮唑蓝比色法检测转染前后SKOV3/SB细胞对顺铂的耐药指数;应用流式细胞仪检测转染前后SKOV3/SB细胞的生长周期,高效液相色谱仪测定转染前后SKOV3/SB细胞中顺铂的药物浓度.实验分为5组:(1)SKOV3组:接种SKOV3细胞;(2)SKOV3/SB组:接种SKOV3/SB细胞;(3)SKOV3/SB阴性对照转染组:接种转染阴性对照序列的SKOV3/SB细胞;(4)SKOV3/SB脂质体转染组:接种转染脂质体的SKOV3/SB细胞;(5)SKOV3/SB siRNA转染组:接种转染siRNA的SKOV3/SB细胞.结果 转染48 h后,SKOV3、SKOV3/SB、SKOV3/SB阴性对照转染、SKOV3/SB脂质体转染、SKOV3/SB siRNA转染组细胞中WWOX Mrna的表达水平分别为0.647±0.025、2.239±0.030、2.392±0.041、2.379±0.047、1.219±0.032,WWOX蛋白的表达水平分别为1.368±0.028、1.639±0.031、1.580±0.025、1.552±0.034、0.653±0.017,SKOV3/SB siRNA转染组与其他各组比较,差异均有统计学意义(P<0.05).转染siRNA前、后,SKOV3/SB细胞对顺铂的耐药指数由5.04降至3.89;细胞周期中G1期由39.2%增至45.6%,S期由50.4%降至37.5%;耐药细胞内顺铂浓度由9.43 ns/L升至23.45 w/L,分别比较,差异均有统计学意义(P<0.05).结论 针对WWOX基因合成的特异性siRNA能明显抑制SKOV3/SB细胞中WWOX Mrna和蛋白的表达,并能在一定程度上恢复其对顺铂的敏感性,推测WWOX基因可能参与了卵巢癌细胞的顺铂耐药过程.     

Overexpression of HER-2/neu is not a risk factor in ovarian clear cell adenocarcinoma

OBJECTIVE: To evaluate the significance of the expression of HER-2/neu as a prognostic factor, we retrospectively examined its overexpression rates chiefly in ovarian clear cell adenocarcinoma (CCA) and their relationships with FIGO stage, lymph node metastasis, and prognosis. METHODS: In addition to 90 specimens of CCA (stage I, 52; stage II, 7; stage III, 28; stage IV, 3) obtained between 1987 and 2002, 19 specimens of serous adenocarcinoma (S), 19 specimens of mucinous adenocarcinoma (M), and 19 specimens of endometrioid adenocarcinoma (E) collected at initial surgery were studied. The expression of HER-2/neu was immunohistologically scored on a scale of 0 to 3+ according to the HercepTest scoring system, and 2+ and 3+ were regarded as overexpression. The relationship between HER-2/neu overexpression and outcome was evaluated by the Kaplan-Meier method and the log-rank test. The relationship with advanced-stage cancer was analyzed by Fisher's exact test. The relationships with lymph node metastasis and histologic types were compared by the t test. RESULTS: The rate of HER-2/neu overexpression in CCA was 45.6% (0, 1+, 2+, and 3+ in 14, 35, 36, and 5 cases, respectively), and no differences in the overexpression rate were noted among histologic types: 52.7% in S, 42.1% in M, and 26.4% in E. In CCA, no association was observed between HER-2/neu overexpression and cumulative survival rate (P = 0.5708), FIGO stage (P = 0.5147), or lymph node metastasis (P = 0.3624). CONCLUSION: The absence in CCA of an association between HER-2/neu overexpression and outcome, stage, or lymph node metastasis indicates that HER-2/neu overexpression is not a prognostic risk factor.     

RNA干扰技术沉默survivin基因逆转卵巢癌细胞株SKOV3/ADM耐药性的研究

目的检测survivin基因在卵巢上皮性癌(卵巢癌)耐药细胞株SKOV3/ADM及其亲本细胞株SKOV3中的表达水平,探讨以survivin基因为靶向的RNA干扰(RNA i)能否有效沉默survivin基因在SKOV3/ADM细胞中的表达,并能否有效逆转SKOV3/ADM细胞对化疗药物的耐药性。方法半定量RT-PCR、免疫荧光法检测survivin mRNA、蛋白在SKOV3/ADM及SKOV3细胞中的表达。以survivin基因为靶向的重组质粒pshRNA-survivin及紫杉醇作用于SKOV3/ADM细胞,将SKOV3/ADM细胞分为4组,即对照组、RNA i组、紫杉醇组、RNA i+紫杉醇组,RT-PCR技术检测各组细胞的survivinmRNA表达水平,吖啶橙/溴乙锭(AO/EB)荧光染色法及末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸标记法(TUNEL)检测各组细胞的凋亡情况。结果survivin mRNA在SKOV3/ADM、SKOV3细胞中的表达水平分别为99.1%、75.3%,SKOV3/ADM、SKOV3细胞的荧光细胞数分别为(59±5)、(42±3)个,两种细胞间比较,差异均有统计学意义(P<0.05)。RNA i后SKOV3/ADM细胞中mRNA的表达水平由99.1%降低至7.9%。AO/EB荧光染色法、TUNEL法检测各组细胞凋亡数,紫杉醇组为(10.2±1.0)、(9.8±0.8)个,RNA i组为(48.5±4.9)、(49.5±4.3)个,RNA i+紫杉醇组为(71.5±6.8)、(68.3±5.7)个,RNA i+紫杉醇组与RNA i组比较,差异有统计学意义(P<0.05),RNA i+紫杉醇组与紫杉醇组比较,差异也有统计学意义(P<0.05)。结论survivin基因在SKOV3/ADM细胞中的表达显著高于SKOV3细胞。以survivin基因为靶向的RNA i可有效抑制SKOV3/ADM细胞中survivin基因的表达,有效增加了SKOV3/ADM细胞对化疗药物紫杉醇的敏感性,显著上调了SKOV3/ADM细胞的凋亡活性。