藏红花素联合顺铂对人宫颈癌HeLa细胞协同抑制作用的研究

目的 探索藏红花素联合顺铂对人宫颈癌HeLa细胞的协同抑制作用及相关调控机制。方法 取对数生长期人宫颈癌HeLa细胞,设置空白对照组（DMSO）、藏红花素组（400 μg·mL^-1）、顺铂组（5 μg·mL^-1）、联合组（藏红花素400 μg·mL^-1+顺铂5 μg·mL^-1）。干预48 h后,CCK-8检测HeLa细胞增殖抑制率,采用CompuSyn软件计算藏红花素与顺铂的联合指数（CI）,Annexin V-FITC染色法检测细胞凋亡,流式细胞术检测细胞周期分布,Western blotting检测激活型半胱氨酸天冬氨酸蛋白酶-3（Cleaved Caspase-3）、B细胞淋巴瘤/白血病-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、细胞周期素D1（Cyclin D1）、周期蛋白依赖激酶2（CDK2）的表达。结果 与顺铂组比较,联合组细胞增殖抑制率显著升高（P<0.05）,CI为0.68,具有中度协同效应;细胞凋亡率显著升高（P<0.01）,G₀/G₁期细胞比例显著升高（P<0.05）,而G₂/M期比例显著降低（P<0.01）,Cleaved Caspase-3、Bax蛋白表达水平及Bax/Bcl-2比值均显著升高（P<0.05）,Cyclin D1、CDK2蛋白表达水平显著降低（P<0.01）。与空白对照组比较,藏红花素组G₀/G₁期细胞比例显著升高而G₂/M期比例显著降低（P<0.01）,Cleaved Caspase-3、Bax表达水平及Bax/Bcl-2比值均显著升高（P<0.01）,Cyclin D1、CDK2表达水平显著降低（P<0.05）。结论 藏红花素联合顺铂可协同抑制人宫颈癌HeLa细胞的增殖和生长,其作用机制可能与调控凋亡相关蛋白的表达从而促进细胞凋亡、阻滞细胞周期进程有关。     

大蒜素联合顺铂对人宫颈癌细胞增殖的抑制作用

目的探讨大蒜素联合顺铂对人宫颈癌HeLa细胞增殖的抑制作用并探讨其机制。方法取对数生长期HeLa细胞，设对照组、大蒜素（50 μg/mL）组、顺铂（5 μg/mL）组、联合给药组（大蒜素50 μg/mL+顺铂5 μg/mL）。药物干预48 h后，通过MTT法检测细胞增殖抑制率，运用CompuSyn软件计算大蒜素与顺铂联合指数（CI），流式细胞术检测细胞周期分布和细胞凋亡水平，Western blotting法检测Cyclin D1、CDK2、P27、Cleaved Caspase-3、Bcl-2、Bax蛋白表达。结果与大蒜素组和顺铂组比较，联合给药组HeLa细胞增殖抑制率显著升高（P<0.05、0.01），CI为0.69（提示大蒜素与顺铂具有中度协同效应）。与对照组比较，大蒜素组和顺铂组G₀/G₁期细胞比例和细胞凋亡率显著升高（P<0.05、0.01），Cyclin D1、CDK2、Bcl-2表达显著下调且P27、Cleaved Caspase-3、Bax表达显著上调（P<0.05、0.01），Bax/Bcl-2比值显著升高（P<0.01）。与大蒜素组和顺铂组比较，联合给药组G₀/G₁期细胞比例和细胞凋亡率显著升高（P<0.05、0.01），Cyclin D1表达显著下调且P27、Cleaved Caspase-3、Bax表达显著上调（P<0.05、0.01），Bax/Bcl-2值显著升高（P<0.01）。结论大蒜素联合顺铂具有协同抑制人宫颈癌HeLa细胞增殖的作用，可能与调节细胞周期和凋亡相关蛋白表达，进而阻滞细胞周期进程并促进细胞凋亡有关。     

儿茶素对骨髓细胞周期及造血生长因子基因表达的作用儿茶素对骨髓细胞周期及造血生长因子基因表达的作用

目的探讨中药鸡血藤Spatholobus suberectus Dunn活性成分儿茶素对小鼠骨髓细胞增殖周期及其脾细胞内造血生长因子IL-6和GM-CSF mRNA表达的影响,以初步阐明其造血调控作用机理。方法用流式细胞仪检测技术观察儿茶素对正常及骨髓抑制小鼠骨髓细胞增殖周期的影响,同时采用RT-PCR技术,检测在儿茶素刺激条件下,小鼠脾细胞内IL-6和GM-CSF mRNA表达的变化。结果儿茶素可促使正常及骨髓抑制小鼠骨髓细胞G₀/G₁期细胞比例下降,S+G₂/M期细胞比例增加,并可使正常及骨髓抑制小鼠脾细胞内IL-6 mRNA和GM-CSF mRNA表达显著上调。结论儿茶素可通过诱导脾细胞内IL-6 mRNA和GM-CSF mRNA的表达,促进正常小鼠骨髓细胞进入增殖周期,并促使骨髓抑制小鼠的骨髓细胞跳出“G₁期阻滞”,进入细胞增殖周期,从而加速造血干/祖细胞的增殖和分化。     

周期蛋白依赖性激酶4/6抑制剂palbociclib

周期蛋白依赖性激酶（CDK）4/6是细胞周期的关键调节因子,能够触发细胞周期从生长期（G₁期）向DNA复制期（S₁期）转变。在雌激素受体阳性（ER⁺）乳腺癌中,CDK4/6的过度活跃非常频繁。palbocicib是一种口服的CDK4/6抑制剂,能够选择性抑制CDK4/6,恢复细胞周期控制,阻断肿瘤细胞增殖。从Ⅱ期临床实验结果来看,palbociclib的临床疗效具有非常明显的优势。     

3-取代芳基氧化吲哚(PH II-7)对肿瘤细胞周期的影响

目的观察3-取代芳基氧化吲哚(PH II-7)对肿瘤细胞周期分布的影响，明确PH II-7的抗敏感肿瘤和耐药肿瘤的共同机制。方法用流式细胞仪检测细胞周期分布，Western印迹分析细胞周期相关蛋白的表达，³H-TdR参入法检测细胞DNA合成，ELISA测定酪氨酸激酶的活性。结果PH II-7对多种肿瘤细胞（包括耐药细胞）的周期分布均有影响，阻滞细胞G₁期至S期的移行，细胞周期相关蛋白CDK2，Rb和c-myc的表达被抑制，Cyclin E的表达升高。PH II-7还可抑制³H-TdR的参入，抑制EGFR的酪氨酸激酶的活性。结论抗耐药肿瘤新药PH II-7是一种细胞周期阻滞剂，可能是通过抑制CDK2而使肿瘤细胞阻滞在G₁期，同时也说明细胞周期阻滞可能是抗耐药肿瘤的新方向。     

肿瘤细胞周期测定方法研究

肿瘤是一类与细胞周期密切相关的疾病。流式细胞术对肿瘤细胞周期的分析可以观察到肿瘤细胞倍体的变化,有助于肿瘤的早期发现和治疗。区分肿瘤细胞所处于的细胞周期,明确地了解每个时相所占细胞周期的比例,对于肿瘤药物的研发具有重要意义。该文着重对G₀期和G₁期,G₂期和M期,以及S期细胞的测定方法进行归纳、总结,并对这些方法的优缺点及适用条件进行阐述和总结,为肿瘤药物的研发及基础研究等方面的科研工作提供参考。     

川芎嗪对人结直肠癌SW480细胞增殖和凋亡的影响及机制研究★

目的 探讨川芎嗪（TMP）对人结直肠癌SW480细胞增殖和凋亡的影响及其作用机制。方法 以DMSO（空白对照组）、不同浓度（50、100、200 μg·mL^-1）的TMP和奥沙利铂（L-OHP，25 μg·mL^-1）干预对数生长期人结直肠癌SW480细胞48 h。采用MTT法和EdU插入实验检测各组SW480细胞增殖率，细胞克隆实验观察SW480细胞克隆形成能力，流式细胞术、Western blotting检测细胞周期、凋亡和蛋白表达。结果 与空白对照组比较，经50、100、200 μg·mL^-1的TMP或25 μg·mL^-1的L-OHP干预可明显降低SW480细胞增殖率，提高细胞凋亡率；经100、200 μg·mL^-1的TMP或25 μg·mL^-1的L-OHP干预可明显抑制细胞克隆形成能力，阻滞细胞周期于G₀/G₁期，提高PTEN、Cleaved Caspase-3、Bax、P27表达量和Bax/Bcl-2比值，降低p-Akt、cyclin D1、Bcl-2表达量和Akt磷酸化率，差异均具有统计学意义（P<0.05）。与L-OHP 25 μg·mL^-1组比较，经TMP 200 μg·mL^-1干预能够明显降低SW480细胞增殖率、提高凋亡率，提高PTEN、Bax、P27表达量和Bax/Bcl-2比值，降低p-Akt、cyclin D1表达量和Akt磷酸化率，差异均具有统计学意义（P<0.05）。结论 TMP可能通过上调PTEN抑制PI3K/Akt通路，进而抑制SW480细胞增殖并促进其凋亡。     

大黄素对人胃癌MNK-45和MGC8-03细胞增殖的影响

目的 观察大黄素对人胃癌MNK-45和MGC8-03细胞增殖的影响。方法 采用MTT法测定MNK-45细胞和MGC8-03细胞增殖抑制率;采用AO/EB双染观察细胞形态学变化;采用流式细胞仪测定细胞周期。结果 大黄素（5～80 μg/mL）对MNK-45和MGC8-03细胞增殖均具有显著抑制作用。P＜0.01或0.05）,且药物剂量越大,作用时间越长,其抑制作用越强（P＜0.01）;大黄素作用于两种细胞后荧光显微镜下观察均显示细胞凋亡特征;大黄素使MNK-45和MGC8-03细胞增殖被阻滞于G₀/G₁期。结论 大黄素能抑制人胃癌MNK-45和MGC8-03细胞增殖,诱导其凋亡,影响其细胞周期时相的分布。     

4-氨基-2-三氟甲基苯基维甲酸酯对K562细胞分化和细胞周期的影响

目的本研究探讨新型维甲酸衍生物4-氨基-2-三氟甲基苯基维甲酸酯(4-amino-2-trifluoromethyl-phenyl retinate,ATPR)对K562细胞株的抑制增殖和诱导分化活性并对其机制进行研究。方法ATPR作用于K562细胞3d后,通过MTT法检测细胞的增殖,NBT还原实验法分析细胞的分化指标,瑞氏染色法在油镜下观察加药前后细胞形态学变化,FCM检测分析细胞周期,RT-PCR法检测cyclinE、cyclinD1、CDK2、CDK4、CDK6、p21cip1、p27kip1、p57kip2和PCNA mRNA的变化情况。Western blot法检测cyclin D1和CDK4蛋白表达的改变。结果ATPR呈浓度依赖性抑制K562细胞增殖的作用。ATPR诱导分化活性表现为NBT阳性细胞率增加,油镜下观察K562细胞有分化成熟的改变,G0/G1期细胞表达量增加,S期细胞表达量减少,呈G1期阻滞。RT-PCR检测发现cyclin E、cyclin D1、CDK2、CDK4、CDK6表达减少,PC-NA、P21cip1、P27kip1改变不明显,P57kip2表达增加。Western blot检测cyclin D1和CDK4蛋白表达减少。结论ATPR有较强的抑制K562细胞增殖并诱导其分化的活性,并通过上调P57kip2的表达,抑制Cyclin-CDK激酶复合物,发挥细胞周期阻滞的作用。     

人参皂苷Rg3对乳腺癌MCF-7细胞增殖和侵袭的影响

目的 观察人参皂苷Rg3对雌激素受体阳性的乳腺癌细胞MCF-7增殖和侵袭的影响,并探讨其可能的作用机制。方法 采用MTT法检测细胞的增殖能力,流式细胞仪分析细胞周期分布以及凋亡比率,通过Transwell小室观察细胞侵袭力,RT-PCR法检测细胞中的MMP-9 mRNA的表达。结果 与对照组相比,人参皂苷Rg3能显著抑制MCF-7细胞的增殖;G₀/G₁期及S期细胞比例减少,而G₂/M期细胞比例显著增加;同时细胞凋亡比率亦明显提升,而细胞侵袭指数降低,且呈现良好的剂量、时间依赖性。同时人参皂苷Rg3还能显著抑制细胞中MMP-9 mRNA的表达水平(P<0.05)。结论 人参皂苷Rg3能抑制MCF-7细胞的增殖和侵袭,其作用机制可能与其能降低MMP-9基因的表达有关。     

Inhibition of cell cycle progression by penta-acetyl geniposide in rat C6 glioma cells

Penta-acetyl geniposide, (Ac)5-GP, the acetylated compound of geniposide, is able to inhibit the growth of rat C6 glioma cells in culture and in the bearing rats. Our recent data indicated that the induction of cell apoptosis and cell cycle arrest at G0/gap phase 1 (G1) by (Ac)5-GP might be associated with the induction of p53 and c-Myc, and mediated via the apoptosis-related bcl-2 family proteins. In this report, we further investigated the mechanism involved in the cell cycle arrest induced by (Ac)5-GP in C6 glioma cells. The inhibitory effect of (Ac)5-GP on the cell cycle progression of C6 glioma cells which arrested cells at the G0/G1 phase was associated with a marked decrease in the protein expression of cyclin D1, and an induction in the content of cyclin-dependent kinase (cdk) inhibitor p21 protein. This effect was correlated with the elevation in p53 levels. Further immunoprecipitation studies found that, in response to the treatment, the formation of cyclin D1/cdk 4 complex declined, preventing the phosphorylation of retinoblastoma (Rb) and the subsequent dissociation of Rb/E2F complex. These results illustrated that the apoptotic effect of (Ac)5-GP, arresting cells at the G0/G1 phase, was exerted by inducing the expression of p21 that, in turn, repressed the activity of cyclin D1/cdk 4 and the phosphorylation of Rb.     

Aryl hydrocarbon receptor-activating polychlorinated biphenyls and their hydroxylated metabolites induce cell proliferation in contact-inhibited rat liver epithelial cells.

Polychlorinated biphenyls (PCBs) exhibit tumor-promoting effects in experimental animals. We investigated effects of six model PCB congeners and hydroxylated PCB metabolites on proliferation of contact-inhibited rat liver epithelial WB-F344 cells. The 'dioxin-like' PCB congeners, PCB 126, PCB 105, and 4'-OH-PCB 79, a metabolite of the planar PCB 77 congener, induced cell proliferation in a concentration-dependent manner. In contrast, the 'non-dioxin-like' compounds that are not aryl hydrocarbon receptor (AhR) agonists, PCB 47, PCB 153, and 4-OH-PCB 187, an abundant noncoplanar PCB metabolite, had no effect on cell proliferation at concentrations up to 10 muM. The concentrations of dioxin-like PCBs leading to cell proliferation corresponded with the levels inducing the expression of cytochrome P450 1A1 mRNA, suggesting that the release from contact inhibition was associated with AhR activation. The effects of PCB 126 and PCB 153 on expression of proteins controlling G0/G1-S-phase transition and S-phase progression were compared. Only PCB 126 was found to upregulate cyclin A and D2 protein levels, and to increase both total cyclin-dependent kinase 2 (cdk2) and cyclin A/cdk2 complex activities. Despite the observed upregulation of cyclin D2, no increase in cdk4 activity was observed. The expression of cdk inhibitor p27Kip1 was not affected by either PCB 126 or PCB 153. These results suggest that dioxin-like PCBs can induce cell proliferation of contact-inhibited rat liver epithelial cells by increasing cyclin A protein levels, a process that then leads to upregulation of cyclin A/cdk2 activity and initiation of DNA replication. This mechanism could be involved in tumor-promoting effects of dioxin-like PCBs.     

高糖抑制内皮细胞增殖的细胞周期调控

目的 研究高浓度葡萄糖(HG)对人脐静脉血管内皮细胞(HUVEC)增殖和细胞周期的影响.方法 以体外培养的HUVEC为模型,采用倒置显微镜观察细胞形态学;噻唑蓝比色法测定细胞的增殖与活性;流式细胞术检测细胞周期以及相关蛋白-增殖性细胞核抗原(PCNA)与细胞周期蛋白D1的表达.结果 HG(20mmol/L、40mmol/L)作用72h后,HUVEC的增殖受到了明显抑制(P＜0.01);48～72h后细胞周期中G0-G1期细胞的百分比增加,与对照组同期比较均有显著差异(P＜0.01);同时PCNA、周期蛋白D1的表达均明显低于对照组(P＜0.01).结论 HG可抑制体外培养的HUVEC增殖,使细胞阻滞于G1期,PCNA及周期蛋白D1表达的下调可能参与了此过程的调控.     

(-)-Syringaresinol inhibits proliferation of human promyelocytic HL-60 leukemia cells via G1 arrest and apoptosis

We examined the effect of (-)-syringaresinol, a furofuran-type lignan isolated from Daphne genkwa, on cell cycle regulation in HL-60 human promyelocytic leukemia cells in vitro. (-)-Syringaresinol decreased the viability of HL-60 cells by inducing G(1) arrest followed by apoptosis in a dose- and time-dependent manner. The G(0)/G(1) phase of the cell cycle is regulated by cyclin-dependent kinases (Cdk), cyclins and cyclin-dependent kinase inhibitors (Cdki). We show by western blot analysis, that the (-)-syringaresinol-induced G(1) arrest was mediated through the increased expression of Cdki proteins (p21(cip1/waf1) and p27(kip1)) with a simultaneous decrease in cdk2, cdk4, cdk6, cyclin D(1), cyclin D(2), and cyclin E expression. The induction of apoptosis after treatment with (-)-syringaresinol for 24 h was demonstrated by morphological changes, DNA fragmentation, altered ratio of Bax/Bcl-2, cleavage of poly(ADP-ribose) polymerase and flow cytometry analysis. (-)-Syringaresinol also induced cytochrome c release and activation of caspase-3 and caspase-9. To our knowledge, this is the first time that (-)-syringaresinol has been reported to potently inhibit the proliferation of human promyelocytic HL-60 cells through G(1) arrest and induction of apoptosis. These findings suggest that (-)-syringaresinol may be a potential chemotherapeutic agent for the treatment of cancer.     

牛磺酸对心肌成纤维细胞细胞周期的影响及其机制的研究

目的研究牛磺酸(taurine,Tau)对血管紧张素Ⅱ(an-giotensinⅡ,AngⅡ)诱导的新生大鼠心肌成纤维细胞(cardiacfibroblast,CFb)增殖的抑制作用,并初步探讨其作用机制。方法用AngⅡ诱导新生大鼠CFb增殖,建立心肌纤维化模型,采用四甲基偶氮唑盐(MTT)法检测细胞增殖;羟脯氨酸测定检测胶原含量;流式细胞仪检测细胞周期及细胞周期蛋白依赖性激酶抑制物p27的变化;免疫细胞化学法测定细胞周期调控蛋白D(cyclin D)及p27蛋白的含量。结果Tau(40、80、160mmol.L-1)可明显抑制AngⅡ诱导的CFb增殖与胶原合成,同AngⅡ组比较差异有显著性(P<0.05或P<0.01),且呈现剂量依赖性。流式细胞仪检测表明Tau在80、160mmol.L-1可促进p27的蛋白表达;细胞G0/G1期百分率随Tau浓度增加而增加,S期细胞百分率随Tau浓度增加而减少,与AngⅡ组相比差异有显著性(P<0.05或P<0.01)。免疫细胞化学染色结合图像分析系统的分析结果也表明Tau可增加p27的蛋白表达,与AngⅡ组相比差异具有统计学意义(P<0.01)。结论牛磺酸通过促进p27的表达,使细胞周期阻滞于G0/G1期,而抑制CFb增殖和胶原含量的增加。     

Destruxin E, a cyclodepsipeptide antibiotic, reduces cyclin D1 levels and inhibits anchorage-independent growth of v-Ki-ras-expressed pMAM-ras-REF cells

Destruxin E (DE), a cyclodepsipeptide isolated from fermentation broths of Metarhizium sp. MA324, inhibited the growth of v-Ki-ras-expressed pMAM-ras-REF (rasREF) cells in the suspension (anchorage-independent) culture (a) more strongly than that in the substratum-attached (anchorage-dependent) culture (b) or that of v-Ki-ras-unexpressed pMAM-ras-REF (REF) cells in the substratum-attached culture (c); the IC(50) values of DE were 0.07 microM (a), 0.4 microM (b), and 1.2 microM (c). DE arrested G1 phase cell cycle progression of rasREF cells in the substratum-attached culture (b). In rasREF cells treated with DE for 72 h in suspension culture (a), the levels of cyclin D1, cyclin A, p27(Kip1), and hyperphosphorylated Rb were decreased, but the levels of cdk4, cdk6, cdk2, p16(INK4a), and p21(Cip1) were not affected. Among these effects, the decrease in cyclin D1 was prominent. DE decreased the level of cyclin D1 in rasREF cells in the suspension culture (a) at 0.1 microM and in the substratum-attached culture (b) at 1 microM, while the level of cyclin D1 in REF cells in the substratum-attached culture (c) was not decreased at 1 microM. The extent of growth inhibition correlated with the decrease in cyclin D1. The level of cyclin D1 mRNA of rasREF cells in the suspension culture (a) was also decreased by DE. DE decreased cyclin D1 mRNA, resulting in inhibition of anchorage-independent growth of rasREF cells.     

Pyrogallol inhibits the growth of human lung cancer Calu-6 cells via arresting the cell cycle arrest

Pyrogallol (PG) is a polyphenol compound and has been known to be an O(2)(-) generator. We evaluated the effects of PG on the growth of human pulmonary adenocarcinoma Calu-6 cells in relation to the cell cycle. DNA flow cytometric analysis indicated that PG induced a G2 phase arrest of the cell cycle in Calu-6 cells at 72h. PG down-regulated the expression of CDKI (p27), CDK2, CDK4 and CDK6 as well as cyclin D1, and increased cyclin A and cyclin B1 proteins. In addition, O(2)(-) levels were significantly increased in PG-treated cells. Treatment with catalase rescued Calu-6 cells from PG-induced apoptosis, and also prevented the growth inhibition as well as a G2 phase arrest by PG, which were accompanied with the down-regulation of O(2)(-) levels. In conclusion, PG inhibited the growth of Calu-6 cells by inducing the cell cycle arrest, accompanied with an increase in O(2)(-) levels.     

An attempt to evaluate the effect of vitamin K3 using as an enhancer of anticancer agents

The possibility of vitamin K3 (VK3) as an anticancer agent was assessed. VK3 dose-dependently diminished the cell viability (measured as esterase activity) with IC50 of 13.7 microM and Hill coefficient of 3.1 in Hep G2 cells. It also decreased the population of S phase and arrested cell cycle in the G2/M phase in a dose-dependent manner. G2/M arrest was regulated by the increment of cyclin A/cdk1 and cyclin A/cdk2 complex, and contrasting cyclin B/cdk1 complex decrease. Finally, combined application demonstrated that VK3 significantly enhanced the cytotoxicity of etoposide, a G2 phase-dependent anticancer agent, whereas it reduced the cytotoxic activity of irinotecan, a S phase-dependent agent. These findings suggest that VK3 induces G2/M arrest by inhibition of cyclin B/cdk1 complex formation, and is thus useful as an enhancer of G2 phase-dependent drugs in hepatic cancer chemotherapy.