PIWIL 2 在人类膀胱尿路上皮癌中的表达及其功能研究

 目的 研究PIWIL2在人类膀胱尿路上皮癌（BTCC）中的表达及siRNA对人类膀胱癌细胞PIWIL2表达的影响。方法 采用反转录聚合酶链反应（RT-PCR）检测BTCC 46例、腺性膀胱炎21例、癌旁组织17例、正常膀胱组织14例中PIWIL2 mRNA的表达;设计合成针对PIWIL2的3个特异性siRNA,转入人类膀胱癌BIU-87细胞,分别采用四甲基偶氮唑蓝、DNA原位末端标记法、RT-PCR和Western blot检测siRNA对BIU-87细胞生长抑制率（IR）、凋亡指数（AI）和PIWIL2 mRNA及其蛋白表达的影响。结果 BTCC 组织中PIWIL2 mRNA的表达率为76.08 %（35／40）,均显著高于腺性膀胱炎组织[42.86 %（9／21）]、癌旁组织[41.17 %（7／17）]和正常膀胱组织[7.14 %（1／14）]（P＝0.008,P＝0.010,P＝0.000）。BTCC组织中PIWIL2的高表达与肿瘤淋巴结转移、病理分级均密切相关。siRNA1～3组细胞的IR[（37.52±8.84）%、（64.36±9.64）%、（62.94±8.43）%]和AI[（26.18±5.42）%、（38.75±6.19）%、（40.02±5.64）%]均分别显著高于对照组[（1.97±0.02）%、（3.35±0.47）%]（均P＝0.000）,PIWIL2 mRNA及其蛋白表达水平均显著低于对照组;其中siRNA 2、3组细胞的IR、AI和对PIWIL2表达的抑制作用均显著高于siRNA 1组。结论 PIWIL2在BTCC中的高表达,提示其与膀胱癌的发生、发展密切相关;体外转录合成的siRNA可抑制BIU-87细胞PIWIL2的表达,诱导肿瘤细胞凋亡,从而抑制肿瘤细胞生长,提示PIWIL2可作为膀胱肿瘤基因治疗的重要靶点。     

Survivin基因siRNA真核表达载体的构建及其对转染膀胱癌细胞Survivin表达的抑制作用

目的构建针对Survivin基因的siRNA(smallinterferenceRNA)真核表达载体,并观察其对转染的膀胱癌细胞中Survivin基因表达和细胞凋亡的影响。方法应用siRNA设计软件设计针对Survivin基因的特异性短链寡核甘酸,化学合成后经退火形成双链siRNA模板,通过克隆到质粒pSilencer1.0-U6构建siRNA真核表达载体并用酶切和测序鉴定;然后将其转染人膀胱癌细胞株BIU-87,以反义Survivin寡核甘酸(ASODN)抑制效果为对照,分别采用噻唑蓝(MTT)比色法和DNA原位末端标记(TUNEL)法检测BIU-87细胞生长抑制率(IR)和凋亡指数(AI),半定量逆转录聚合酶链反应(RT-PCR)和Westernblot检测BIU-87细胞中SurvivinmRNA及其蛋白的表达。结果酶切和测序证实siRNA真核表达载体构建成功。siRNA对BIU-87细胞的IR和AI(62.14%和33.77%)均分别显著高于ASODN(39.33%和23.98%)和空白对照组(1.98%和3.75%),P均<0.05,SurvivinmRNA及其蛋白相对表达水平均显著低于ASODN和空白对照组。结论构建的siRNA真核表达载体能有效地抑制Sur-vivinmRNA的转录和表达,诱导BIU-87细胞凋亡和抑制细胞生长,为膀胱肿瘤的基因治疗提供新的方法和手段。     

靶向生存素基因siRNA诱导骨肉瘤细胞株U-2OS的凋亡

目的:体外转录合成生存素(survivin)基因siRNA,观察其在骨肉瘤细胞株U-2OS中抑制survivin基因后细胞增殖及细胞凋亡情况.方法:将U-2OS细胞分为A组(空白对照组)、B组(非特异性转染组)、C组(特异性转染组),在荧光显微镜下观察转染前后细胞形态的改变,用RT-PCR和Western blot法分别检测转染前后survivin基因mRNA和蛋白的干涉效果,MTT法检测细胞增殖,流式细胞仪检测细胞凋亡情况,并比较各组结果.结果:荧光显微镜下可见转染后细胞增殖变慢,凋亡细胞增加;RT-PCR结果显示survivin siRNA明显抑制survivin基因mRNA表达.Western blot法检测C组蛋白阳性表达率比A、B两组显著降低(P＜0.01);特异性转染组的细胞增殖缓慢,与其它两组比较有显著性差异(P＜0.01).流式细胞仪检测C组细胞凋亡率与其他两组比较有显著性差异(P＜0.01).结论:体外转录合成特异性survivin siRNA能有效抑制U-2OS细胞中survivin mRNA及蛋白表达,从而抑制细胞增殖,诱导细胞凋亡.RNA干扰技术为骨肉瘤的基因治疗提供了一种新策略.     

锰卟啉光动力学疗法对人膀胱癌BIU-87细胞Bcl-2表达的影响

[目的]研究锰卟啉的光动力学疗法(PDT)对人膀胱癌BIU-87细胞Bcl-2表达的影响,探讨其诱导BIU-87细胞凋亡的机制.[方法]合成水溶性金属卟啉化合物5,10,15,20.四(N-甲基-4-吡啶基)锰卟啉四碘盐(锰卟啉)后,分别采用膜联蛋白(Annexin-V),碘化丙锭(PI)双标记流式细胞术、逆转录聚合酶链反应(RT-PCR)和Western-blotting检测锰卟啉在不同时间(0、10、20、30min)的PDT对人膀胱癌BIU-87细胞的凋亡率和Bcl-2 mRNA及其蛋白质表达水平的影响.[结果]1μmol/L锰卟啉的PDT作用20min和30min后均能诱导BIU-87细胞凋亡,凋亡率分别为9.08%±1.55%、19.15%±2.39%;在诱导细胞凋亡过程中,锰卟啉的PDT能显著抑制BIU-87细胞中Bcl-2 mRNA和相应蛋白质的表达,且锰卟啉的PDT作用对细胞凋亡和Bcl-2表达的影响有显著的光照剂量依赖性.[结论]适宜光照剂量锰卟啉的PDT可以通过抑制凋亡抑制基因Bcl-2的表达来诱导BIU-87细胞发生凋亡,为金属卟啉PDT的临床应用提供了实验依据.     

三氧化二砷诱导K562／ADM细胞凋亡过程中对bcl-2、survivin、ROS表达的影响

[目的]探讨As2O3诱导白血病K562／ADM耐药细胞凋亡的分子机制。[方法]采用MTT比色法检测K562／ADM增殖活性：细胞形态学和Annexin Ⅴ／PI双染色检测细胞凋亡：RT—PCR检测bcl-2、survivin和caspase-3基因mRNA的表达水平；流式细胞法（FCM）测定bcl-2、caspase-3蛋白表达。激光共聚焦显微镜（LCSM）观察细胞内活性氧（ROS）产生情况。[结果]As2O3显著抑制K562／ADM细胞的增殖；经As2O3处理后细胞形态上出现典型的凋亡改变，AnnexinⅤ／PI双染显示凋亡细胞明显增加：凋亡抑制基因bcl-2、survivin mRNA及bcl-2蛋白表达下调，caspase-3 mRNA表达和caspase-3活性显著增强，ROS活性下降。[结论]As2O3诱导K562／ADM耐药细胞凋亡，与bcl-2和survivin表达下调，caspase-3活化有关，而与活性氧的氧化损伤无关。     

VEGF（165） RNA干涉诱导人宫颈癌HeLa细胞凋亡的研究

目的：利用RNA干涉技术抑制肿瘤细胞VEGF165基因表达,探讨其对肿瘤细胞凋亡的诱导作用。方法：设计针对VEGF165基因的小干扰RNA,合成DNA模板,体外转录合成siRNA（small interfering RNA）,采用lipofectamine2000转染人宫颈癌HeLa细胞。通过Hoechst33258染色、流式细胞术、RT-PCR检测人宫颈癌HeLa细胞体外凋亡情况。在体内实验中建立子宫颈癌荷瘤裸鼠模型,将VEGF siRNA直接注入瘤体,观察siRNA对肿瘤生长的影响,蛋白印迹试验检测VEGF siRNA在体内条件下对VEGF、bcl-2蛋白表达的影响。结果：所设计的两个siRNA均能有效诱导细胞凋亡产生凋亡小体,并使细胞周期阻滞于G0/G1期;VEGF165和bcl-2在mRNA及蛋白水平的表达也受到有效抑制,明显减少。结论：针对人VEGF165基因体外转录合成的siRNA在体内外均可诱导HeLa细胞发生凋亡。     

T7体外转录siRNA靶向干扰Pim-2效应观察

[目的]观察T7RNA聚合酶介导体外转录合成siRNA靶向干扰Pim-2基因表达的效应。[方法]利用T7RNA聚合酶介导的体外转录合成靶向Pim-2的siRNAⅠ、Ⅱ、Ⅲ．将其转染入人结肠癌细胞株SW480。利用RT-PCR和Western blot观察Pim-2基因在RNA和蛋白质水平的表达变化。[结果]与对照细胞相比，转染siRNAⅠ、Ⅱ、Ⅲ48h后，Pim-2基因mRNA的抑制率分别为65.4％（P〈0．05），46．2％（P〈0．05）和56．1％（P〈0．05）；转染72h后，Pim-2基闵蛋白表达抑制率分别为61．6％（P〈0．05），45．8％（P〈0．05）和55．6％（P〈0．05）。[结论]T7体外转录合成siRNA，能有效而特异地抑制人原癌基因Pim-2的表达．     

siRNA沉默S100P基因对胰腺癌细胞的生长抑制作用

[目的]探讨小干扰RNA（siRNA）沉默S100P基因后对胰腺癌SW1990细胞株生长的影响。[方法]化学合成针对S100P基因的siRNA,用脂质体转染试剂将siRNA体外转染至人胰腺癌SW1990细胞中,48h后收集细胞,分别提取细胞总RNA和蛋白。用实时定量RT-PCR测定S100P基因mRNA水平的表达;免疫印迹测定S100P蛋白的表达;MTT法测定细胞的生长曲线。[结果]转染siRNA后,S100P基因在mRNA水平上表达减少了77%,蛋白表达降低了74%,均显著性低于对照组。siRNA-S100P明显抑制细胞生长。[结论]应用RNAi技术沉默S100P基因可以降低人胰腺癌SW1990细胞株中S100P表达,抑制细胞生长。     

靶向survivin基因siRNA对宫颈癌细胞放射敏感性影响的实验 

 目的 探讨survivin表达对宫颈癌放射敏感性的影响。 方法 RT-PCR和Western blot法分析survivin基因mRNA和蛋白的表达, 平板集落形成实验检测细胞 增殖,流式细胞术检测细胞凋亡率。 survivin siRNA转染HeLa细胞,比较survivin siRNA对细胞凋亡、增殖以及放射敏感性的 影响。 结果 在宫颈癌HeLa细胞中存在survivin的表达,survivin siRNA可诱导HeLa细胞凋亡并抑制增殖 。 结论 survivin siRNA可通过影响细胞凋亡和增殖来增加HeLa细胞的放射敏感性     

靶向notch1的siRNA对U87-EGFRvⅢ胶质瘤细胞株凋亡和放射敏感度的影响

目的 探讨靶向notch1的siRNA对过表达EGFRvⅢ的U87胶质瘤细胞株U87-EGFRvⅢ凋亡和放射敏感度的影响。方法 利用脂质体转染合成的siRNA转染U87-EGFRvⅢ细胞。用RT-PCR和免疫印迹法来分别检测 notch1的蛋白和mRNA的表达水平;用AnnexinⅤ-FITC/PI流式细胞术检测细胞凋亡情况;用平板集落形成实验检测转染后细胞的放射敏感度。结果 合成的notch1 siRNA可有效抑制notch1在mRNA和蛋白水平的表达（P<0.05）。转染notch1 siRNA作用后细胞凋亡率比对照组明显增加（P<0.05）。平板集落形成实验初步证明notch1 siRNA可提高U87-EGFRvⅢ细胞的放射敏感度。结论 下调notch1基因可促进U87-EGFRvⅢ细胞的凋亡和提高其放射敏感度,为研究notch1基因在肿瘤放疗中的作用及其靶向联合治疗胶质瘤提供基础。     

Prognostic Significance of Inflammation-associated Blood Cell Markers in Nonmetastatic Clear Cell Renal Cell Carcinoma

ObjectivesOur objective was to evaluate the effect of the neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), lymphocyte/monocyte ratio (LMR), and red blood cell distribution width (RDW) on the survival outcomes of nonmetastatic clear cell renal cell carcinoma (ccRCC).Materials and MethodsWe accessed our single-center, urologic-oncologic registry to extract the data for patients who had undergone nephrectomy for nonmetastatic ccRCC. The optimal cutoff for these markers was determined using X-tile software, and survival analyses using Cox regression were performed.ResultsA total of 687 patients had undergone nephrectomy. The optimal cutoffs for NLR, PLR, LMR, and RDW were 3.3, 210, 2.4, and 14.3%, respectively. The NLR, PLR, LMR, and RDW were significantly associated with a larger pathologic tumor size, and stage, more aggressive Fuhrman grade, and the presence of tumor necrosis. After adjusting for age, baseline Eastern Cooperative Oncology Group, pathologic tumor and nodal stage, and Fuhrman grade, only PLR remained an independent prognostic marker for both cancer-specific survival (hazard ratio, 2.69; 95% confidence interval, 1.36-5.33; P = .004) and overall survival (hazard ratio, 2.19; 95% confidence interval, 1.36-3.50; P = .001). When the PLR was included with the Leibovich score and University of California, Los Angeles, integrated staging system, the Harrell’s c-index increased from 0.854 to 0.876 and 0.751 to 0.810, respectively, for cancer-specific survival at 5 years after nephrectomy. When risk stratified by the Leibovich risk group and UCLA integrated staging system, PLR was a significant prognostic factor only within the intermediate- to high-risk groups.ConclusionsPLR is a robust prognostic marker in nonmetastatic ccRCC that clearly outperforms other inflammatory indexes in those who had undergone nephrectomy. However, its prognostic effect was limited in the low-risk category of ccRCC.     

Cell Size Following Irradiation in Relation to Cell Cycle

The cell volume of Ehrlich ascites tumour cells was studied following a radiation dose of 5.0 Gy. The cell volume increased 12 to 30 hours after irradiation by about 20 per cent, was normal at about 50 hours, and increased again at 72 hours. In order to explain these changes the composition of the cells in cell cycle was studied. In addition, the cell volume of irradiated cells from the various parts of the cell cycle, separated by centrifugal elutriation, was measured. The changes in the mean cell volume of unseparated cells could be explained by variations in the cell cycle composition of the cell population. Irradiated cells from the various parts of the cell cycle did not deviate from the volume of non-irradiated cells. The cell volume doubled during the cell cycle. This increase was, however, not linear.     

Changes in Cell Characteristics Due to Culture Conditions in Cell Lines From Human Small Cell Lung Cancer

Eight cultured cell lines were established from human smallcell lung cancers. Every cell line showed the morphologicaland biochemical characteristics of small cell cancer. Changesin cell characteristics were observed in many of these celllines when culture conditions were changed: "oat cell type"changed to "intermediate cell type" and vice versa when serum-freemedium was changed to serum-supplemented medium; a deficiencyof vitamin A in the medium caused a change to squamous cellsand vice versa; and a tumor promoter (teleocidin B) enhancedthe adherence of these cells to the surface of plastic culturedishes. These findings provide evidence that many small celllung cancer cell lines can change their morphology with changesin the environment of the cells.     

High Occurrence of Non-Clear Cell Renal Cell Carcinoma in Oman

It is conventionally accepted that renal cell carcinoma (RCC) occurs in older patients and the clear cell type is the most common histology. However, ethnic variations exist and this study was carried out to determine the epidemiological pattern of RCC in Oman. Ninety RCC patients who presented to a tertiary care center in the Sultanate of Oman from 2010 to 2014 were studied. The main findings were that the median age of presentation was low, more patients presented with localized stage, and there was a higher incidence of non-clear (especially papillary) histology. Data from other Gulf countries and possible reasons for the different profile are discussed.     

Cell Death Induced by Baicalein in Human Hepatocellular Carcinoma Cell Lines

We examined the action of baicalein, a flavonoid contained in the herbal medicine sho-saiko-to (TJ-9), on three cell lines of human hepatocellular carcinoma (HCC). Treatment with baicalein strongly inhibited the activity of topoisomerase II and suppressed the proliferation of all three HCC cell lines. But the mode of cell death induced by baicalein differed according to the cell line. Baicalein induced apoptosis in a concentration-dependent mannner in only one cell line, and an increased concentration of baicalein produced cell death via necrosis in the other two lines. These results suggest that the inhibition of topoisomerase II is not by itself sufficient for induction of apoptosis, and that there is a more important mechanism which can account for the difference in susceptibility of cells to apoptosis induced by baicalein.     

Expressions of Cell Cycle Regulators in Human Colorectal Cancer Cell Lines

To study the altered mechanisms of cell cycle regulation in colorectal cancer, the expressions of cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, p53 and retinoblastoma (Rb) protein were analyzed by western blotting in a series of human colorectal cancer cell lines. The colorectal cancer cell lines exhibited various expression patterns of cell cycle regulators, which may reflect differences in the biological characteristics of cancer cells and in the genetic backgrounds of carcinogenesis. A correlation was found between p53 gene alteration and p21 expression, suggesting that p53 gene mutation usually suppresses p21 expression, though p21 expression could be induced via both ap53 -dependent and a p53 -independent pathway in colorectal cancer. None of the cell lines studied expressed p16 protein, suggesting that inactivation of p16 may be a common alteration in colorectal cancer. Moreover, all the D-type cyclins, especially D2 and D3, were expressed at a high level in most of the cell lines. Loss of p16 expression and increased expression of D-type cyclins promote CDK-mediated Rb phosphorylation. All of the colorectal cancer cell lines studied herein expressed Rb protein, but the growth-suppressive properties of Rb may be inactivated by the loss of p16 expression and increased expressions of D-type cyclins. In view of the pivotal role of Rb in cell cycle regulation, loss of p16 expression and overexpression of D-type cyclins may be critical alterations in colorectal cancer.     

膀胱肿瘤细胞对T淋巴细胞功能抑制的研究

 目的　了解膀胱肿瘤细胞对T淋巴细胞功能的影响。方法　将膀胱肿瘤细胞株EJ、T2 4 ,接受致死性射线的EJ、T2 4 ,人正常膀胱移行上皮分别与由正常人外周血中分离所得的T淋巴细胞共培养 ,2 4小时后分离共培养的T淋巴细胞 ,行T淋巴细胞增殖反应 (MTT法 )及T淋巴细胞凋亡检测(TUNEL法 )。结果　EJ组和T2 4组分别与接受致死性射线的EJ组和T2 4组相比 ,T淋巴细胞的增殖反应降低 (P <0 .0 1) ;TUNEL法检测T淋巴细胞凋亡结果的比较 :EJ组和T2 4组分别与接受致死性射线的EJ组和T2 4组比较 ,T淋巴细胞凋亡指数均降低 (P <0 .0 5 )。结论　膀胱肿瘤细胞株EJ、T2 4可通过某种机制抑制T淋巴细胞的功能 ,增加T淋巴细胞凋亡。