在肾癌细胞系GRC—1细胞周期不同阶段Wnt—5A基因的？…

Ｗｎｔ基因所编码的蛋白质与许多生长因子一样具有分泌型生长因子的结构特点,其家族成员Ｗｎｔ－５Ａ是许多恶性肿瘤的自分泌生长因子,在肾细胞癌中表达显著升高。为研究在细胞周期的不同阶段生长因子ＷＮＴ－５Ａ在转录水平的表达情况,我们采用胸腺嘧啶双阻断及高压笑气处理的方法,使肾细胞癌细胞系ＧＲＣ－１细胞同步化。用半定量反转录多聚酶链反应对处于细胞周期不同阶段的细胞ｃＤＮＡ进行扩增,Ｓ期与Ｇ１,Ｍ期Ｗｎｔ－５     

PTEN基因诱导人胚肾293细胞凋亡和细胞周期停滞

为了研究抑癌基因PTEN过表达对HEK293细胞凋亡和细胞周期停滞的作用,以野生型PTEN和PTEN突变子(T910G)表达质粒分别转染无PTEN表达的人胚肾293细胞,采用细胞质梯度DNA方法检测细胞凋亡,以流式细胞仪分析细胞周期.发现PTEN过表达能够诱导人胚肾293细胞质中出现梯度DNA,293细胞发生凋亡,PTEN过表达改变细胞周期分布,G0/G1期细胞增加13%,S期细胞下降15%.PTEN突变子对细胞凋亡和G1细胞停滞的影响略弱于野生型PTEN.PTEN基因过表达明显下调血小板衍生生长因子(PDGF)诱导的蛋白激酶B(PKB)和p42,p44-促分裂原活化蛋白激酶(MAPK)磷酸化水平,PTEN突变子对p42,p44-MAPK磷酸化水平的调节作用略弱于野生型PTEN.PTEN通过抑制细胞增殖,诱导细胞凋亡而影响细胞生长.     

Wnt-5a抑制上皮性卵巢癌中β-catenin和MMP-26的表达及肿瘤细胞的迁移

Wnt-5a是Wnt信号转导途径中一个重要的成员,可影响Tcf/Lef转录因子,调控特定基因的表达．令人费解的是,它在不同肿瘤中具有截然不同的促进或抑制肿瘤作用．目前关于Wnt-5a在卵巢癌中的表达与功能尚不十分清楚．免疫组织化学检测显示,Wnt-5a在卵巢癌组织中的表达低于正常卵巢组织．蛋白质免疫印迹法检测揭示,Wnt-5a在卵巢癌细胞株A2780中表达低于正常卵巢细胞株TC-1,不同浓度Wnt-5a作用下 A2780细胞内β-catenin表达降低．实时荧光定量PCR检测mRNA揭示,不同浓度Wnt-5a作用下A2780细胞内β-catenin和MMP-26表达降低．细胞划痕、Tanswell法显示,Wnt-5a可抑制A2780细胞迁移．本研究结果提示,Wnt-5a在卵巢癌中扮演抑制肿瘤的角色,与抑制β-catenin和MMP-26表达、肿瘤的细胞运动能力有关．     

PTEN基因诱导人胚肾293细胞凋亡和细胞周期停滞

为了研究抑癌基因PTEN过表达对HEK293细胞凋亡和细胞周期停滞的作用,以野生型PTEN和PTEN突变子(T910G)表达质粒分别转染无PTEN表达的人胚肾293细胞,采用细胞质梯度DNA方法检测细胞凋亡,以流式细胞仪分析细胞周期.发现PTEN过表达能够诱导人胚肾293细胞质中出现梯度DNA,293细胞发生凋亡,PTEN过表达改变细胞周期分布,G0/G1期细胞增加13%,S期细胞下降15%.PTEN突变子对细胞凋亡和G1细胞停滞的影响略弱于野生型PTEN.PTEN基因过表达明显下调血小板衍生生长因子(PDGF)诱导的蛋白激酶B(PKB)和p42,p44-促分裂原活化蛋白激酶(MAPK)磷酸化水平,PTEN突变子对p42,p44-MAPK磷酸化水平的调节作用略弱于野生型PTEN.PTEN通过抑制细胞增殖,诱导细胞凋亡而影响细胞生长.     

RASSF1A对食管癌细胞增殖影响的研究

目的:观察外源性RASSF1A基因对食管癌细胞的增殖作用并探讨机制.方法:将包含RASSF1A基因的质粒转染食管癌细胞EC9706,建立稳定转染细胞克隆,Western blot检测RASSF1A基因表达,通过细胞生长曲线检测细胞生长活性和增殖能力、流式细胞仪检测对细胞周期的影响、裸鼠移植瘤实验检测转染细胞的体内成瘤特性.结果:稳定表达RASSF1A基因的EC9706细胞中RASSF1A蛋白表达增加;细胞生长速度明显减慢;细胞周期中G1/G0期比例明显增加,S期比例减少;EC9706细胞的裸鼠致瘤能力被抑制.结论:RASSF1A基因能显著抑制食管癌细胞在体内外的生长,其机制可能与该基因诱导凋亡、抑制增殖作用有关.     

应用组织芯片检测大肠癌组织中p27和cyclin D1的表达及意义

目的 应用组织芯片检测大肠癌组织中p27和cyclinD1的表达及意义.方法 应用免疫组织化学技术检测人大肠癌组织芯片150芯(包括70例大肠癌组织和5例癌旁组织)中p27和cyclinDl的表达.采用多光谱成像系统对免疫组织化学结果进行图像分析,并用SPSS13.0软件对各组染色分析后的数据做单因素方差分析和SNK(q)检验,检验水准α为0.05.结果 大肠癌组织中P27呈低表达,癌旁组织中呈高表达;大肠癌组织中cyclinD1呈高表达,癌旁组织中cyclinD1呈低表达,2组比较差异均有统计学意义(均P ＜0.05).结论 在大肠癌细胞周期G1/S期调控机制中,cyclinD1作为细胞周期G1／S期调控的正问因子促进细胞增殖;p27具有抑制cyclinD1的作用,从而阻止细胞周期GI/S期的转变,抑制细胞增殖. 统对免疫组织化学结果进行图像分析,并用SPSS13.0软件对各组染色分析后的数据做单因素方差分析和SNK(q)检验,检验水准α为0.05.结果 大肠癌组织中P27呈低表达,癌旁组织中呈高表达;大肠癌组织中cyclinD1呈高表达,癌旁组织中cyclinD1呈低表达,2组比较差异均有统计学意义(均P ＜0.05).结论 在大肠癌细胞周期G1/S期调控机制中,cyclinD1作为细胞周期G1／S期调控的正问因子促进细胞增殖;p27具有抑制cyclinD1的作用,从而阻止细胞周     

LncRNA MIR31HG通过诱导细胞周期阻滞抑制食管鳞癌细胞增殖活性

本研究探讨lnc RNA MIR31HG对食管鳞癌细胞增殖活性的影响.利用定量PCR检测MIR31HG在食管鳞癌标本及其癌旁组织、人食管上皮细胞系Het-1A和食管鳞癌细胞系Eca-109、EC-1、KYSE30中的表达;采用过表达质粒pc DNA3.1-MIR31HG在食管鳞癌细胞系中过表达MIR31HG;MTT法和SRB法检测细胞增殖率;细胞周期分析试剂盒检测细胞周期进程;Caspase3活性检测试剂盒分析Caspase3活性;PCR和Western blot法检测p53、Caspase3及Bcl-2的m RNA和蛋白质表达水平.结果显示,食管癌组织中MIR31HG表达水平显著低于癌旁组织(P0.05);与Het-1A细胞相比,Eca-109、EC-1、KYSE30细胞中MIR31HG的表达均显著下调(P0.05),提示MIR31HG可能介导食管癌的发生发展.转染pc DNA3.1-MIR31HG可显著上调食管癌细胞中MIR31HG的m RNA表达(P0.01),且MIR31HG过表达可显著抑制食管癌细胞增殖活性(P0.05),减少S期细胞数(P0.05),增加G1期细胞数(P0.05),提示MIR31HG可能通过阻碍细胞周期G1期~S期进程抑制食管癌细胞增殖活性.此外,MIR31HG过表达显著增加Caspase3活性,增加Caspase3和p53的m RNA和蛋白质表达水平,同时抑制Bcl-2 m RNA和蛋白质表达水平.这表明,MIR31HG可通过抑制食管癌细胞的增殖活性阻碍食管癌的发生发展,这可能为食管癌的诊断和治疗提供新策略.     

G6PD通过细胞周期蛋白D1/D2调控人黑色素瘤细胞周期的进程

葡萄糖-6-磷酸脱氢酶(G6PD)在人皮肤黑色素瘤A375细胞中处于高表达与高活性状态, 但G6PD在黑色素瘤发生发展过程中的作用及其具体机制尚不明确.本文在前期运用 siRNA方法构建G6PD敲减的黑色素瘤A375稳转细胞(A375-G6PDΔ)基础上,构建表达载体pBabe-puro-G6PDWT在A375-G6PDΔ细胞中过表达野生型的G6PD基因,从而构建G6PD表达恢复的稳转细胞(A375-G6PDΔ-G6PDWT).3株细胞A375-WT、A375-G6PDΔ和 A375-G6PDΔ-G6PDWT经G6PD酶活性测定、MTT测定、克隆形成实验、流式细胞仪分析细胞周期和Western 印迹检测.结果显示,A375-G6PDΔ-G6PDWT细胞的G6PD蛋白表达量 (0.847 ± 0.080)及其活性(0.394 ± 0.029)分别是A375-G6PDΔ的3.28倍(P＜0.01) 和7.34倍(P＜0.01),分别是A375-WT细胞的91-57%和2.12倍(P＜0.05).与A375-WT细 胞相比,A375-G6PDΔ细胞G0/G1期细胞数增加,S期细胞数减少,增殖指数PI降低了25-70%（P＜0.05）,细胞周期蛋白D1/D2、细胞周期蛋白E表达分别下降37.4%、54.3% (P＜0.01)和17.3%;而A375-G6PDΔ-G6PDWT细胞呈现G1/S期阻滞解除,细胞周期蛋白D1/D2蛋白分别恢复到A375-WT细胞的89.5%和87.6%,细胞周期蛋白E表达未见 恢复,呈现生长增殖和克隆形成率的恢复并接近于A375-WT细胞. 结果提示,G6PD通 过细胞周期蛋白D1/D2调控人皮肤黑色素瘤A375细胞G1期向S期转换的进程,这为黑色 素瘤发病机制的研究提供了新的思路.     

低氧诱导因子在肾透明细胞癌中作用的研究进展

VHL基因具有调节转录、稳定细胞生长相关基因和调节细胞周期的功能,其突变、缺失、重排和超甲基化与肾细胞癌(RCC)的发生密切相关。VHL基因产物VHL肿瘤抑制蛋白(p VHL)是泛素连接酶的成分,具有调节低氧诱导因子(HIF)稳定性的作用。HIF家族主要包含三种HIFα因子(HIF1α、HIF2α、HIF3α)和两种HIFβ因子(HIF1β、HIF2β)。HIF1α位于14q染色体上,在肾透明细胞癌中该染色体经常缺失,且这种14q的缺失常伴有预后不良。HIF2α的表达异常促进了p VHL缺陷型肾透明细胞癌中的发生。目前,抑制HIF2α及其下游的血管内皮生长因子(VEGF)的药物都处于临床试验的不同阶段,已有四种VEGF抑制剂获准用于肾透明细胞癌的治疗。选择抑制HIF或具有HIF靶基因抑制选择性的药物进行研究,可能为肾透明细胞癌的治疗提供新的方法。本文就HIF在VHL蛋白缺陷型肾透明细胞癌中作用的研究进展进行了综述。     

中药固真方对UMR106细胞周期蛋白表达及细胞周期的影响

应用流式细胞技术和 Western blot技术 ,观察了中药固真方对大鼠成骨肉瘤细胞 UMR1 0 6细胞的细胞周期及细胞周期蛋白 cyclin E和 cyclin A表达的影响 .结果显示 :固真方处理 UMR1 0 6细胞后 2 4 h,S期细胞百分比达高峰 ,占 49.6% ,而对照组 30 h时 ,S期细胞才增高 ,达 43.2 % .cy-clin E的表达在固真方处理 8h时增高 ,而对照组在 1 6h时 ,cyclin E表达才达高峰 .cyclin A的表达在给药组处理 2 4 h时最高 ,而对照组在 30 h时 ,cyclin A表达才增高 .揭示固真方可缩短细胞周期的时程 ,加速细胞周期的运行 ,从而促进细胞增殖 .     

Characterization of the effects of butyric acid on cell proliferation, cell cycle distribution and apoptosis

We examined concentration-dependent changes in cell cycle distribution and cell cycle-related proteins induced by butyric acid. Butyric acid enhanced or suppressed the proliferation of Jurkat human T lymphocytes depending on concentration. A low concentration of butyric acid induced a massive increase in the number of cells in S and G2/M phases, whereas a high concentration significantly increased the accumulation of cells in G2/M phase, suppressed the accumulation of cells in G0/G1 and S phases, and induced apoptosis that cell cycle-related protein expression in Jurkat cells treated with high levels of butyric acid caused a marked decrease in cyclin A, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4 and CDK6 protein levels in G0/G1 and S phases, with apoptosis induction, and a decrease in cyclin B, Cdc25c and p27KIP1 protein levels, as well as an increase in p21CIP1/WAF1 protein level, in the G2/M phase. Taken together, our results indicate that butyric acid has bimodal effects on cell proliferation and survival. The inhibition of cell growth followed by the increase in apoptosis induced by high levels of butyric acid were related to an increase in cell death in G0/G1 and S phases, as well as G2/M arrest of cells. Finally, these results were further substantiated by the expression profile of butyric acid-treated Jurkat cells obtained by means of cDNA array.     

mTOR及其底物在HeLa细胞的细胞周期不同时相中的表达

为探讨细胞生长的机制 ,用RT PCR、Western印迹及蛋白激酶活性测定等方法对同步化的HeLa细胞的细胞周期不同时相中mTOR(mammaliantargetofrapamycin) ,p70S6激酶 (p70S6K)的α1 、α2 、β1 、β2 不同亚型及起始因子 4E结合蛋白 1 (4EBP1 )的表达进行了检测 .RT PCR的结果表明 :在G1 、S1 、G2 、M1 、M2 几个细胞周期时相中 ,mTOR的mRNA表达无明显变化 .mTOR的底物P70S6K的亚型α1 、α2 、β1 、β2 在M期表达均有明显增加 .4EBP1的表达在M期明显减少 .免疫印迹的结果与RT PCR的一致 ,即M期p70S6K的α1 、α2 、均有增加 ,4EBP1在M期减少 .活性测定表明 ,G2 期、M期mTOR较其它期有明显增加 ,4EBP1在M期活性有所下降 .研究结果表明 :mTOR、p70S6K、4EBP1很可能在HeLa细胞的生长中起重要的调节作用     

Cell cycle-dependent expression of cyclooxygenase-2 in human fibroblasts.

The purpose of this investigation is to determine whether the levels of cyclooxygenase-2 (COX-2) expression are cell cycle dependent. We used a serum-starved human foreskin fibroblast model to determine changes in COX-2 mRNA, protein, and promoter activity in response to stimulation with interleukin-1b (IL-1b) and phorbol 12-myristate 13-acetate (PMA) at G0, G1, S and G2/M phases of the cell cycle. IL-1b (1 ng/ml) and PMA (100 nM) induced robust COX-2 expression in the G0 cells, and the level of COX-2 expression declined progressively after the cells had entered the cell cycle. The COX-2 mRNA level at G1, S and G2/M phases of the cell cycle was 76%, 46%, and 30% of that at G0, respectively. A 5-flanking promoter fragment of COX-2 constructed into a luciferase expression vector was transfected into cells. The promoter activity in response to PMA stimulation was significantly higher in G0 than in S phase cells. These results imply that G0 cells are the key players in inflammation and other COX-2-dependent pathophysiological processes. When the cells are in the proliferative phase, COX-2 inducibility becomes restrained probably by an endogenous control mechanism to avoid COX-2 mediated oxidative DNA damage.     

顺铂靶向食管癌细胞周期Cx43表达的研究

目的:为研究顺铂治疗食管鳞癌细胞的靶向作用。方法:本研究使用流式细胞技术双变量分析检测顺铂对食管癌细胞周期进程和癌细胞周期的连接蛋白43(connexin 43,Cx43)表达的影响。结果:顺铂对食管鳞癌细胞周期的影响主要作用于S期的DNA复制,细胞阻滞于S期,G2/M期减少。顺铂诱导食管鳞癌细胞周期S和G2/M期的Cx43表达的大幅度改变。低浓度顺铂(由0~2μmol/L),Cx43表达增强;顺铂渐高浓度(2~12μmol/L),细胞Cx43表达由强逐渐变弱,特别是G2/M期细胞的Cx43表达活跃,易受顺铂影响。结论:我们的研究表明以顺铂处理食管鳞癌细胞,癌细胞周期的S期和G2/M期的Cx43表达与S期的DNA复制一样可作为的潜在治疗靶标。顺铂靶向作用细胞周期S和/或G2/M期细胞的特性可能减少或避免对非分裂细胞的影响。     

Cell cycle function of a Medicago sativa A2-type cyclin interacting with a PSTAIRE-type cyclin-dependent kinase and a retinoblastoma protein

In plants multiple A-type cyclins with distinct expression patterns have been isolated and classified into three subgroups (A1-A3), while in animal somatic cells a single type of cyclin A is required for cell-cycle regulation from the S to M phases. We studied the function of an A2-type cyclin from Medicago sativa (Medsa;cycA2) which, in contrast to animal and most plant A-type cyclins, was expressed in all phases of the cell cycle. Using synchronized alfalfa cell cultures and anti-Medsa;CycA2 polyclonal antibodies, we showed that while the mRNA level increased steadily from the late G1 to the G2-M phase, the protein level after a rapid increase in S-phase reached a plateau during the G2 phase. In the yeast two-hybrid system, the Medsa;CycA2 protein interacted with the PSTAIRE-motif-containing cyclin-dependent kinase Cdc2MsA and with the maize retinoblastoma protein. Unexpectedly, the CycA2-associated kinase activity was biphasic: a first activity peak occurred in the S phase while the major one occurred during the G2/M transition, with no apparent dependence upon the actual levels of the Medsa;CycA2 and Cdc2MsA proteins. Immunohistological localization of the cyclin A2 protein by immunofluorescence and immunogold labelling revealed the presence of Medsa;CycA2 in the nucleus of the interphase and prophase cells, while it was undetectable thereafter during mitosis. Together these data suggest that Medsa;CycA2 plays a role both in the S phase and at the G2/M transition.     

Cell cycle control of Wnt/β-catenin signalling by conductin/axin2 through CDC20

Wnt/β-catenin signalling regulates cell proliferation by modulating the cell cycle and is negatively regulated by conductin/axin2/axil. We show that conductin levels peak at G2/M followed by a rapid decline during return to G1. In line with this, Wnt/β-catenin target genes are low at G2/M and high at G1/S, and β-catenin phosphorylation oscillates during the cell cycle in a conductin-dependent manner. Conductin is degraded by the anaphase-promoting complex/cyclosome cofactor CDC20. Knockdown of CDC20 blocks Wnt signalling through conductin. CDC20-resistant conductin inhibits Wnt signalling and attenuates colony formation of colorectal cancer cells. We propose that CDC20-mediated degradation of conductin regulates Wnt/β-catenin signalling for maximal activity during G1/S.