目的 探讨小干扰RNA(siRNA)沉默核糖核苷酸还原酶M2(RRM2)基因表达对入骨肉瘤细胞Saos-2生物学影响及分子机制.方法 利用小干扰RNA技术,观察沉默Saos-2细胞中RRM2基因的表达,用实时荧光定量PCR(Real time-PCR)及Western blot技术检测人骨肉瘤细胞Saos-2和人正常成骨细胞hFOB1.19的mRNA及蛋白的表达；用CCK-8方法检测si-RRM2对Saos-2细胞增殖的影响；用Transwell细胞迁移系统检测si-RRM2对Saos-2细胞迁移能力的影响；用酶联免疫吸附试验(ELISA法)检测Saos-2细胞凋亡；采用Western blot技术检测细胞周期调控因子细胞周期素D1(Cyclin D1)和抗凋亡蛋白B细胞淋巴瘤/白血病-2(Bcl-2)蛋白水平变化.结果 Saos-2细胞RRM2 mRNA和蛋白比hFOB1.19细胞高表达；用si-RRM2转染Saos-2细胞后,Real time-PCR结果证实能时间及剂量依赖性下调RRM2基因表达；CCK-8方法结果显示下调RRM2基因会时间及剂量依赖性抑制Saos-2增殖,而人正常成骨细胞增殖抑制没有显著变化；Transwell细胞迁移系统检测显示下调RRM2基因后,显著抑制了Saos-2的迁移能力；si-RRM2联合化疗药物阿霉素促进Saos-2细胞凋亡；Western blot结果显示沉默RRM2后细胞周期调控因子Cyclin D1和抗凋亡蛋白Bcl-2均下调.结论 RRM2的过表达与人骨肉瘤细胞Saos-2的高增殖和迁移能力有关,抑制RRM2的功能是治疗人骨肉瘤的一个潜在的治疗策略.

Objective To investigate the effect of small interference RNA(siRNA) targeting RRM2 on the biological behavior of human osteosarcoma cell line Saos-2 and the molecular mechanisms.Methods RRM2 expression was knocked down in human osteosarcama cell line Saos-2 by RRM2 siRNA.The expression of RRM2 mRNA and protein was determined in human osteosarcoma cell line Saos-2 and human osteoblast-like cell line hFOB1.19 by real time-PCR and Western blot.The cell proliferation was detected by CCK-8.The migration was observed by using transwell system.The apoptotic rate was observed by ELISA.The expression of CyclinD1 and Bcl-2 proteins were detected by Western blot.Results The expression of RRM2 mRNA and protein was higher in Saos-2 than in hFOB1.19.siRRM2 could down-regulate the expression of RRM2 in Saos-2 cells in a time-and concentration-dependent manner.CCK-8 assay showed that si-RRM2 could inhibit the proliferation ability of Saos-2 cells in a time-and concentrationdependent manner,but had no

目的：探讨非小细胞肺癌组织乳腺癌易感基因1(breast cancer susceptibility gene1，BRCA1)、切除修复交叉互补基因1(excision repair cross-complementing gene1，ERCC1)、核苷酸还原酶 M1亚基(ribonucleotide reductase M1， RRM1)蛋白表达与肿瘤体外药敏的相关性，为非小细胞肺癌个体化治疗提供预测指标。方法：收集32例非小细胞肺癌患者手术切除的新鲜标本，采用ATP-TCA方法检测紫杉醇、顺铂、吉西他滨体外药敏；采用免疫组化PV方法检测BR-CA1、ERCC1、RRM1蛋白表达。结果：BRCA1高表达18例(56.2％)，低表达14例(43.8％)，BRCA1高表达组紫杉醇有效13例(72.2％)，BRCA1低表达组有效4例(28.6％)(P<0.05)；ERCC1高表达13例(40.6％)，低表达19例(59.4％)，ERCC1高表达组顺铂有效2例(15.4％)，ERCC1低表达组有效11例(57.9％)(P<0.05)；RRM1高表达15例(46.9%)，低表达17例(53.1%)， RRM1高表达组吉西他滨有效3例(20.0%)，RRM1低表达组有效10例(58.8%)(P<0.05)。结论：BRCA1、ERCC1、RRM1蛋白表达水平和紫杉醇、顺铂、吉西他滨体外药物敏感性具有相关性，联合检测为非小细胞肺癌筛选化疗药物提供更加可靠的依据。

Obj ective: To explore the relationship between BRCA1、ERCC1、RRM1 protein expressions of non-small-cell lung cancer and sensitivity of the cancer to chemotherapeutic drug in vitro, and provide prognostic markers to guide individu-al chemotherapy in non-small-cell lung cancer field. Methods: 32 fresh operative non-small-cell lung cancer samples were collected and immediately were used to be tested the sensitivity to paclitaxel,cisplatin,gemcitabine with the method of ATP-tumor chemosensitivity assay;detected expressions of BRCA1, ERCC1, RRM1 in 32 cases of non-small-cell lung cancer paraffin-embedded pathological specimens by means of immunohistochemical analysis(PV method). Results: There were 18 (56.3%)cases with the high level of BRCA1 protein expression, and still another 14 cases(43.8%) with the low level of BR-CA1 protein expression, there were 13 cases(72.2%) with sensitive to paclitaxel in the high level of BRCA1 protein expres-sion group,and 4 sensitive cases (28.6%)

切除修复交叉互补基因1(exsicion repair cross-complementing group 1,ERCC1)和核糖核苷酸还原酶M1(ribonu?cleotide reductase M1,RRM1)的表达水平可预测非小细胞肺癌(non-small cell lung cancer,NSCLC)对化疗的敏感性.旨在评价免疫组织化学检测的蛋白水平与液相芯片检测的mRNA水平的临床应用价值.方法:采用免疫组织化学和液相芯片方法分别检测42例非小细胞肺癌组织中ERCC1、RRM1的蛋白表达和mRNA表达水平,非参数相关性分析二者表达的相关性.结果:两种方法检测的非小细胞肺癌组织中ERCC1和RRM1蛋白表达与mRNA表达的一致率分别为52.3%(22/42)和76.2%(32/42);两种方法检测RRM1和ERCC1蛋白表达和mRNA表达呈正相关(r=0.778,P=0.010;r=0.277,P=0.076).结论:免疫组织化学检测的RRM1和ERCC1的蛋白水平与液相芯片检测的mRNA水平具有较好的一致性和相关性,二种方法同时检测可以增加临床应用的敏感性和特异性.

Objective:With the treatment of non-small cell lung cancer (NSCLC) entering the era of individualization through the detection of expression levels of promising biomarkers in NSCLC tumor samples, tailored chemotherapy, and prognosis evaluation for NSCLC patients are now possible. This study focuses on two commonly utilized detection methods:immunohistochemistry (IHC) and suspension biochip. This study aims to determine the differences between IHC and suspension biochip with regard to the expres-sion levels of biomarkers. Methods:Forty-two NSCLC patients were enrolled into the study from April 2011 to June 2012. The expres-sion levels of excision repair cross-complementing group 1(ERCC1) and ribonucleotide reductase (RRM1) in the tumor samples were tested through IHC and suspension biochip, respectively. The statistical methods used in the analysis were were performed with the SPSS (version 17.0) statistical software;P<0.05 was considered statistically significant. Results:The

目的 探讨药物相关分子靶标检测在儿童颅外恶性实体瘤个体化治疗中的作用和意义.方法 将2011年4月至2013年4月期间于上海交通大学医学院附属新华医院治疗的38例难治性儿童颅外恶性实体瘤的手术标本及外周血,通过免疫组织化学、酶联免疫吸附试验、荧光原位杂交、聚合酶链式反应扩增和测序的方法检测肿瘤药物相关分子靶标.共检测15种化疗药物和2种靶向药物的基因表达或突变.结果 入组的38例标本中,35例为手术标本,其中3例为二次手术标本、3例为外周血标本送检肿瘤药物相关分子靶标.检测了肿瘤标本化疗药物分子靶点DNA拓扑异构酶ⅡA(TopoⅡA)34例、微管蛋白β3(Tubulinβ3) 32例、人切除修复交叉互补基因1(ERCC1) 28例、DNA拓扑异构酶Ⅰ(TOPO Ⅰ)28例、DNA修复蛋白O6-甲基鸟嘌呤-DNA-甲基转移酶(MGMT) 20例、胸苷酸合成酶(TS) 13例、核糖核苷酸还原酶M1 (RRM1)6例.68.75％对氟尿嘧啶及培美曲赛敏感,66.67％对吉西他滨敏感,56.25％对长春碱类敏感;而67.65％对蒽环类/依托泊苷敏感性低,64.29％对铂类药物、拓扑替康/伊立替康敏感性低,63.64％对替莫唑胺敏感性低.外周血标本化疗药物分子靶点33例CYP2C9*3、14例二氢叶酸还原酶(DHFR C829T)基因多态性PCR测序,结果显示100.00

Objective To identify the significance of genomic markers of drug sensitivity for individualized therapy in children with extracranial malignant solid tumors.Methods The surgical specimens and peripheral blood samples in 38 children with refractory extracranial malignant solid tumors were collected between Apri.2011 and Apri.2013.The genomic markers of anticancer drug sensitivity were examined,including gene expression or mutation of 15 chemotherapeutic agents and two targeted drugs by methods of immunohistochemical staining,enzyne linked immunosorbent assay,fluorescence in situ hybridization,polymerase chain reaction amplification and sequencing.Results Among all of 38 samples,35 were firstly surgical specimens and 3 secondary surgical specimens,and 3 of them were peripheral blood samples.The tumor tissue of the targets for chemotherapeutic agents such as topoisomerase Ⅱ A (TOPO Ⅱ A) in 34 cases,Tubulinβ3 in 32 cases,excision repair cross-complementing 1 (ERCC1) in 28 cas