宫颈鳞状细胞癌ATP-TCA法体外药敏试验与耐药相关蛋白表达的关系

目的:探讨代表不同耐药机制的P糖蛋白(P-gp)、谷胱甘肽S-转移酶(GST-π)、DNA拓扑异构酶Ⅱ(TopoⅡ)、胸苷酸合成酶(TS)在原发宫颈鳞癌组织中的表达及其与临床病理参数的关系;分析三磷酸腺苷生物荧光法体外药敏试验(ATP-TCA)结果与耐药蛋白表达的关系。方法:收集32例宫颈鳞癌患者的新鲜癌组织制成单细胞悬液,采用ATP-TCA法成功检测上述细胞对11种化疗药物(共16种组合)的体外敏感性;采用EnVision二步法免疫组化检测32例宫颈鳞癌组织中P-gp、GST-π、TopoⅡ、TS蛋白的表达,并分析其与临床分期、肿瘤分化程度的关系。结果:耐药蛋白P-gp、GST-π、TopoⅡ、TS在宫颈鳞癌组织中的表达与临床分期、肿瘤分化程度无相关性;耐药蛋白P-gp表达与体外PTX+CBP耐药正相关(P=0.040);耐药蛋白TS表达与体外5-FU、5-FU+MMC、5-FU+DDP耐药正相关(P=0.015,0.012,0.006);TopoⅡ,GST-π蛋白表达与宫颈鳞癌体外耐药均无相关性。结论:体外药敏试验联合免疫组化检测,有可能用于宫颈鳞癌化疗前药敏预测。     

人卵巢上皮性癌顺铂耐药细胞系3AO/cDDP模型的建立及耐药机理的实验研究

目的：模拟临床卵巢上皮性癌化疗的用药特点建立顺铂耐药细胞系3AO／CDDP,检测LRP、MRP、P－gp、GSTπ和TopeⅡ表达。方法：以临床卵巢上皮性癌化疗用药的最低剂量换算终浓度,采用大剂量顺铂（10μg／ml）反复间歇24h暴露法建立顺铂耐药细胞系;用FCM法检测LRP、MRP、P－gp、GSTπ和TopoⅡ表达。结果：历时4．5个月建成顺铂耐药株3AO／cDDP,耐药性稳定,耐药指数1．62,与临床耐药倍数相近。3AO／cDDP细胞MRP、P－gp表达与3AO相比无明显变化（P＞0．05）,LPR、GSTπ表达明显升高（P＜0．005及P＜0．05）,Tope-Ⅱ含量明显降低（P＜0．05）。结论：3A0／cDDP是模拟临床用药特点建立的、研究顺铂耐药的理想模型;顺铂耐药与LRP、GSTπ表达升高及TopoⅡ含量降低有关,与MRP、P-gp无关。     

白细胞介素6诱导卵巢上皮性癌细胞对化疗药物产生耐药的机制研究

目的 探讨白细胞介素6(IL-6)诱导卵巢上皮性癌(卵巢癌)细胞对化疗药物产生耐药的机制及相关信号传导通路.方法 应用外源性重组IL-6短期处理或将正义IL-6基因(ssIL-6)稳定转染至A2780细胞(不表达IL-6但表达其受体、对顺铂和紫杉醇敏感),同时将反义IL-6基因(asIL-6)稳定转染至SKOV3细胞(高表达IL-6及其受体、对顺铂和紫杉醇耐药),应用四甲基偶氮唑蓝(MTT)比色法、逆转录PCR(RT-PCR)技术及蛋白印迹法(western blot)观察IL-6是否影响卵巢癌细胞对顺铂和紫杉醇的敏感性,并对可能的信号传导通路进行研究.结果 (1)外源性重组IL-6处理A2780细胞后,对顺铂和紫杉醇的耐药倍数分别为6.25和7.31倍;ssIL-6转染A2780细胞后(内源性过表达IL-6),IL-6低、中、高表达细胞对顺铂的耐药倍数分别为7.13、7.47和8.34倍,对紫杉醇的耐药倍数分别为7.61、8.27和10.70倍;而asIL-6转染SKOV3细胞后(抑制内源性IL-6表达),IL-6中、高抑制表达细胞对顺铂的耐药倍数分别为0.15和0.10倍,对紫杉醇的耐药倍数分别为0.10和0.08倍,可恢复SKOV3细胞对顺铂和紫杉醇的敏感性.(2)外源性IL-6作用后,上调了A2780细胞中耐药相关基因MDR1和GST-π以及凋亡抑制基因bcl-2、bcl-xL和XIAP mRNA和蛋白的表达;与此相一致,ssIL-6转染的A2780细胞中MDR1、GST-π及bcl-2、bcl-xL、XIAP mRNA和蛋白的表达水平增加,而asIL-6转染的SKOV3细胞则明显降低.(3)有丝分裂原活化的蛋白激酶-细胞外信号调节激酶(MEK)抑制剂--PD98059和磷脂酰肌醇3激酶(PI3K)抑制剂--wortmannin能分别阻断IL-6诱导的卵巢癌细胞细胞外信号调节激酶(ERK)和蛋白激酶B(Akt)的活化作用,且两者均能阻断IL-6诱导的卵巢癌细胞对顺铂和紫杉醇产生的耐药,细胞生长明显减少.结论 IL-6诱导的卵巢癌细胞化疗耐药很可能与其上调卵巢癌细胞耐药相关基因MDR1、GST-π和凋亡抑制基因bcl-2、bcl-xL、XIAP的表达以及活化MEK/ERK和PI3K/Akt信号传导通路有关.     

子宫颈癌新辅助化疗影响因素与药物敏感性基因

新辅助化疗（neoadjuvant chemotherapy,NACT）是局部晚期宫颈癌的综合治疗方式之一,部分相关化疗药物敏感性基因对肿瘤耐药、化疗副反应的预测作用取得了进展。宫颈癌肿瘤大小是影响新辅助化疗近期疗效的独立因素;核苷酸切除修复交叉互补组1（ERCC1）、Ⅲ微管蛋白（TUBB3）、核糖核苷酸还原酶M1（RRM1）基因阳性或过度表达可作为铂类、抗微管类和吉西他滨药物耐药的预测标志,但不推荐临床常规检测及应用,尿苷二磷酸葡萄糖醛酸转移酶1A1（UGT1A1）多态性与伊立替康毒副反应有关,为预测宫颈癌新辅助化疗近期疗效及个体化治疗提供参考。     

卵巢恶性肿瘤中GST-π表达与肿瘤内在性耐药及患者预后关系的研究

目的：探讨谷胱甘肽Ｓ－转移酶π型（ＧＳＴ－π）在卵巢恶性肿瘤组织中的表达及其与肿瘤内在性耐药和患者预后的关系。方法：应用免疫组化ＳＰ法检测４５份卵巢恶性肿瘤组织和１０份正常卵巢组织中ＧＳＴ－π的表达，结合肿瘤细胞体外药物敏感试验结果和患者２ａ随访结果。结果：１．４５份卵巢恶性肿瘤组织ＧＳＴ－π阳性表达率为１００％，１０份正常卵巢组织仅２份呈弱阳性。２．１８份卵巢恶性肿瘤组织ＧＳＴ－π染色程度（＋）、（）、（）三组之间阿霉素对肿瘤细胞的抑制率有统计学差异（Ｐ＜０．０５），其中（）组抑制率明显低于（＋）组（Ｐ＜０．０５）。其余５种药物对肿瘤细胞抑制率差异无显著性（均Ｐ＞０．０５）。３．２８例Ⅲ期卵巢上皮性癌患者２ａ存活率为３９．３％，存活时间≥２ａ和＜２ａ两组ＧＳＴ－π表达差异有显著性（Ｐ＜０．０１），前者染色程度明显弱于后者。结论：ＧＳＴ－π表达在卵巢恶性肿瘤对阿霉素内在性耐药中起重要作用，而与顺铂、卡铂、５－ＦＵ、更生霉素、长春新碱内在性耐药无关。ＧＳＴ－π表达高提示卵巢癌患者预后差，可供临床评价患者预后时参考。     

卵巢肿瘤中谷胱甘肽S转移酶的表达及其生物学意义

谷胱甘肽S转移酶(GSTs)是一组与人体解毒功能有关的同功酶系,目前对其亚型GST-π的研究最为广泛。体外试验显示GSTs与顺铂、烷化剂和阿霉素的耐药性相关。应用免疫组化法和生化法分析人体卵巢肿瘤,发现卵巢恶性肿瘤中GST-π的表达水平明显高于良性肿瘤,化疗药物可诱导卵巢癌细胞中GST-π表达水平升高。卵巢恶性肿瘤的GST-π表达与临床分期、残余瘤、组织学类型和分化程度均无关,而与耐药性和预后的关系目前尚有争议。     

苦参碱联合顺铂对子宫颈癌SiHa细胞中TSLC1基因表达的影响

目的探讨苦参碱联合顺铂对人宫颈癌SiHa细胞肺癌肿瘤抑制因子1（TSLC1）基因的表达及SiHa细胞对顺铂敏感性的影响,以期对治疗宫颈癌提供依据。方法采用四甲基偶氮唑蓝比色法（MTT）测定不同浓度苦参碱组（50、100、150、200、250μg／ml）、顺铂组（5、10、15、20、25μg／ml）和联合组（15μg／ml顺铂联合5种浓度苦参碱）干预SiHa细胞后的抑制率;采用实时荧光定量PCR（FQ—PCR）技术检测TSLC1 mRNA表达水平的变化。结果苦参碱及顺铂呈剂量依赖方式抑制SiHa细胞的生长,且随着各组浓度的增高,TSLC1 mRNA表达上调（P〈0．01）。不同浓度联合组对细胞的抑制率分别为39．35％、63．51％、68．74％、72．04％和74．31％,TSIC1 mRNA的表达量分别为8．59±0．01、15．33±0．01、26．38±0．07、61．34±0．01和92．33±0．01,与苦参碱组或顺铂组比较,差异均有统计学意义（P〈0．05）。结论苦参碱联合顺铂抑制人宫颈癌SiHa细胞生长的机制可能与TSLC1 mRNA的表达上调有关,且苦参碱能够增加SiHa细胞对顺铂的敏感性。     

GST-π和DNATopoⅡ在晚期宫颈癌组织中的表达

目的：探讨谷胺甘肽Ｓ－转移酶、脱氧核糖核酸拓扑Ⅱ的表达与晚期宫颈癌化疗的关系。方法：应用免疫组化Ｓ－Ｐ法检测２８份晚期宫颈癌组织化疗前后ＧＳＴ－π和ＤＮＡＴｏｐｏⅡ的表达。结果：化疗前２８份宫颈癌组织中ＧＳＴ－π过表达１７份（６０．７１％),ＤＮＡ ＴｏｐｏⅡ低表达１６份（５７．１４％）;化疗后ＧＳＴ－π过表达２４份（８５．７１％）,ＤＮＡ ＴｏｐｏⅡ低表达１８例（６４．２８％）。化疗前后ＧＳＴ－π的表达差异有显著性（Ｐ＜0．05）,ＤＮＡ ＴｏｐｏⅡ的表达差异无显著性（Ｐ＞０．０５）,二者表达有相关性（Ｐ＝０．０１９）。ＧＳＴ－π过表达和ＤＮＡＴｏｐｏⅡ低表达与患者的化疗反应呈负相关（Ｐ＝０．０１４,Ｐ＝０．０３８）。结论：ＧＳＴ－π过表达与宫颈癌内在性耐药和获得性耐药有关,ＤＮＡ TｏｐｏⅡ低表达只与宫颈癌内在性耐药有关。ＧＳＴ－π和ＤＮＡ TｏｐｏⅡ的表达均能很好地预测晚期宫颈癌患者的化疗耐药与反应。     

紫杉醇化疗在宫颈癌中的临床应用

紫杉醇为广谱有效的抗实体瘤药物,用于宫颈癌患者治疗已进行广泛临床研究,证实其可靠有效。从单药、双药及三药化疗等方面,综述近年紫杉醇治疗宫颈癌的临床应用。紫杉醇（T）联合顺铂（P）或卡铂（C）化疗方案优于紫杉醇单药或三药化疗,安全有效。尤其在新辅助化疗中;与顺铂相比,卡铂肾毒性小,无需水化,TC方案相对简单;TP或TC配合放疗疗效显著;紫杉醇周疗与3周疗法比较疗效类似,不良反应较小。     

紫杉醇化疗在宫颈癌中的临床应用

紫杉醇为广谱有效的抗实体瘤药物,用于宫颈癌患者治疗已进行广泛临床研究,证实其可靠有效。从单药、双药及三药化疗等方面,综述近年紫杉醇治疗宫颈癌的临床应用。紫杉醇（T）联合顺铂（P）或卡铂（C）化疗方案优于紫杉醇单药或三药化疗,安全有效。尤其在新辅助化疗中;与顺铂相比,卡铂肾毒性小,无需水化,TC方案相对简单;TP或TC配合放疗疗效显著;紫杉醇周疗与3周疗法比较疗效类似,不良反应较小。     

紫杉醇联合顺铂、5-FU新辅助化疗晚期宫颈癌323例疗效对比观察

目的:对比观察紫杉醇联合顺铂(DDP)+5-氟尿嘧啶(5-FU)化学治疗晚期宫颈癌的疗效及毒副作用。方法:观察组323例:化疗第1天:紫杉醇150～180mg静脉滴注3h;第2～第6天每天经动脉导管推注5-FU 500mg、DDP20mg;对照组176例:在第1～第7天每天经动脉导管推注5-FU 500mg、DDP 20mg。两组均3～4周为1疗程,连续2～3个疗程。结果:化疗结束后4周检查,观察组中78例完全缓解,213例部分缓解,27例病情稳定,5例恶化,有效率(完全缓解+部分缓解)90．1%;54例因病灶明显缩小,接受了宫颈癌根治术;观察组总的5年生存率71%。对照组化疗结束后4周检查,176例中17例完全缓解,131例部分缓解,26例病情稳定,2例恶化,有效率为84．1%;14例因病灶明显缩小,接受了宫颈癌根治术;对照组总的5年生存率40%。两组近期疗效比较,差异无统计学意义(P＞0．05．);但两组5年后总生存状况比较,差异有显著统计学意义(P＜0．01)。结论:紫杉醇联合DDP、5-FU治疗晚期宫颈癌的疗效明显优于DDP+5-FU。     

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.     

三磷酸腺苷-肿瘤体外药敏试验联合耐药基因检测预测原发性卵巢癌的化疗敏感性

目的 评价三磷酸腺苷-肿瘤体外药敏试验(ATP-TCA)联合耐药基因检测预测原发性卵巢癌的化疗敏感性,为临床治疗提供参考.方法 选择2008-2009年间收治的接受肿瘤细胞减灭术的原发性卵巢癌患者47例,采用ATP-TCA检测卵巢癌患者对顺铂、卡铂、紫杉醇、紫杉醇+卡铂4种方案的体外药物敏感性,采用实时逆转录(RT)PCR技术检测其中46例(1份冻存的癌组织标本因降解较严重未予检测mRNA)卵巢癌患者的癌组织中耐药基因--BRCA1、ERCC1 mRNA的表达水平.采用x2检验分析ATP-TCA检测结果 与卵巢癌患者临床病理指标的相关性;采用ATP-TCA联合耐药基因检测预测卵巢癌患者对紫杉醇+卡铂方案的化疗敏感性.结果 (1)卡铂、顺铂、紫杉醇+卡铂3种方案的ATP-TCA检测结果 与临床疗效均有明显的相关关系(P<0.05),其中紫杉醇+卡铂方案的ATP-TCA检测结果 与临床疗效的符合率最高,达83%(39/47);而紫杉醇方案的ATP-TCA检测结果 与临床疗效无明显相关关系(P>0.05).紫杉醇+卡铂方案的ATP-TCA检测结果 预测临床疗效的灵敏度、特异度、阳性预测值、阴性预测值分别为90%、71%、84%、80%.紫杉醇+卡铂方案的ATP-TCA检测结果 与残瘤灶直径(P=0.023)、有无新辅助化疗(P=0.011)有明显相关关系.(2)临床化疗敏感和耐药患者BRCA1 mRNA的表达水平分别为0.673±2.143和-1.436±2.594,两者比较,差异有统计学意义(P=0.008);ERCC1 mRNA的表达水平分别为-0.529±1.982和-3.188±2.601,两者比较,差异也有统计学意义(P=0.001);BRCA1和ERCC1 mRNA的表达与临床疗效有明显的相关关系(P<0.01).(3)ATP-TCA联合耐药基因检测,预测临床应用紫杉醇+卡铂方案可能会发生耐药的准确率达到8/9.结论 ATP-TCA是一种比较理想的体外药敏检测方法,可以有效地预测临床化疗敏感性;ATP-TCA联合耐药基因检测可以提高预测卵巢癌临床化疗敏感性的准确率,指导卵巢癌的个体化治疗.

Abstract：

Objective To predict clinical chemotherapy sensitivity of primary ovarian cancer by jointing adenosine triphosphate(ATP) - tumor chemo-sensitivity assay(TCA) method in vitro and detection of drug resistance genes, provide reference for clinical treatment. Methods Forty-seven primary epithelial ovarian tumor samples were collected from the patients who received cytoreductive surgery. Viable ovarian cancer cells obtained from malignant tissue were tested for their sensitivity to carboplatin (CBP), cisplatin (DDP), paclitaxel(PTX) and CBP + PTX using ATP-TCA method in vitro; at same time, real-time quantitative PCR was used to analysis BRCA1 and ERCC1 mRNA relative expression in forty-six specimens (1 frozen tumor samples mRNA were not detected due to serious degradation). The relationship between ATP-TCA test results, clinical indicators, and the effectiveness of the joint prediction on clinical chemosensitivity by combining these two methods were statistically analyzed using chi-square test. Results (1)The results showns that three programs of DDP,CBP and PTX + CBP were significantly related with clinical results(P<0.05) in vitro, in which the compliance rate in PTX + CBP program was the highest 83%(39/47) ,and the predictive sensitivity, predictive specificity, positive predictive value, negative predictive value and predictive accurate rate were 90%,71%,84% and 80% ,respectively.PTX + CBP combined in vitro test results was also related with residual tumor size and neoadjuvant chemotherapy, which was more prone to drug resistance with residual tumor larger than 2 cm (P = 0. 023) and with neoadjuvant chemotherapy (P = 0.011). (2) BRCA1 mRNA expression levels in the clinical-resistant group and the clinical-sensitive group was 0.673 ± 2.143 and - 1.436 ± 2.594 (P=0.008), ERCC1 mRNA expression levels in the clinical-resistant group and the clinical-sensitive group was -0.529 ± 1.982 and - 3.188 ±2.601 (P =0.001). There were also significant correlation among the expression levels of BRCA1 ,ERCC1 mRNA and clinical efficacy (P<0.01). (3)ATP-TCA and detection of drug resistance genes combined to predict the clinical application of PTX + CBP resistance may occur in 8/9 cases. Conclusions ATP-TCA may be an ideal method of in vitro drug sensitivity testing method, which could effectively predict clinical chemotherapy sensitivity. Combination of the drug-resistant associated genes detection method and the ATP-TCA method can increase the predictive effectiveness of ovarian cancer chemosensitivity and guide individual chemotherapy of ovarian cancer.     

Reversal of multidrug resistance by small interfering double-stranded RNAs in ovarian cancer cells

OBJECTIVE: Cisplatin (DDP) resistance is a major barrier to overcome before chemotherapy can become curative for most patients presenting with ovarian cancer. In this study, we investigated the effect of siRNAs on expression of p-gp, GST-pi mRNA and protein in cisplatin-resistant human ovarian cancer cells in order to restore sensitivity to DDP. METHODS: Small interfering double-stranded RNAs (siRNA) were designed to target p-glycoprotein (p-gp) and glutathione S-transferases (GST) mRNA as a strategy to inhibit both resistant gene expression at the mRNA level. Using Real-Time PCR and western blotting assay the changes of the RNA and protein levels of both drug resistant genes were studied. RESULTS: Transfection of MDR-1 and GST siRNAs into human multi-drug resistance (MDR) ovarian cancer cell lines, COC1/DDP and SKOV3/DDP, resulted in a time-dependent inhibition of both gene expressions with the decline of the IC(50) values but had no effect on the expression of a-Tubulin. Inhibition of P-gp and GST expression by siRNA enhanced the intracellular accumulation of and restored sensitivity to DDP. CONCLUSIONS: These studies suggest that p-gp and GST siRNAs are effective inhibitors of MDR gene expression and reverse the resistance of ovarian carcinomas. Our studies may provide a new insight to develop siRNAs as a novel therapeutic tool for the treatment of ovarian carcinomas.     

In vitro studies of 5-FU sensitivity on uterine cervical cancer cell lines--comparison between squamous cell carcinoma and adenocarcinoma

In order to improve the postoperative survival rate of patients with cervical cancer, we have treated them with adjuvant chemotherapy (oral Tegafur) and proved this treatment to be useful. However, the prognosis of cervical adenocarcinoma cases has not been improved yet. In this study, 5-FU sensitivity, morphological changes and DNA metabolism of cultured cervical cancer cells were examined using OMC-1 and OMC-4 cell line originating from cervical squamous cell carcinoma and adenocarcinoma, respectively. The EC 50 (Effective Concentration for 50% Cell Kill) of 5-FU on OMC-1 and OMC-4 cells after 96 hours of incubation with 5-FU was 0.13 micrograms/ml and 9.1 micrograms/ml, respectively. The morphological changes were more prominent in OMC-1 cells than in OMC-4 cells after 192 hours of incubation with 0.1 micrograms/ml of 5-FU. The incorporation of 3H-deoxyuridine into the DNA was inhibited more significantly in OMC-1 cells than in OMC-4 cells even at a low concentration of 5-FU. The intracellular FdUMP and thymidylate synthetase (TS) inhibition rate of OMC-1 and OMC-4 after 96 hours of incubation with 0.1 microgram/ml of 5-FU was 4.7 pmol/g and less than 2.6 pmol/g, 58.7% and 60.0%, respectively. These results suggest that 5-FU sensitivity of cervical adenocarcinoma cell line (OMC-4) is lower than that of cervical squamous carcinoma cell line (OMC-1) and it may owe much not to the TS inhibition rate but to the intracellular FdUMP.     

RNAi抑制宫颈癌HeLa细胞survivin基因及其对顺铂敏感性影响的研究

目的:利用RNA i技术抑制宫颈癌HeLa细胞株Survivin基因,观察HeLa细胞株的细胞凋亡率、survivin蛋白表达及对顺铂(DDP)药物敏感性的变化。方法:以培养的宫颈癌HeLa细胞株为体外研究模型,设脂质体组、对照组、RNA i组进行实验。(1)半定量RT-PCR,W estern blot法检测Survivin mRNA、蛋白在各组细胞中的表达;(2)用流式细胞术分析各组细胞周期及细胞凋亡率的影响;(3)用MTT法分析DDP对各组细胞存活率作用的浓度。结果:(1)RT-PCR检测结果显示,Survivin扩增条带(206bp)的亮度差异显著,RNA i组明显弱于脂质体组及对照组。RNA i组Survivin mRNA相对含量较质脂体组明显下降,差异有统计学意义(P<0.01);W estern blot检测各实验组Survivin蛋白的表达,脂质体组与对照组Survivin蛋白条带明显存在,且基本一致,而RNA i组同样位置的条带基本消失;(2)RNA i组细胞阻滞在G0/G1期,G/M期减少,与脂质体组和对照组分别比较,差异有统计学意义(P<0.01);(3)MTT比色法检测结果显示,不同浓度顺铂处理后脂质体组、对照组、RNA i组的IC50分别为(0.298±0.035)、(0.147±0.031)、(0.012±0.001),RNA i组HeLa细胞对顺铂的药物敏感性增高。两者相比差异有统计学意义(P=0.000)。结论:RNA i明显抑制了Survivin在转录及翻译水平的表达;RNA i抑制Survivin基因表达,能明显提高宫颈癌HeLa细胞对顺铂的药物敏感性。     

卵巢恶性肿瘤组织谷胱甘肽S转移酶的表达及其与化疗耐药的关系

目的检测卵巢肿瘤组织中谷胱甘肽Ｓ转移酶（ＧＳＴ－π）的表达,并探讨其与化疗耐药的关系。方法采用免疫组化ＳＰ法对５３例卵巢恶性肿瘤、２０例卵巢良性肿瘤和１７例正常卵巢组织进行ＧＳＴ－π检测,并对相关的临床病理因素进行分析。结果（１）卵巢恶性肿瘤组织中ＧＳＴ－π表达阳性率为６０．４％,卵巢良性肿瘤组织中ＧＳＴ－π表达阳性率仅为１０．０％,而正常卵巢组织中则无一例ＧＳＴ－π表达阳性;（２）不同病理类型、临床分期和术后残留灶大小的恶性肿瘤组织中ＧＳＴ－π表达阳性率相比较,差异无显著性;（３）恶性肿瘤中,初治和复发的ＧＳＴ－π表达阳性率分别为４７．２％和８８．２％;（４）ＧＳＴ－π表达阳性的患者对化疗的有效率低于ＧＳＴ－π表达阴性的患者,前者为３７．５％（１２／３２）,而后者为７６．２％（１６／２１）;（５）ＧＳＴ－π表达阳性的患者治疗后１年及３年生存率低于ＧＳＴ－π表达阴性者。结论卵巢恶性肿瘤组织中ＧＳＴ－π表达与其化疗耐药有关。ＧＳＴ－π表达的检测对临床判断化疗敏感性和预后有一定的参考价值。     

65例早期宫颈癌耐药基因产物与临床预后关系的初步探讨

目的:探讨耐药基因产物P-糖蛋白(P-gp)、谷胱甘肽-s-转移酶(GST-π)、拓扑异构酶Ⅱ(TopoⅡ)、胸苷酸合成酶(TS)在宫颈癌中的表达及临床意义.方法:应用免疫组织化学方法检测65例宫颈癌中P-gp、TopoⅡ、GST-π、TS的表达,并分析其与临床预后之间的关系.结果:P-gp的表达与肿瘤分化程度、淋巴结转移有关(P<0.05),与UICC分期、局部复发无关(P>0.05);TopoⅡ的表达与淋巴结转移、局部复发有关(P<0.05),与肿瘤分化程度、UICC分期无关(P>0.05);GST-π的表达与各个指标之间均有关(P<0.05);TS与淋巴结转移、UICC分期有关(P<0.05),与局部复发、肿瘤分化无关(P>0.05)。经多因素Cox比例风险模型分析显示:局部复发、淋巴结转移及TopoⅡ、GST-π的表达与宫颈癌患者术后生存率有关(P<0.05)。结论:联合检测宫颈癌中P-gp TopoⅡ、GST-π、TS对判断宫颈癌的预后十分必要;局部复发、淋巴结转移及TopoⅡ、GST-π的表达可作为宫颈癌患者独立的预后因素。     

Chemotherapy of ovarian cancer based on in vitro (HTCA) and in vivo (nude mice) chemosensitivity testing

The human tumor clonogenic assay (HTCA) and the human tumor xenograft system implanted in nude mice were performed simultaneously in an ovarian cancer patient as chemosensitivity testing. Eight anticancer drugs (5-FU, MMC, VCR, ACD, BLM, VLB, CDDP, and ADM) were applied to the HTCA and the human tumor xenograft system. In the HTCA, 5-FU and MMC were sensitive, VCR was moderately sensitive, and ACD, BLM, VLB, CDDP, and ADM were resistant. In the human tumor xenograft system, MMC, VCR, and ADM showed tumor regression (++), and CDDP, VLB, BLM, 5-FU, and ACD exhibited no response (-). Two of the three drugs, which were classified as sensitive or intermediately sensitive in the HTCA, showed tumor regression (++) in the human tumor xenograft system. And four of the five drugs, which were resistant in the HTCA, exhibited no response (-) in the human tumor xenograft system. Clinically, PVB therapy (CDDP, VLB, and BLM) was applied to the present patient, but after recurrence, 5-FU + MMC therapy was applied on the basis of the results of the HTCA. In addition, ADM was added with reference to the results of the human tumor xenograft system. As a result of this therapy, the tumor growth was inhibited. It is possible from the present data that simultaneous chemosensitivity testing of the HTCA and the human tumor xenograft system implanted in nude mice is very useful when choosing sensitive anticancer drugs.