HBx蛋白羧基端30个氨基酸缺失对肝癌细胞生物学行为的影响

目的:探讨羧基端缺失了30个氨基酸的乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)突变体ΔHBx和野生型HBx对肝癌细胞Huh7生物学行为的影响。方法:脂质体介导pcDNA3ΔHBx和pcDNA3HBx重组体转染人HBV(-)的肝癌细胞Huh7。PCR扩增Neo基因及Western blot检测转染重组体。运用细胞生长曲线绘制、平板克隆形成、流式细胞仪和裸鼠成瘤实验对转染pcDNA3ΔHBx、pcDNA3HBx和pcDNA3的细胞生物学活性进行检测。结果:pcDNA3ΔHBx组细胞生长速度较pcDNA3HBx组和pcDNA3组减慢,其倍增时间延长;pcDNA3ΔHBx组克隆形成率较pcDNA3HBx组和pcDNA3组下降;细胞周期检测显示pcDNA3ΔHBx表达使Huh7细胞G0/G1期→S期的进程明显减慢。结论:HBx羧基端缺失了30个氨基酸的突变体比野生型HBx具有明显抑制细胞增殖的能力,提示该区域对于HBx功能发挥起着至关重要的作用。     

HBx蛋白羧基端缺失对肝癌细胞生物学行为的影响

背景与目的:肝癌组织内普遍存在着截短表达的HBx蛋白,这种蛋白可能与肝癌的发生有关,本研究探讨截短表达的HBx蛋白和野生型HBx对人肝癌细胞Huh7生物学行为的影响。方法:脂质体法介导HBx突变体和野生型HBx重组体转染HBV(-)的Huh7细胞。PCR方法扩增Neo基因检测质粒DNA片段插入。通过MTT、平板克隆形成实验、流式细胞仪和裸鼠成瘤实验检测稳定转染细胞的生物学活性。结果:HBx3′-20和HBx3′-40组细胞生长速度较HBx3′-30组明显增快(P<0.05);HBx3′-20组[(17.34±2.77)%]和HBx3′-40组[(18.36±2.61)%]克隆形成率明显较HBx3′-30组[(7.31±1.44)%]和pcDNA3组[(6.87±2.38)%]高(P<0.05)。细胞周期检测结果显示,对比于野生型HBx组,HBx3′-20组和HBx3′-40组蛋白的表达能加速Huh7细胞由G0/G1期到S期的进程[S期:(36.96±1.82)%vs.(46.20±3.23)%,(53.99±4.02)%,P<0.05],相反,HBx3′-10和HBx3′-30组则出现G1期阻滞,而HBx组与pcDNA3空载体组间[(38.60±1.15)%]在S期无明显变化。裸鼠成瘤实验显示,HBx3′-40组[(3.19±0.34)cm3]成瘤体积明显大于HBx3′-30组[(1.58±0.27)cm3]、HBx组[(1.75±0.15)cm3]和pcDNA3组[(1.67±0.12)cm3],各组瘤重存在显著性差异(P<0.01)。结论:对比野生型HBx组,HBx3′-20和HBx3′-40对Huh7细胞的生长具有明显促增殖作用,HBx3′-30组显示出抑制作用。HBx通过缺失突变修饰其生物学功能,在原发性肝癌发生、发展过程中起着重要作用。     

吲哚美辛抗人结肠癌HCT116细胞双向电泳蛋白质表达谱的差异分析

目的分析吲哚美辛(indomethacin,IN)对人结肠癌细胞系HCT116蛋白质表达谱的影响,为研究IN抗结肠癌作用的相关蛋白提供新的方法.方法应用固相pH梯度(immobilized pH gradient,IPG)双向凝胶电泳技术(two-dimensional gel electrophoresis,2-DE)分离IN-处理组和未处理组HCT116细胞的总蛋白,银染显色,ImageMaster 2D图像分析软件比较分析IN-处理组和未处理组HCT116细胞系的双向凝胶电泳图谱并识别差异表达的蛋白质.结果获得了背景清晰、分辨率高、重复性好的HCT116细胞系的双向凝胶电泳图谱,IN-未处理组与处理组蛋白质点数为:1256±50,1169±36;匹配率为:93.0%,90.6%.IN-未处理组的3块胶在蛋白质点位置上有较好的重复性,不同胶间蛋白质点在等电聚焦(isoelectric focusing,IEF)方向的偏差是0.896±0.177 mm,在十二烷基磺酸钠聚丙烯酰胺凝胶电泳(sodiumdo-decylsufate-polyacrylamide gelelectrophoresis,SDS-PAGE)方向的偏差为1.106±0.289 mm.比较分析IN-处理组和未处理组HCT116细胞的双向凝胶电泳图谱,发现IN-处理组和未处理组共有45个差异点,IN-处理组有9个蛋白质点表达上调,34个蛋白质点表达下调,2个蛋白质点仅在IN-未处理组表达.结论首次建立了IN-处理和未处理HCT116细胞的双向凝胶电泳图谱,识别了45个差异表达的蛋白质点,对这些差异蛋白质点的鉴定将为揭示IN抗结肠癌作用的机理提供实验依据.     

nm23-H1基因转染前后人高转移大细胞肺癌细胞株的比较蛋白质组学研究

背景与目的肿瘤转移抑制基因nm23-H1基因调控肺癌细胞转移潜能的确切机理尚未明了,本研究旨在探讨人高转移大细胞肺癌细胞株(L9981)和转染nm23-H1基因的人高转移大细胞肺癌细胞株(L9981-nm23-H1)的差异表达蛋白,为阐明肺癌转移的分子机制、发现早期诊断肺癌转移的分子标志和新的治疗靶点提供实验依据。方法应用固相pH梯度双向凝胶电泳分离L9981和L9981-nm23-H1细胞株的总蛋白,对25个差异明显的蛋白质点进行质谱鉴定和生物信息学分析。结果研究观察到nm23-H1基因转染使人L9981细胞株蛋白质组的表达谱发生了明显变化:5个蛋白质表达缺失,9个新的蛋白表达,16个蛋白质表达下调,12个蛋白质表达上调。这些蛋白质主要涉及细胞骨架蛋白、信号转导蛋白、细胞代谢相关蛋白、发育增殖相关蛋白及肿瘤侵袭相关蛋白。结论 nm23-H1基因转染L9981后,蛋白质表达谱发生了显著的变化,这些差异蛋白质可能是逆转肺癌侵袭转移的生化基础,本研究结果可能为阐明肺癌转移的分子机制提供线索。     

RNA干扰PNAS-2后对细胞周期相关基因和蛋白表达的影响

目的:研究RNA干扰抑制白血病细胞株U937中PNAS-2基因表达后,对细胞周期相关基因和蛋白质表达的影响.方法:将已构建的靶向抑制PNAS-2的干扰组质粒和对照组质粒分别转染U937细胞;FCM法检测2组间细胞周期的变化;采用基因芯片和蛋白质芯片检测比较2组细胞在基因水平和蛋白质表达水平上的差异.结果:FCM检测结果显示干扰组细胞出现G2/M期阻滞,与对照组比较差异有统计学意义;基因芯片筛选结果提示共有9条与细胞周期相关的基因发生变化,上调6条,下调3条;蛋白质芯片筛选发现有29条与细胞周期相关的蛋白表达发生变化,上调25条,下调4条.结论:RNA干扰抑制PNAS-2基因后,U937细胞周期出现G2/M期阻滞.基因芯片和蛋白质芯片检测结果均显示cyclin A表达上调;p53只有蛋白表达上调,而cyclin B则蛋白表达不变,提示这些变化可能与细胞周期的阻滞有关.     

HBx基因转染对Huh7细胞中基因转录的影响

目的：研究且酞基因对肝癌细胞Huh7中基因表达的影响，为探讨且戳在肝癌发生发展中的分子机制提供依据。方法：以pcDNA3．1b质粒为载体，将HBx转入Huh7细胞中，同时将pcDNA3．1b作空白对照。用Western印迹法证实基因转染成功。应用Affymetrix公司的人类全基因组寡核苷酸基因芯片分析转染HBx基因前后，Huh7细胞的基因表达谱。随机选择转染前后有明显差异的6个基因（3个上调，3个下调），用荧光定量PCR技术进行验证。结果：从基因芯片结果分析中发现，与对照组相比,HBx转染后Huh7细胞中明显上调的基因有88个，下调的基因有111个，这些差异基因的功能涉及多个方面。结论：HBx基因对肝癌细胞的影响是双向的，既有转录激活的作用，同时又有转录抑制的功能。这些数据对于理解HBx基因在肝癌发生发展中的作用具有重要的参考意义。     

肺癌骨转移血清蛋白质组学研究

目的:应用蛋白质组学双向荧光差异凝胶电泳和质谱技术筛选肺癌骨转移血清标志.方法:收集肺癌伴骨转移、无骨转移各24例血清样本.同组血清等量混合,用除白蛋白试剂盒除去血清中的白蛋白,再经除盐浓缩后,将4组处理后的血清样品用不同的CyDye染料交叉标记后进行双向荧光差异凝胶电泳(2-D DIGE).利用DeCyder软件分析,将肺癌伴骨转移组与肺癌无骨转移组比较的差异蛋白质点行基质辅助激光解析离子化飞行时间质谱(MALDI-TOF-MS)鉴定.结果:双向荧光差异凝胶电泳显示,肺癌骨转移组血清蛋白中有16个斑点,与肺癌无骨转移组相比差异有统计学意义.其中11个蛋白质表达水平显著增加,选取5个蛋白质表达水平显著下降.5个差异蛋白点进行胶内原位酶解、肽质指纹图谱分析,成功得到4个蛋白质肽质指纹图,并查询数据库初步鉴定该4个蛋白质分别为视黄醇结合蛋白-甲状腺素运载蛋白复合物(RBP-TTR)、结合殊蛋白(Hp)、S-亚硝基血红蛋白(SNO-Hb)和de-sArg141 α Hb.结论:双向荧光差异凝胶电泳结合质谱鉴定是血清差异蛋白质组学研究的可靠平台和有力工具.所鉴定出的蛋白质可能与肺癌骨转移的发生、发展有关,有助于肺癌骨转移发病机制的了解.     

结直肠癌化疗敏感性相关蛋白的蛋白质组学初步研究

目的:本研究应用蛋白质组学技术建立化疗敏感性不同的结直肠癌组织总蛋白双向凝胶电泳(two-dimensional gel electrophoresis,2-DE)图谱,并鉴定部分差异表达蛋白,以发现与结直肠癌化疗敏感性有关的蛋白.方法:收集临床诊断为晚期结直肠癌的病例,肠镜活检获取新鲜结直肠癌标本后液氮保存备用,根据肿瘤药物敏感实验分为化疗高敏感组和化疗低敏感组.提取组织总蛋白,采用双向凝胶电泳技术得到各组凝胶图谱;采用PD-quest 7.3软件进行图像的合成、对比和差异分析,识别化疗高敏感组和化疗低敏感组之间差异表达的蛋白斑点;选取差异蛋白质点,胶内酶解后行肽指纹图分析及网上数据库检索,鉴定差异蛋白质;应用Western印迹法检测部分差异蛋白的表达情况.结果: 建立了化疗高敏感组和化疗低敏感组的双向凝胶电泳图谱,多数蛋白质点集中于pH 4～8、相对分子质量为(20～100)×10~3.高敏感组和低敏感组的电泳图谱中平均蛋白质点数分别为(842±23)个和(793±19)个,2组平均匹配率为90.7%,2组间差异表达蛋白质点数为(79.00±13.56)个;选择30个差异蛋白质点进行质谱分析,经数据库查询鉴定出9个差异表达蛋白.结论:在化疗敏感性不同的结直肠癌中存在蛋白质表达的差异,这些差异表达蛋白可能与化疗敏感性有关,并可能用于化疗敏感性的预测.     

放疗敏感性不同宫颈癌组织的蛋白质组学比较研究

目的:比较放疗敏感性不同的早期宫颈癌组织之间蛋白质组的差异,为确定宫颈癌放疗敏感性相关标志物提供依据。方法:收集进行治疗前的早期宫颈癌组织标本。根据放疗后WHO实体瘤疗效判断标准,选择其中对放疗敏感和不敏感的标本分为高敏感组和低敏感组。提取组织总蛋白,进行双向凝胶电泳得到凝胶图谱,采用PD-quest 7.0软件进行匹配和差异分析,识别两组之间表达差异蛋白点。将部分差异蛋白点进行胶内原位切割、酶解后进行MALDI-TOF-MS分析,获取肽质量指纹图,数据库搜索鉴定蛋白质。应用Western印迹技术验证筛选的部分差异表达蛋白的表达。结果:建立了分辨率高、重复性好的宫颈癌放疗高敏感组和低敏感组的双向凝胶电泳图谱。高敏感组蛋白点(754±64)个(n=5),低敏感组蛋白点(777±48)个(n=5),组间平均匹配率为87.6%。选择部分差异在2倍以上的蛋白斑点进行质谱分析,8个差异表达蛋白被成功鉴定,其中5个蛋白质在高敏感组高表达,3个蛋白质在高敏感组低表达。Western印迹结果与蛋白质组筛选结果基本一致。结论:放疗高敏感组和低敏感组宫颈癌组织间存在蛋白表达的差异,这些差异表达的蛋白可能与放疗敏感性有关。     

Compliance with guidelines and correlation with outcome in patients with advanced germ-cell tumours

PurposeTo evaluate prior compliance with guidelines in patients treated with salvage chemotherapy for advanced germ-cell tumours (GCT).Patients and methodsData concerning the initial management of patients requiring salvage chemotherapy for GCT at Institut Gustave Roussy between 2000 and 2010 were obtained and correlated with recommendations for treatment. Criteria of non-compliance were defined based on guidelines. Compliance with guidelines, predictive factors for non-compliance and the impact on outcome were analysed.ResultsAmong 82 patients treated in the salvage setting, guidelines to initial treatment were followed in only 41 cases (50%). The most common non-compliance criteria were non-adherence to the planned dose (16%), an inappropriate interval between first-line chemotherapy cycles (16%), the lack of post-chemotherapy surgery (16%) and a long interval to post-chemotherapy surgery (48%). Compliance with standard care was better in cancer centres than in other hospitals (private or public) (Odd Ratio (OR): 6.9, P = 0.001). A poor-risk status according to the International Germ Cell Cancer Collaborative Group (IGCCCG) was also predictive of compliance in univariate but not in multivariate analysis. No significant difference in outcome after salvage chemotherapy was observed. Patients relapsing after non-compliant first-line therapy tended to be more easily salvaged, which is consistent with the fact that their initial treatment was inadequate. Some of these relapses were therefore probably not due to true biologically refractory disease.ConclusionGuidelines for first-line treatment are adhered to in only half the patients requiring salvage chemotherapy. As the only predictive factor for non-compliance was the treating centre, centralisation of patients with GCT in well-trained hospitals should be recommended.     

Living with secondary breast cancer: coping with an uncertain future with unmet needs

JOHNSTON S.R.D. (2010) European Journal of Cancer Care 19 , 561–563 Living with secondary breast cancer: coping with an uncertain future with unmet needs     

Costs associated with complications are lower with capecitabine than with 5‐fluorouracil in patients with colorectal cancer

BACKGROUND:

Capecitabine, an oral alternative to 5‐fluorouracil (5‐FU) in patients with colorectal cancer (CRC), has equal clinical efficacy and a favorable safety profile; however, its use may be limited because of unit cost concerns. In this study, the authors measured the cost of chemotherapy‐related complications during treatment with capecitabine‐ and 5‐FU–based regimens.

METHODS:

Patients with CRC who received at least 1 administration of capecitabine or 5‐FU during 2004 and 2005 were identified from the Thomson MarketScan research databases. Monthly frequency and cost for 23 complications were recorded. Logistic regression was used to predict complication probability. General linear models were used to predict monthly complication cost and total monthly expenditure.

RESULTS:

In total, 4973 patients with CRC met the inclusion criteria for this analysis. Although the most frequently observed complications were the same between capecitabine and 5‐FU (nausea and vomiting, infection, anemia, neutropenia, diarrhea), each was observed with greater frequency in 5‐FU–based regimens. The mean predicted monthly complication cost was significantly higher (by 136%) with 5‐FU monotherapy than with capecitabine monotherapy (difference, $601; 95% confidence interval [95% CI], $469‐$737). In addition, the mean predicted monthly complication cost for 5‐FU+oxaliplatin was higher than the cost with capecitabine plus oxaliplatin (difference, $1165; 95% CI, $892‐$1595). When acquisition, administration, and complication costs were taken into consideration, there were no significant differences in the total cost between capecitabine regimens and 5‐FU regimens.

CONCLUSIONS:

Capecitabine compared well with 5‐FU–based therapy in patients with CRC and was associated with lower complication rates and associated costs. Cancer 2009. © 2009 American Cancer Society.     

Experience with Impedance Phlebography Compared with Venography

Venography is a particularly reliable method for the diagnosis of deep venous thrombosis but is not suitable as a screening test. Impedance phlebography represents another attempt to discover a simple, non-invasive and reliable method of detecting deep venous thrombosis. It does not, however, meet these criteria.     

再放疗并同步使用Docetaxel在鼻咽癌放疗后复发病人中的应用

目的:不能手术切除的鼻咽癌放疗后再复发的病人,其治疗困难,化疗疗效差,而单独再放疗只能挽救一小部分病人,本文探讨再放疗并同步使用多西紫彬醇(Docetaxel)在鼻咽癌首次放疗后复发病人中可行性及毒副反应,并评价其疗效。方法:对11例鼻咽癌足量放疗后经组织病理学证实复发、而无法行手术及腔内放疗的患者进行了同步放化疗。放疗采用三维适形放疗,外照射鼻咽部,分次量为1.8Gy,总剂量为36Gy-39.6Gy。化疗采用Docetaxel,15mg/m2,每周一次,静脉滴注。结果:10%、33%的患者分别出现Ⅲ度、Ⅳ度皮肤反应,18%、10%的病人分别出现Ⅲ度、Ⅳ度黏膜反应,18%患者出现Ⅲ度恶心呕吐,27%的患者出现Ⅲ度-Ⅳ度白细胞下降,10%患者出现Ⅲ度血小板下降。1例患者因严重的黏膜反应致使治疗延迟2周。治疗结束后,9例(82%)患者达到CR,2例(18%)达到PR,反应率为100%。结论:对于放疗后局部复发的鼻咽癌患者,采用同步放化疗,3D-CRT同时每周使用Docetaxel是可行的,其毒性反应在可以接受的范围内,短期疗效显著。