缺氧对胎盘滋养层细胞细胞周期和凋亡的影响

目的 观察缺氧时滋养层细胞细胞周期及凋亡变化.方法 体外培养胎盘绒毛膜癌滋养层细胞株(BeWo细胞),二氯化钴(CoCl2)处理模拟化学缺氧.噻唑蓝法测定不同浓度(0、125、250、500μmol/L)CoCl2作用不同时相(6、12、24 h)后细胞活力;流式细胞术检测250 μmol/L CoCl2处理细胞不同时间(6、12、24 h)后细胞周期和凋亡改变.结果 125 μmol/L CoCl2对细胞活力无明显影响(P＞0.05);250μmol/L CoCl2作用时细胞活力呈下降趋势(P＞0.05);500μmol/L CoCl2处理6、12、24 h后细胞活力均有明显下降(P＜0.01).250 μmol/L CoCl2诱导构建滋养层细胞缺氧模型.250μmol/L CoCl2作用12、24 h后,G2/M期细胞比例增多(分别为0.24±0.13和0.31±0.01)(P＜0.05或P＜0.01).250 μmol/L CoCl2作用细胞24 h后细胞凋亡呈增加趋势(P＞0.05).结论 成功构建了滋养层细胞体外缺氧培养模型.缺氧诱导滋养层细胞周期向G2/M期进展,增加细胞凋亡.     

芹黄素对体外人大肠癌Lovo细胞生物学活性的影响及机制研究

目的探讨芹黄素对体外人大肠癌Lovo细胞生物学活性的影响及其可能的作用机制。方法不同浓度的芹黄素作用于体外培养的人大肠癌Lovo细胞24、48或72 h,采用光学显微镜和透射电镜分别观察芹黄素对Lovo细胞形态和超微结构的影响;采用四甲基偶氮唑盐比色法(MTT法)和平板克隆形成实验分别检测芹黄素对Lovo细胞增殖和克隆形成能力的影响;采用PI染色法、Annexin V FITC/PI双染法,流式细胞术分别检测芹黄素对Lovo细胞周期分布和凋亡的影响;采用Western blot法检测芹黄素对Bcl-2、pro-caspase-3、cleaved caspase-3表达的影响。结果芹黄素可浓度-时间依赖性的抑制人大肠癌Lovo细胞的增殖及克隆形成,作用48、72 h时的半数抑制浓度IC50分别为98.05μmol·L-1及33.91μmol·L-1;细胞周期分布发生改变,主要表现在G0/G1期细胞比例减少、G2/M期细胞比例增加,细胞周期阻滞于G2/M期;细胞凋亡率增加;Bcl-2、pro-caspase-3表达下调;cleaved caspase-3表达上调;差异具有统计学意义(P<0.05)。结论芹黄素可通过阻滞细胞周期于G2/M期抑制Lovo细胞增殖,通过下调Bcl-2、pro-caspase-3表达,上调cleaved caspase-3表达,诱导Lovo细胞凋亡,是一种有开发前景的大肠癌化疗药物。     

酒石酸锑钾对肝L-02细胞的抑制和凋亡诱导影响

目的 探讨酒石酸锑钾(PAT)对人胚肝L-02细胞的生长、细胞周期和诱导凋亡的影响.方法 采用改良的噻唑蓝(MTT)比色法检测不同浓度PAT处理24、48、72、96及120 h对L-02细胞的生长抑制效应,用PI单染流式细胞术检测不同浓度PAT处理24 h诱导细胞周期的改变情况,用ArmexinV-FITC/PI双染流式细胞术检测不同浓度PAT作用24 h对细胞凋亡的影响.结果 PAT染毒24、48、72、96及120 h,随PAT染毒剂量的增大,L-02细胞生长抑制率明显升高;浓度200μanol/L PAT作用24 h使细胞发生S期阻滞,≥400 μmol/L PAT使细胞发生G1期阻滞;浓度200μmol/L PAT作用24h诱导细胞凋亡,≥400μmol/L PAT可导致细胞坏死.结论 在一定剂量下,PAT可以显著抑制L-02细胞的生长增殖,低剂量PAT主要引起L-02细胞发生S期阻滞和细胞凋亡,高剂量PAT主要引起L-02细胞发生G1期阻滞和细胞坏死.     

全反式维甲酸联合索拉菲尼对人SMMC-7721肝癌细胞株的作用

目的观察全反式维甲酸联合索拉菲尼对SMMC-7721细胞增殖及凋亡的影响。方法选用ATRA(10-5mol/L)及不同浓度的索拉菲尼(6μmol/L、9μmol/L、12μmol/L),并选用10-5mol/L的ATRA分别联合索拉菲尼(6μmol/L、9μmol/L、12μmol/L)作用于SMMC-7721肝癌细胞24h、48h、72h;期间药物对SMMC-7721肝癌细胞生长的抑制阻滞作用通过MTT比色法进行观测,同时细胞生长过程中利用倒置显微镜观察形态变化,其后药物作用48h时SMMC-7721细胞的周期分布和凋亡的情况利用流式细胞术分析。结果不同浓度的索拉菲尼单药、全反式维甲酸单药及联合用药均对SMMC-7721肝癌细胞的生长明显抑制并改变细胞的形态,促进肝癌细胞的凋亡比例增加。其凋亡作用有随剂量-时间改变的依赖性。两药联合应用相比单用全反式维甲酸或索拉菲尼其细胞凋亡作用更为显著。12μmol/L索拉菲尼、9μmol/L索拉菲尼分别与ATRA联合作用于细胞48h及72h相比较,其抑制作用无明显差异。两药联合作用时可见细胞周期阻滞在S期的肝癌细胞较单药应用时显著增多(P<0.01),凋亡率增加。结论 ATRA、索拉菲尼均有阻滞肝癌SMMC-7721细胞增殖及促凋亡的作用,联合索拉菲尼作用增强并具协同作用。     

姜黄素抑制人神经胶质瘤U251细胞增殖的机制

目的 探讨姜黄素对人神经胶质瘤U251细胞增殖的抑制作用及其相关机制.方法 应用0、10、20、40、60、80 μmol/L的姜黄素处理人神经胶质瘤U251细胞,检测细胞增殖、细胞周期、细胞凋亡及葡萄糖转运蛋白-3(GLUT-3)表达情况.结果 10、20、40、60、80 μmol/L姜黄素作用于U251细胞吸光度(OD)值低于0μmol/L组(P＜0.05).姜黄素组作用72 h时人神经胶质瘤U251细胞G1期细胞比例少于对照组,G2/M期细胞比例多于对照组(P＜0.05).两组细胞凋亡率比较差异无统计学意义(P＞0.05).姜黄素组GLUT-3表达水平低于对照组(P＜0.05).结论 姜黄素可以抑制人神经胶质瘤U251细胞增殖,其作用可能是通过下调GLUT-3的表达水平来实现的.     

吲哚美辛对宫颈癌Hela细胞增殖和凋亡作用的影响

目的:研究吲哚美辛对宫颈癌Hela细胞增殖、凋亡的影响。方法:以体外培养的Hela细胞为试验对象,分别用0(空白对照)、200、400、600、800、1 000μmol/L吲哚美辛作用24、48、72 h后,采用MTT法检测吲哚美辛对Hela细胞的增殖抑制率;用0(空白对照)、400、600、800μmol/L吲哚美辛作用24 h后,采用倒置显微镜观察细胞形态的改变,并用流式细胞仪检测细胞周期分布和细胞凋亡情况。结果:600、800、1 000μmol/L吲哚美辛可抑制Hela细胞的增殖,增殖抑制率与药物浓度和作用时间呈正相关。与空白对照比较,吲哚美辛可使Hela细胞形态由多角形逐渐向圆形转变且细胞呈现凋亡和坏死状态;其中细胞G0/G1期比例增加、S期比例减少,细胞凋亡率增加。结论:吲哚美辛可抑制Hela细胞增殖,诱导其凋亡,主要阻滞细胞于G0/G1期。     

半边旗多糖对肺腺癌SPCA-1细胞的诱导凋亡作用

目的研究蕨类植物半边旗多糖对人肺腺癌SPCA-1细胞的体外杀伤及诱导凋亡作用。方法用MTT法测定细胞成活率：用流式细胞术测定细胞周期时相分布和凋亡率，并进行形态学观察。结果半边旗多糖对肺腺癌SPCA-1细胞有强烈的杀伤作用，具明显的时间和剂量效应(0.09375-3μg／ml，24h-72h)；作用肺腺癌SPCA-1细胞24h后，G0／G1期细胞比例明显升高，S期、G2/M期细胞比例下降明显。细胞凋亡形态明显。结论半边旗提取物对肺腺癌SPCA-1细胞有强烈的杀伤作用，明显影响细胞周期时相分布，可引起SPCA-1凋亡。     

Cpd5对人卵巢癌SKOV3细胞增殖抑制和凋亡诱导作用

目的：观察Cpd5[2-（2-巯基乙醇）-3-甲基-1,4-萘醌,Compound5]对人卵巢癌SKOV3细胞增殖和凋亡的影响。方法：MTT法观察Cpd5对SK-OV3细胞的生长抑制作用,AnnexinV/PI双标记流式细胞术检测Cpd5对SKOV3细胞的凋亡诱导,Hoechst-33258染色荧光显微镜观察细胞凋亡形态。结果：5、10、20、30、40、50、60μmol/LCpd5处理SKOV3细胞48h,生长抑制率分别为2.77%、5.19%、10.61%、41.15%、71.37%、82.90%、89.81%。不同时间点（12、24、48和72h）检测,细胞生长抑制率存在剂量-时间依赖关系。流式细胞术检测,30μmol/LCpd5处理12、24和48h后,细胞凋亡率分别达9.25%、20.07%、56.16%;50μmol/L组的细胞凋亡率明显高于30μmol/L,且随时间增加而显著增加。用40、50和60μmol/LCpd5处理细胞12h,光镜观察即可见明显的形态学改变。荧光显微镜观察：AnnexinV-EGFP/PI双染显示,实验组比阴性对照组细胞凋亡率明显增多;Cpd5作用24、48h后,Hoechst-33258染色可见细胞出现明显的凋亡形态,20μmol/L浓度组凋亡率增加,30、40、50、60μmol/L各组细胞凋亡率显著增加。结论：Cpd5能以剂量-时间依赖的方式抑制SKOV3细胞增殖并诱导凋亡,是潜在的抗卵巢癌新化合物。     

他克莫司对人肾小球系膜细胞增殖的影响

目的探讨他克莫司对体外培养的人肾小球系膜细胞增殖的影响及可能机制。方法采用四甲基偶氮唑盐法(MTT)检测不同浓度的他克莫司处理后系膜细胞的增殖情况;用流式细胞仪检测不同浓度的他克莫司作用48、72h后系膜细胞细胞周期的改变;用流式细胞仪检测不同浓度的他克莫司作用72h后系膜细胞发生凋亡的情况。结果①浓度为1μmol/L的他克莫司对系膜细胞的增殖无明显抑制作用;作用24、48和72h后,浓度为5～20μmol/L的他克莫司能显著抑制系膜细胞的增殖;随着他克莫司浓度的增大,抑制作用更显著,呈一定的剂量—效应关系;随着他克莫司作用时间的延长,抑制作用更显著,呈一定的时间—效应关系。②5μmol/L的他克莫司作用48、72h,G0/G1期的系膜细胞百分比增高,S期的系膜细胞百分比降低,细胞周期阻滞于G0/G1期。③5μmol/L的他克莫司作用72h,对系膜细胞的凋亡无明显影响。结论他克莫司显著抑制体外培养的人肾小球系膜细胞增殖,且呈剂量和时间依赖性;其可能机制是使系膜细胞的细胞周期阻滞于G0/G1期。     

5-氮杂-2'-脱氧胞苷对OS-RC-2肾癌细胞生物学行为的影响

目的 研究5-氮杂-2'-脱氧胞苷(5-Aza-CdR)对OS-RC-2肾癌细胞株增殖、凋亡的影响.方法 用不同浓度10-7、10-6、10-5、10-4mol/L的特异性DNA甲基转移酶抑制剂5-Aza-CdR处理OS-RC-2肾癌细胞株,未经药物处理细胞作对照(对照组).透射电镜观察5-Aza-CdR处理OS-RC-2肾癌细胞株前后细胞形态学变化.MTT检测OS-RC-2肾癌细胞株抑制率.流式细胞仪检测OS-RC-2肾癌细胞株凋亡.以甲基化特异性PCR(MSP)检测细胞处理前后γ-catenin基因的甲基化状态.结果 用药后细胞器肿胀、线粒体空化、溶酶休增多,核质不均匀.5-Aza-CAR明显抑制OS-RC-2肾癌细胞的增殖,而且与药物浓度及作用时间在一定范围内成正相关(P<0.05).10-7,10-6,10-7mol/L5-Aza-CdR处理细胞后,细胞凋亡率增高[(3.74±0.34)%,(7.85±0.59)%;(12.93±1.32)%vs.对照组(0.86±0.08)%],成剂量依赖性(P<0.01);作用3 d后,在10-6mol/L时细胞周期中处于G0/G1期的显著增多(P<0.05),细胞周期阻滞于G0/G1期.未经5-Aza-CdR处理的OS-RC-2细胞中γ-catenin基因启动子区域高甲基化,经5-Aza-CdR105mol处理72 h后,γ-catenin基因启动子区域高甲基化得到逆转.结论 5-Aza-CdR能消除γ-catenin基因启动子甲基化状态,使其重新表达而抑制OS-RC-2肾癌细胞株的生长,使细胞周期阻滞于G0/G1期,并促进其凋亡.     

树突状细胞对肿瘤细胞株的直接杀伤活性

目的 对比分析干扰素－γ（Interferon-γ,IFN-γ)或脂多糖（lipoplysaccharide,LPS)刺激后的树突状细胞（dendritic cell,DC)对肿瘤细胞杀伤活性的差异。方法 分离健康供者外周血单核细胞，用粒单细胞集落刺激因子和白介素－4诱导为DC。于培养液中加入LPS或IFN－γ培养12h，作为LPS激活的DC（LPS－DC）及IFN－γ激活的DC（IFN－DC）。用流式细胞仪检测DC表面共刺激分子的改变，以明确LPS或IFN－γ对DC的不同刺激作用；同时，以恶性血液病细胞株HL－60 Jurkat及Daudi为靶细胞，用不同效靶比与DC共同培养18h，采用^51Cr释放试验检测LPS－DC及IFN－DC抗肿瘤活性的差异。结果 ①LPS及IFN－γ可不同程度的上调DC表面CD86、CD80、CD83及CD1a的表达，以LPS刺激组明显。②IFN－γ和LPS可分别增强DC对HL60及Daudi的杀伤活性，在效靶比为20：1及10：1时杀伤率与未加刺激因子对照组（medium-DC)相比差异有显著意义（P＜0．05）。相反，IFN－γ－DC对Daudi、LPS－DC对HL－60无明显杀伤活性，但两者对Jurkat均具杀伤作用。结论 LPS及IFN－γ激活的DC对肿瘤细胞的杀伤活性具有相对肿瘤特异性。     

Nitric oxide, cell signaling and cell death

Nitric oxide (NO) is an important bioregulatory molecule in the nervous, immune and cardiovascular systems. NO participates in the regulation of the daily activities of cells as well as in cytotoxic events. It possesses a controversial effect on cell viability by acting both as a protection against apoptogenic stimuli, or by inducing apoptosis when produced at elevated concentrations. The mechanisms of NO in regulating these biological functions can be either through cyclic guanylate cyclase (cGMP)-dependent or cGMP-independent pathways. The purpose of this review is to highlight the implication of NO in cell signalling, synaptic transmission, and cell death. We focus also on the protective role as well as the toxicity of NO. Finally, the adverse effects of inhaled nitric oxide are also depicted in this review.     

Mast cell regulatory effect on lymphoid cell proliferation.

It has been shown rat mast cells (MC) can modulate lymphocyte proliferation in vitro. Depending on concentrations tested both serosal MC and their supernatants enhanced the spontaneous and T-mitogen-induced proliferation of spleen and lymph node cells. In addition T-mitogen-induced thymocyte proliferation was also increased. The enhancing effect of MC on lymphoid cell proliferation appeared after MC and lymphocytes were cocultured for 24, 48 or 72 h. The highest enhancing action of MC was observed when MC and lymphocytes were plated simultaneously. In contrast, when MC were added 24 or 48 h after the start of lymphocyte culture, the enhancing action of MC decreased or was abolished, respectively. No dependence was found between histamine concentration in MC supernatants and the enhancing activity of supernatants. After chromatographic separation of MC supernatants the fractions with molecular weights between 1-6 KDa augmented lymphoid cell proliferation.     

Pharmacotherapy for treating metastatic clear cell renal cell carcinoma

Introduction: Over the past decade metastatic renal cell carcinoma (RCC) treatment landscape has dramatically evolved from the era of cytokines-based immunotherapy (which benefited very few patients, at the expenses of high toxicities) to the present era of targeted agents and novel immunotherapeutics, greatly improving the prognosis of our patients.

Areas covered: Here we have reviewed the present status of the medical treatment of metastatic RCC. To do this, we interrogated the Medline database, as well as the proceedings of the main Oncological and Urological conferences for the relevant trials coducted so far.

Expert opinion: Despite all the advances made in these relatively few years, further improvements are needed, since none of the available agents proved able to cure even a sigle metastatic RCC patient. In particular, advances are awaited from the results of ongoing trial of combinations of different immune checkpoint inhibitors and of immune checkpoint inhibitors with anti-VEGF/VEGFRs agents. Furthermore, a better understanding of the molecular escape pathways used by the tumor to overcome VEGFR blockade or immune activation will hopefully bring soon to the clinic more active, tailored treatments, to be used in second line and beyond.     

Rapid fluorometric assay for cell viability and cell growth using nucleic acid staining and cell lysis agents

The purpose of this study was to establish a new method for rapidly and simply assessing cell viability and growth with objective validation if the assay system proceeded under suitable conditions of cell culture. In this method, a cell lysis agent was combined with a fluorescent probe for nucleic acid which exclusively passes through the disrupted membranes of dead cells but not intact membranes of viable cells. The distinctive feature of this probe is to possess a large fluorescence enhancement (460-fold) on binding to nucleic acid despite very low intrinsic fluorescence. In this fluorometric assay based on cell lysis and staining (FACLS), the fluorescence intensity was linearly related to total tumour cell number. This FACLS was also used to evaluate the chemosensitivity of MOLT-4 human leukaemia cells and to measure cell viability. The results were similar to those obtained by MTT colorimetric and trypan blue exclusion assays. The main advantage of this assay is its ability to measure simultaneously both cell viability and cell growth rapidly (within about 5 min) and simply (two steps) with validation of cell culture conditions in each microplate. This method could be widely applicable to cytotoxic evaluation of anticancer drugs and other chemicals.     

T cell targeted immune enhancement yields effective T cell adjuvants

Given the critical role of cell-mediated immunity (CMI) in defense against attack from pathogens that establish chronic infections, it has become abundantly clear that current vaccine methodology will not be sufficient to develop the appropriate immune response for protection and/or clearance of infection. By extension, this logic also applies to cancer vaccines where T cell immune-mediated destruction is a critical mechanism for control of the disease. This review describes our current thoughts on the events associated with immune activation and evaluates the various approaches to achieve successful immune activation with defined or targeted antigens as opposed to using inactivated or attenuated organisms. The advantages and disadvantages of the current adjuvants for antigens that focus on mimicking the infection events via the innate immune system or antigen uptake are described in the context of generation of T cell specific responses. A central theme of the discussions is the importance of cytokines in modulating the immune response towards T cell immunity, either by adjuvant modulation or use of natural cytokine mixtures targeted towards the site of immune activation. Also discussed is the possibility that thymomimetic agents such as thymosin alpha1, levamisole and methyl inosine monophosphate (MIMP) may be useful in enhancing the T cell mediated arm of the immune response.     

Stem cell pharmacogenomics

Therapeutic stem cell applications represent a newly evolving approach for the treatment of several genetic and degenerative diseases. The advent of pharmacogenomics too, holds promise for an individualized, optimal treatment regime for a large variety of medical conditions. A combination of the benefits of these two technologies creates a new niche in therapeutic medicine research viz. that of stem cell pharmacogenomics (SCP). The development of this approach requires the application of existing technologies in genomics, proteomics and bioinformatics to resolve the various issues involved in advancing the therapeutic applications of stem cell medicine. In this brief overview of the subject, we attempt to provide fresh insights into the exclusive niche of stem cell pharmacogenomics and discuss some of the priority issues that need to be targeted, based on the existing principles of pharmacogenomics, stem cell characteristics and transplantation medicine. Advances in these areas are imperative in realizing the dream of stem cell therapies contributing towards the improvisation of the quality of human life.