蟾蜍灵诱导人肺腺癌细胞凋亡作用及其机制

目的 研究中药蟾酥有效成分蟾蜍灵(bufalin)对人肺腺癌细胞的作用及其机制。方法 MTT法检测蟾蜍灵对细胞的增殖抑制作用;瑞氏 吉姆萨染色法观察细胞形态学的变化;流式细胞术分析细胞凋亡和线粒体跨膜电位;Western blot法检测Bcl-2、Caspase-3蛋白的表达。结果 (1) 蟾蜍灵明显抑制A549细胞增殖,48h及72h的IC50分别为(56.14±6.72)nmol/L、(15.57±4.28)nmol/L。(2) 蟾蜍灵能诱导肺癌细胞凋亡,形态学表现为出现凋亡小体及流式细胞仪检测到的亚二倍体凋亡峰。(3) 蟾蜍灵可以明显降低细胞线粒体跨膜电位(ΔΨm)。(4) 蟾蜍灵诱导细胞凋亡过程中,下调Bcl-2蛋白表达(P<0.01),同时活化Caspase-3。结论 线粒体途径是蟾蜍灵诱导肺腺癌A549细胞凋亡的主要通路之一。     

Livin基因反义寡核苷酸诱导肺腺癌A549细胞的凋亡

目的探讨Livin基因反义寡核苷酸(antisense oligodeoxynucleotides,ASODN)对肺腺癌细胞A549增殖和凋亡的影响.方法用阳离子脂质体介导Livin ASODN转染A549细胞后,MTT法检测对A549细胞增殖抑制率的影响;RT-PCR法和细胞免疫荧光化学检测Livin ASODN转染前后,A549细胞中Livin mRNA和蛋白表达的变化;吖啶橙/溴乙锭(acridine orange/ethidium bromide,AO/EB)复合染色检测A549细胞的凋亡率.结果在A549细胞中同时有Livin α和Livin β的表达,Livin蛋白在细胞质和细胞核中均有分布.Livin基因ASODN可显著抑制A549细胞的增殖(P＜0.01).且作用具有剂量依赖性.终浓度为400 nmol/L的Lip-ASODN转染后,A549细胞Livin mRNA和Livin蛋白的表达均被明显抑制,细胞凋亡率达(31.25 ±5.75)%,与对照组(3.23±1.98)%相比差异有统计学意义(P＜0.01).结论Livin ASODN可下调Livin基因表达,有效抑制A549细胞的增殖,并促进A549细胞凋亡.因此,Livin基因有望成为肺癌基因治疗的新靶点.     

多柔比星诱导肺腺癌细胞凋亡机制的探讨

目的:探讨多柔比星(ADM)诱导肺腺癌细胞系A549细胞凋亡机制.方法:应用噻唑蓝(MTT)比色法检测ADM对A549细胞增殖抑制作用,透射电镜观察细胞形态学改变;细胞电泳仪测定A549细胞表面电荷的变化;流式细胞仪(FCM)测定A549细胞Fas、Bcl-2蛋白水平变化;比色法检测Caspase-3活性的变化.结果:ADM可抑制细胞增殖,作用24、48及72 h的IC<,50>值分别为5.2、4.8和4.2 mg/L,电镜下见到明显的凋亡细胞;细胞表面电位在4 h开始下降,4～24 h期间下降最明显,>24 h下降趋势降低;Fas蛋白表达明显上调,Bcl-2蛋白表达下降,Caspase-3活性明显增强.结论:ADM能抑制并诱导A549细胞凋亡,细胞表面电荷下降可能是ADM诱导细胞凋亡的早期事件,其作用可能与上调Fas蛋白、下调Bcl-2蛋白表达有关.     

蟾蜍毒素联合紫杉醇抑制人胃癌MGC803细胞增殖和诱导凋亡的作用

目的探讨蟾蜍毒素联合紫杉醇抑制人胃癌MGC803细胞增殖及诱导凋亡的作用。方法应用MTT法检测不同浓度蟾蜍毒素及紫杉醇单药及联合对胃癌细胞MGC803增殖抑制作用;流式细胞术分析细胞凋亡和周期阻滞;Westernblot法检测C-FLIPL、Caspase-8蛋白的表达。结果(1)蟾蜍毒素及紫杉醇单药均抑制MGC803细胞增殖,呈剂量-时间依赖效应,且联合用药的细胞增殖抑制率明显高于单药组,差异有统计学意义(P<0.05)。24、48及72 h联合指数(CI)分别为0.77、0.86、0.72;(2) 40 nmol/L蟾蜍毒素、25 ng/ml紫杉醇单药及联合作用细胞24 h,流式细胞仪检测凋亡率分别为14.13%、19.74%及53.30%,联合组与单药组比较差异有统计学意义(P<0.05);(3)Western blot结果显示,蟾蜍毒素、紫杉醇单药组及联合组作用细胞24 h后,C-FLIPL蛋白表达依次下降至对照组的78.38%、64.71%、46.73%,Caspase-8蛋白表达上调至对照组的150.25%,156.86%,180.58%。结论蟾蜍毒素协同紫杉醇诱导胃癌MGC803细胞凋亡,且可能与下调C-FLIP、上调Caspase-8蛋白有关。     

抑制NF-κB通路增强蟾蜍灵诱导的HL-60细胞凋亡

目的:观察蟾蜍灵对HL-60细胞活力和凋亡的影响,对NF-κB通路的作用,探讨NF-κB通路在蟾蜍灵诱导HL-60细胞凋亡过程中的作用.方法:锥虫蓝染色检测细胞活力;流式细胞仪技术检测细胞凋亡和周期分布;采用Western Blot技术检测IKK的磷酸化.结果:蟾蜍灵以时间和剂量依赖性方式诱导HL-60细胞凋亡.24,48和72h抑制细胞活力的IC50浓度分别为26.3,7.8和2.0nmol/L.对照组HL-60细胞有pIKK表达,蟾蜍灵可诱导IKK的快速活化.NF-κB通路的特异性抑制剂Bay 11-7082增强蟾蜍灵的凋亡诱导作用.结论:蟾蜍灵可诱导HL-60细胞凋亡,促进IKK活化与NF-κB通路调节相关,而且与NF-κB通路抑制剂有协同作用.     

抑制NF—κB通路增强蟾蜍灵诱导的HL-60细胞凋亡

目的：观察蟾蜍灵对HL-60细胞活力和凋亡的影响,对NF—κB通路的作用,探讨NF—KB通路在蟾蜍灵诱导HL-60细胞凋亡过程中的作用。方法：锥虫蓝染色检测细胞活力;流式细胞仪技术检测细胞凋亡和周期分布;采用WesternBlot技术检测IKK的磷酸化。结果：蟾蜍灵以时间和剂量依赖性方式诱导HL-60细胞凋亡。24,48和72h抑制细胞活力的Ic50浓度分别为26．3,7．8和2．0nmol／L。对照组HL-60细胞有pIKK表达,蟾蜍灵可诱导IKK的快速活化。NF—κB通路的特异性抑制剂Bay11—7082增强蟾蜍灵的凋亡诱导作用。结论：蟾蜍灵可诱导HL-60细胞凋亡,促进IKK活化与NF—κB通路调节相关,而且与NF—κB通路抑制剂有协同作用。     

蟾蜍灵在诱导K562细胞凋亡过程中下调TOPO-Ⅱα蛋白表达

目的探讨蟾蜍灵(bufalin)诱导K562细胞凋亡的分子机制。方法用流式细胞仪和形态学检测细胞凋亡;用免疫组化SP法检测TOPO-Ⅱα蛋白表达。结果蟾蜍灵在诱导K562细胞凋亡过程中,下调TOPO-Ⅱα蛋白表达;细胞外信号转导激酶抑制剂(extra-cellularsignal-regulatedkinase,ERK)PD98059能协同蟾蜍灵下调TOPO-Ⅱα蛋白表达;而四癸酰佛波酯(TPA)却拮抗蟾蜍灵的作用。结论蟾蜍灵诱导K562细胞凋亡可能与抑制ERK信号途径以及下调TOPO-Ⅱα蛋白表达有关。     

蟾蜍灵对胃癌组织MGC-803细胞周期作用机制的研究

目的探讨蟾蜍灵对胃癌MGC803细胞周期及周期蛋白的影响。方法采用台盼蓝拒染法测定细胞增殖活力;瑞士姬姆萨染色观察细胞形态学的变化;流式细胞仪检测细胞周期;免疫组织化学方法检测细胞周期相关蛋白p16、p21、pRb的表达。结果1)蟾蜍灵抑制胃癌MGC803细胞的增殖,24、48、72h的IC50分别为0.086、0.048和0.036μmol/L。2)蟾蜍灵在0.01～0.1μmol/L时作用24～72h,形态学上可见细胞体积增大,出现双核、多核细胞。3)流式细胞仪显示蟾蜍灵浓度为0.01～0.1μmol/L时可明显诱导细胞周期G2/M期阻滞。4)蟾蜍灵作用于胃癌MGC803细胞,观察到p16、p21、pRb蛋白的表达明显上调。结论蟾蜍灵引起胃癌MGC803细胞的周期阻滞可能与p16、p21、pRb蛋白的上调有关。     

Livin反义寡核苷酸对人乳腺癌细胞株MCF-7增殖和凋亡影响的研究

目的：探讨Livin mRNA反义寡核苷酸（ASODN）对人乳腺癌MCF-7细胞抑制增殖及诱导凋亡的影响。方法：设计合成特异性的Livin硫代磷酸ASODN及其对照错义寡核苷酸（MSODN），脂质体转染至培养的MCF-7细胞。采用MTT法检测Livin ASODN对MCF-7细胞的增殖抑制作用，RT-PCR检测Livin mRNA的表达水平，电镜、流式细胞仪与吖啶橙／溴化乙锭（AO／EB）细胞染色法检测细胞凋亡水平和形态学改变。结果：Livin ASODN在终浓度为600nmol／L作用MCF-7细胞48h时，能明显地抑制其增殖（IC50=604.4），降低Livin mRNA的表达;电镜、流式细胞仪与A0／EB细胞染色法则表明MCF-7细胞在形态学上出现明显的凋亡改变，细胞凋亡率显著增加，而对照组寡核苷酸未见抑制效应。结论：Livin ASODN能够特异性下调MCF-7细胞中Livin基因表达，在诱导肿瘤细胞凋亡、抑制增殖中发挥重要作用。     

盐酸埃克替尼对人肺癌HCC827细胞Akt通路活化和凋亡相关蛋白表达的影响

目的:探讨酪氨酸蛋白激酶抑制剂盐酸埃克替尼(Icotinib)对人非小细胞肺癌(NSCLC)HCC827细胞Akt通路的活化和凋亡相关蛋白PARP、Caspase-3和Caspase-8表达的影响。方法:将体外培养的人肺癌HCC827细胞分为Icotinib处理组和未处理的对照组,采用四甲基偶氮唑盐法(MTT)检测Icotinib对细胞增殖的抑制作用,采用Western blot检测蛋白表达,应用SPSS 13.0进行统计学分析。结果:Icotinib处理HCC827细胞48h的IC50为0.65μmol/L;选用0.001、0.01μmol/L和0.1μmol/L的Icotinib分别作用HCC827细胞48h,细胞凋亡率分别为(3.35±1.77)%、(8.95±3.18)%和(20.4±3.12)%(P<0.05)。与对照组相比,Icotinib明显抑制Akt的磷酸化水平,诱导PARP、Caspase-3和Caspase-8活化裂解。结论:Icotinib通过抑制Akt信号通路的活化、进一步诱导PARP、Caspase-3和Caspase-8裂解,进而抑制人NSCLC细胞增殖及诱导细胞凋亡。     

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.     

Inhibition of Glioma Cell Proliferation by Neural Stem Cell Factor

Summary Neural stem cells (NSC) have unique differentiation-, proliferation-, and motility properties. To investigate whether they secrete factors that interfere with the proliferation of glioma cells, we grew glioma cells in conditioned medium (CM) obtained from cultures of neurospheres including neural stem / progenitor cells (NSPC) isolated from embryonic (E14)- or adult mouse brain or fetal human brain. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and BrdU-labeling assays showed that CM from NSPC (NSPC/CM) contained factor(s) that inhibited the proliferation of glioma cells by 28–87%. Filter-fractionation of NSPC/CM revealed that the 50,000–100,000 nominal molecular weight limit (NMWL) fraction contained the inhibitory activity. On the basis of these observations we transplanted 203G glioma cells and/or NSPC into the intrathecal space of the cisterna magna of mice to investigate whether NSPC interfere with the proliferation of glioma cells in vivo. Mice transplanted with both 203G and NSPC survived significantly longer than did mice transplanted only with 203G. We concluded that NSPC secrete factor(s) that may control glioma cell proliferation.     

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.     

Inhibition of Natural Killer Cell Cytotoxicity by Cell Growth-related Molecules

Certain MHC class I molecules on target cells are known to inhibit the cytotoxic action of NK cells. By using monoclonal antibody (mAb) Cho-1, we have found inhibitory non-MHC class I cell surface molecules that are noncovalently-associated with 200 kDa and 40 kDa antigens. Poly I-C-induced rat NK cells were not cytotoxic to rat fetus-derived fibroblast WFB cell line. In contrast, NK cells were cytotoxic to H- ras oncogene-induced transformants of WFB, W14 and W31. FACS analysis indicated that mAb Cho-1 reacts with WFB, but not with W14 and W31 cells. Thus, this antigen may disappear concomitantly with cell growth and transformation. Cho-1 antigens were also expressed on other NK-resistant lines, such as mouse BALB3T3 fibroblast, EL-4 lymphoma and human fibroblast HEPM. However, they were not expressed on NK-sensitive mouse YAC-1 and H- ras transformant (Brash) of BALB3T3 cells. Furthermore, treatment of target cells with IFN-γ clearly induced the cell surface expression of Cho-1 antigens, and conferred a resistance to NK cytolysis on target cells. These data strongly suggest that Cho-I antigen expression may correlate with target cell susceptibility to NK cells. Indeed, treatment of NK-resistant WFB as well as HEPM cells with F(ab')₂ fragments of mAb Cho-1 resulted in the acquisition of susceptibility to NK cytolysis. Cho-1 antigens may be novel molecules that regulate the NK resistance of cells.     

人干扰素对HL60,K562细胞生长及细胞周期的影响

用人干扰素(α和γ)与HL60、K562细胞共同培养后,对细胞生长有不同程度抑制作用。IFN_r对细胞生长抑制作用强于IFN_r,IFN_r和IFN_r联合应用有协同作用,在K562细胞,细胞在细胞周期中的分布也发生改变,G_0/G_1期细胞比例减少,S期细胞比例增高,在HL60细胞则无明显的细胞周期再分布情况。提示细胞在S期的堆积是细胞生长受抑的原因之一。     

Cell kinetics

Cell kinetic concepts have pervaded radiation therapy since the early part of the 20th century and have been instrumental in the development of modern radiotherapy. In this review, the fundamental radiobiological concepts that have been developed based on cell kinetic knowledge will be revisited and discussed in the context of contemporary radiation therapy. This will include how the proliferation characteristics, variation in sensitivity during the cell cycle and the extent of radiation-induced cell cycle delay translate into a variable time for the expression of damage, how cell kinetics interacts with hypoxia and how the response to fractionated radiation schedules is influenced by cell kinetics in terms of repair, redistribution, reoxygenation and repopulation. The promise of combining radiation with new biologically targeted agents and the potential of non-invasive positron emission tomography imaging of proliferation are areas where cell kinetics will continue to influence radiotherapy practice.     

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.