熊果酸对口腔鳞状细胞癌Tca8113细胞周期及凋亡的实验研究

目的探讨熊果酸对口腔鳞状细胞癌(Tca8113)的生长抑制情况及其对细胞周期和凋亡的作用。方法用浓度0、10、20、30、40、50μmol/L的熊果酸对口腔鳞状细胞癌进行干预,MTT法检测细胞增殖活性,流式细胞仪分析细胞周期,Annexin-V-FITC/PI双染流式细胞仪定量检测细胞凋亡。结果 (1)熊果酸对Tca8113细胞的增殖抑制具有时间--剂量依赖性,各实验组细胞增殖抑制率与对照组比较差异具有统计学意义(P<0.05)。(2)各浓度组熊果酸将细胞阻滞于S期,浓度30、40、50μmol/L的熊果酸总凋亡率分别为30.76%、43.77%及70.15%,与对照组比较差异有统计学意义(P<0.05)。结论熊果酸在体外抑制口腔鳞状细胞癌生长,呈时间--剂量依赖性,并阻滞细胞周期,促进细胞凋亡。     

丹参酮衍生物对K562细胞的生长抑制及诱导凋亡作用研究

目的研究丹参酮衍生物对人红白血病细胞株K562的生长抑制以及诱导凋亡作用。方法MTT比色法检测不同浓度丹参酮1、丹参酮2和丹参酮B对K562细胞的增殖抑制作用;PI单染法检测3者对K562细胞周期的影响;An-nexin V/PI双染法检测3者对K562细胞诱导凋亡的作用。结果丹参酮1和丹参酮B能够抑制K562细胞增殖,IC50分别为5.22和15.11μmol·L-1,且分别在2.5~10μmol·L-1和10~40μmol·L-1剂量范围内,G0/G1期细胞比例的增加及早期凋亡细胞百分率的提高均呈剂量相关性;丹参酮2在100μmol·L-1的剂量下,对K562细胞的抑制率仅为27.8%,无诱导其凋亡作用,但可以使G0/G1期细胞增多。结论丹参酮1和丹参酮B抑制K562细胞增殖,阻滞其于G0/G1期并诱导细胞凋亡;丹参酮2对K562细胞没有明显生长抑制及诱导凋亡作用,但可将其阻滞于G0/G1期。     

去甲斑蝥素诱导人卵巢癌SK-OV-3细胞发生有丝分裂期阻滞与凋亡

目的考察去甲斑蝥素(NCTD)对人卵巢癌SK-OV-3细胞生长的抑制作用,探究其诱导细胞发生有丝分裂期阻滞及凋亡的过程及相关性。方法 NCTD 30,60,120和240μmol.L-1分别作用人卵巢SK-OV-3细胞24,48和72 h后,MTT法检测细胞存活率;NCTD 60μmo.lL-1分别作用SK-OV-3细胞0,6,12和24 h后,流式细胞术测定细胞周期的变化;NCTD 60μmol.L-1分别作用SK-OV-3细胞0,6,12和24 h,倒置显微镜下检测其对细胞形态学变化的影响;Giemsa染色检测细胞核的变化;间接免疫荧光术结合激光扫描共聚焦显微镜技术检测细胞内微管分布及有丝分裂期纺锤体形成的影响。NCTD 60μmol.L-1分别作用12和36 h后,流式细胞术检测细胞凋亡情况;再将NCTD 60μmo.lL-1作用12 h后的细胞,采用温和的机械振荡法分离为悬浮细胞和贴壁细胞,继续作用24 h,流式细胞术及Giemsa染色检测分析两个时相两种细胞凋亡的影响。结果 MTT结果显示,NCTD 30～240μmol.L-1对SK-OV-3细胞的生长抑制作用存在明显的时间(P<0.05)和浓度依赖性(P<0.05);NCTD作用SK-OV-3细胞24,48和72 h的IC50分别为(261.3±2.4)μmo.lL-1,(48.3±1.7)μmol.L-1和(10.9±1.0)μmol.L-1。NCTD 60μmol.L-1分别作用于SK-OV-3细胞0,6,12和24 h,SK-OV-3细胞逐渐表现出G2/M期阻滞,呈时间依赖性;倒置相差显微镜下观察,NCTD引起SK-OV-3细胞逐渐收缩变圆,与周围细胞分离;Giemsa染色可见处于有丝分裂期的细胞显著增多,多个核细胞比例增多;免疫荧光染色发现,NCTD组细胞的微管系统受到干扰,有丝分裂期细胞纺锤体形成异常。NCTD作用细胞12和36 h凋亡率分别达20.4%和62.3%;将NCTD作用12 h的细胞,人工振荡分离为悬浮细胞和贴壁细胞,检测凋亡情况,同时检测继续作用24 h后两组细胞凋亡情况,发现处于有丝分裂间期的SK-OV-3细胞凋亡率大于处于有丝分裂期的细胞。结论 NCTD主要通过诱导人卵巢癌SK-OV-3细胞G2/M期阻滞和细胞凋亡抑制细胞生长。干扰细胞有丝分裂过程中微管的组装和纺锤体的形成可能是NCTD诱导SK-OV-3细胞M期阻滞的原因之一。NCTD诱导的SK-OV-3细胞发生凋亡可不依赖细胞M期阻滞。     

土槿皮乙酸B不通过Fas途径诱导人乳腺癌MCF-7细胞凋亡和M期周期阻滞

目的初探土槿皮乙酸B(PAB)诱导人乳腺癌MCF-7细胞凋亡和周期阻滞。方法MTT法测定PAB对MCF-7细胞的生长抑制情况;相差显微镜观察细胞形态变化;荧光显微镜观察经Hoechst33258染色的DNA变化;流式细胞仪检测用PI染色后MCF-7细胞的周期分布;免疫印迹实验检测相关蛋白的表达。结果PAB时间剂量依赖性的抑制MCF-7细胞生长。4μmol.L-1PAB在24h使DNA皱缩,在36和48h使DNA皱缩的细胞死亡。4μmol.L-1PAB时间依赖性的促进PARP〔poly-(ADP-ribose)polymerase〕的剪切。在36hPAB剂量依赖性的增加cdc2和核内cyclinB1的表达。Fas拮抗性抗体UB2不影响PAB诱导的凋亡,但Fas激动性抗体CH11促进PAB诱导的凋亡。并且UB2不影响PAB诱导的细胞周期阻滞,Fas激动性抗体CH11不影响G1和S期,但促进凋亡特征性的亚二倍体峰生成。结论4μmol.L-1的PAB通过凋亡方式可明显地抑制MCF-7细胞生长,并促进M期阻滞。Fas途径不参与PAB诱导的凋亡和周期阻滞。     

三氯乙酸染毒对肝L-02细胞的增殖作用及对DNA甲基化水平的影响

目的 检测三氯乙烯(TCE)主要代谢产物三氯乙酸(TCA)对人肝L-02细胞染毒24 h后的增殖作用,以及对DNA总体甲基化水平的影响,探索TCE对肝L-02细胞的表型遗传毒性.方法 选择0.1-0.9 mmol/L浓度TCA作为合适的染毒剂量对肝L-02细胞染毒24 h后分别进行下列试验:以CCK-8试剂盒观测细胞的生长曲线;以抗5-mC抗体进行免疫荧光检测细胞核中胞嘧啶甲基化总体水平(代A 0.9 mmol/L染毒24 h);以流式细胞仪(FCM)检测其对细胞周期的影响及凋亡情况;提取细胞DNA进行琼脂糖电泳分析其对DNA的损伤作用.以上试验必要时设置5-氮杂胞苷(5-aza-dC5 μmol/L)处理的L-02细胞为基因组DNA低甲基化的对照以及肝癌细胞作为细胞周期分析的对照.结果 TCE在0.1～0.9 mmol/L浓度下染毒24 h后可以促进肝L-02细胞生长,并在0.9 mmol/L浓度作用下24 h可致肝L-02细胞DNA总体甲基化水平降低,荧光强度和正常细胞比较明显减弱;细胞周期分析发现TCA处理24 h后再用正常培养基继续培养24 h后可使细胞S G2期中细胞比例增加(与肝癌细胞接近);DNA梯状电泳分析未发现DNA损伤性改变.结论 TCA染毒24 h可促进细胞生长,可诱导基因组DNA低甲基化,并造成细胞周期的改变.提示TCA对肝细胞的早期细胞毒性作用与表型遗传机制有关.     

吴茱萸碱对人肝癌细胞HepG2的生长抑制及诱导凋亡作用

目的探讨吴茱萸碱对人肝癌细胞HepG2的生长抑制、诱导凋亡和对细胞周期的影响。方法体外试验MIT法测存活率,DAPI染色观察细胞凋亡形态,流式细胞术、彗星电泳技术分析药物对DNA作用。结果吴茱萸碱能抑制人肝癌细胞HepG2的生长。用64、16、4、1、0.25μmol.L-1浓度的吴茱萸碱处理HepG2细胞72 h的抑制率分别为74.0%、69.0%、60.5%、44.0%、16.4%。DAPI染色后吴茱萸碱组癌细胞均表现出较为典型的细胞凋亡特征。流式细胞仪检测1μmol.L-1吴茱萸碱作用24和36 h出现亚二倍体凋亡峰,细胞周期阻滞于G2/M期。凋亡率对照组为4%,1μmol.L-1吴茱萸碱作用12、24、36 h的凋亡率分别为4.4%、18.0%、30.3%。彗星电泳显示1μmol.L-1吴茱萸碱作用24和48 h后,细胞后面形成长的拖尾,平均光密度值较阴性对照组降低,彗星尾距较阴性对照组增加,且二者的改变与作用时间相关。结论吴茱萸碱能抑制人肝癌细胞HepG2的生长及诱导其凋亡。     

槲皮素对肝癌HepG2细胞生长影响的体外研究

目的 观察槲皮素对肝癌HepG2细胞生长及hTERT基因表达的影响.方法 以台盼蓝拒染法计数肝癌细胞的生长抑制率,用透射电镜从形态变化方面了解凋亡的发生,流式细胞术检测细胞周期变化,Western-blot检测hTERT基因表达改变,PCR-TRAP法检测端粒酶活性.结果 槲皮素抑制肝癌HepG2细胞增殖的作用明显,且呈浓度和时间依赖性,槲皮素处理48 h后的半数抑药浓度（IC5o）为25.5μmol/L;形态学检测显示出了细胞凋亡的特征变化,经10-20 μm/L的槲皮素处理,肝癌HepG2细胞周期阻滞于G0/G1期,且HepG2细胞hTERT蛋白降低,端粒酶活性被抑制.结论 槲皮素能呈时间、剂量依赖性地抑制肝癌细胞的生长,能诱导HepG2细胞发生凋亡,其抑制增生与诱导凋亡的机制可能与下调hTERT基因表达、抑制端粒酶活性、破坏端粒稳定性有关.     

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。     

依地福新对Hela细胞生长的抑制作用及其机制研究

目的:观察依地福新对人宫颈癌Hela细胞的体外生长抑制作用,并进一步探讨其作用机制。方法:细胞中分别加入不同浓度的依地福新(0.5、1.0、5.0、10.0μmol.L-1)处理96h,并设立未加依地福新的对照组。应用MTT法检测细胞增殖活性;流式细胞仪检测细胞周期分布;染色法检测细胞凋亡率。结果:与对照组比较,不同浓度依地福新处理Hela细胞24～96h后,细胞增殖显著受到抑制,并呈浓度及时间依赖性;1.0、5.0、10.0μmol.L-1浓度依地福新处理72h后,Hela细胞G0/G1期细胞数量显著增加,S期细胞数量显著降低(P<0.01);各浓度组细胞凋亡率均显著增加(P<0.01)。结论:依地福新对Hela细胞具有生长抑制作用,其机制可能与阻滞细胞周期及诱导凋亡有关。     

紫杉醇对肝癌细胞HepG2和大鼠原代培养肝细胞增殖抑制和诱导凋亡作用的研究北大核心CSCD

目的探究紫杉醇对肝癌细胞HepG2和大鼠原代培养肝细胞增殖抑制和凋亡的影响。方法将肝癌细胞HepG2和原代肝细胞分别分为阴性对照组、不同浓度(5、10、20、40、80μg·L^(-1))紫杉醇组。MTT法检测不同浓度紫杉醇组作用肝癌细胞HepG2和原代肝细胞24、48、72 h的增殖抑制率;通过Hoechst 33258染色,在荧光倒置显微镜下观察细胞凋亡的形态学改变;Annexin V-FITC/PI双染,用流式细胞术检测不同浓度的紫杉醇对肝癌细胞HepG2和原代肝细胞细胞周期变化和凋亡率的影响。结果 MTT结果表明,紫杉醇对肝癌细胞HepG2和原代肝细胞剂量依赖性地抑制增殖(P<0.05)。Hoechst 33258染色结果显示,紫杉醇诱导肝癌细胞HepG2和原代肝细胞24 h后呈现凋亡形态学改变。流式结果提示,紫杉醇作用肝癌细胞HepG2和原代肝细胞24 h均能改变细胞周期,与阴性对照组相比,均能提高G_2/M期细胞比例(P<0.05),且阻滞作用随着药物浓度的增加而增强。其中紫杉醇能明显提高肝癌细胞HepG2 G_2/M期比例(P<0.05),G_0/G_1期细胞比例降低(P<0.05);S期细胞变化不大(P>0.05)。同时紫杉醇均能诱导两种细胞发生凋亡。但凋亡的程度不同,紫杉醇诱导肝癌细胞HepG2凋亡率明显高于原代肝细胞,且随着紫杉醇浓度的增高,细胞的凋亡率逐渐增加。结论紫杉醇能够抑制肝癌细胞HepG2和原代肝细胞的活力,是通过诱导细胞凋亡和阻滞细胞周期在G_2/M期完成的。     

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.