环六亚甲基双乙酰胺对胃癌不同细胞周期两条信号系…

环六亚甲基双乙酰胺（ＨＭＢＡ）对ＭＧｃ８０－３不同时相细胞内ｃＡＭＰ－ＰＫＡ与ＤＡＧ－ＰＫＣ两大系统不仅具有正负调控作用,而且其作用具有周期特异性。其中Ｇ１期是最敏感的调控时相,与对照组相比,ｃＡＭＰ水平上升１０２．３％,ＰＫＡ活性升高３４８％,ＤＡＧ含量下降５１．４％,ＰＫＣ活性降低３２．３％;次敏感时相为Ｇ２期;Ｍ期基本没受影响,Ｓ期变化规律不同于其他时相。     

c-ski对大鼠皮肤成纤维细胞增殖的调节作用及机制

c-ski是成纤维细胞增殖的复杂调节子,它对中胚层来源的皮肤成纤维细胞增殖的作用还不清楚。在观察正常成纤维细胞周期c-ski表达的时相特点的基础上,通过体外转染c-ski,观察它对细胞增殖活性、细胞周期进展以及周期蛋白表达的影响。结果显示：c-ski mRNA表达在加入血清后开始升高,在细胞周期G,期的高峰期达到峰值,S期显著下降,在G2／M期维持在较低的水平：转染的c-ski可以以剂量依赖的方式增加细胞的增殖活性,并且可以逆转Smad3对细胞增殖活性的抑制作用;C-ski使成纤维细胞提前达到G0／G1期的最低点,进入S期：同时细胞G1期周期蛋白cyclinD的表达增加。这些结果表明：C-ski是皮肤成纤维细胞G1期的调节子,通过加快G1期进展促进增殖,抑制Smad3活性,促进cyclinD的表达可能与这一作用的分子机制有关。     

蛋白激酶C_ζ对小鼠受精卵发育早期基因组转录活化的影响

为研究蛋白激酶Cζ (proteinkinaseCζ ,PKCζ)在小鼠受精卵细胞早期发育过程中对胚胎基因组活化影响 ,采用免疫印迹和细胞免疫荧光的方法 ,观察PKCζ的抑制剂对小鼠受精卵 1 细胞期G1和G2 不同时期小鼠受精卵基因组活化的影响 .小鼠 1 细胞期受精卵蛋白激酶C (PKC)的活性不断增加 ,并在G2 期达到最高 .PKC的抑制剂calphostinC可以明显抑制PKC的活性达 4 7% .同时calphostinC对受精卵 1 细胞期基因组的早期活化具有显著的抑制作用 (P <0 0 1) .在小鼠 1 细胞期受精卵的G2 期 ,具有活性的磷酸化PKCζ的含量明显多于G1期和卵母细胞MⅡ期 ,分别比它们高2 7%和 110 % .PKCζ的特异性抑制剂可以抑制受精卵 1 细胞期基因的转录和活化 (P <0 0 5 ) .实验结果表明 ,PKCζ参与了小鼠受精卵基因组早期转录的调控     

孕激素诱导cyclin G1在小鼠子宫内膜上皮细胞的表达及其对细胞增殖的影响

本文在体外培养条件下研究卵巢激素诱导小鼠子宫内膜上皮细胞cyclin G1的表达及细胞增殖和细胞周期进程的变化,以探讨孕激素依赖的细胞周期调控因子cyclin G1对子宫内膜上皮细胞增殖的负调控作用.原代培养小鼠子宫内膜上皮细胞,待其生长汇合后分为4组:对照组(C组)、雌激素组(E组)、孕激素组(P组)、雌、孕激素共同作用组(EP组).加入相应激素作用24 h后,用细胞免疫化学方法检测各组细胞cyclin G1的表达水平:四甲基偶氮唑蓝(MTT)比色法检测各组细胞活力,间接观察子宫内膜上皮细胞的增殖情况;用流式细胞仪检测分布在细胞周期各时相的子宫内膜上皮细胞所占百分数.细胞免疫化学结果显示,cyclin G1在C组和E组子宫内膜上皮细胞上无明显表达,而在P组和EP组子宫内膜上皮细胞中表达明显,且定位于细胞核内.MTT法结果显示,与C组相比,E组细胞活力明显增高,而P组和EP组的细胞活力均明显下降,表明雌激素能促进子宫内膜上皮细胞增殖,而孕激素则具有抑制子宫内膜上皮细胞增殖的作用.流式细胞术检测显示,与C组相比,E组中处于S期的子宫内膜上皮细胞百分数增多;P组与EP组中处于S期的子宫内膜上皮细胞百分数明显减少,而处于G1期的细胞百分数和G2/M期的细胞百分数则明显增加.上述结果提示,孕激素依赖的cyclin G1可能通过阻滞细胞周期进程来参与孕激素对子宫内膜上皮细胞增殖的负调控作用.     

两种人胃癌细胞M期与间期时相质膜表面Con A受体复合物分布及侧向运动

利用荧光标记和荧光漂白恢复方法研究了两种人胃腺癌细胞M期与间期时梠中,细胞膜表面ConA受体复合物分子的分布与侧向运动.结果表明:MGC80-3细胞M期时相与SGC7901细胞间期时相膜表面ConA受体复合物分布近似,其侧向运动方式呈扩散型;SGC 7901 M期时相细胞膜表面ConA受体复合物的分布与MGC80-3细胞间期时和相基本类似,其侧向运动主要是流动型.凡是受体复合物是流动型运动的细胞.其膜上可动分子的百分比都寓于扩散型运为的细胞,P值小于0.01.     

两种人胃癌细胞M期与间期时相质膜表面Con A受体复合物分布及侧向运动

利用荧光标记和荧光漂白恢复方法研究了两种人胃腺癌细胞M期与间期时梠中,细胞膜表面ConA受体复合物分子的分布与侧向运动.结果表明:MGC80-3细胞M期时相与SGC7901细胞间期时相膜表面ConA受体复合物分布近似,其侧向运动方式呈扩散型;SGC 7901 M期时相细胞膜表面ConA受体复合物的分布与MGC80-3细胞间期时和相基本类似,其侧向运动主要是流动型.凡是受体复合物是流动型运动的细胞.其膜上可动分子的百分比都寓于扩散型运为的细胞,P值小于0.01.     

鳗鲡繁殖生物学研究 Ⅲ.鳗鲡性腺发育组织学和细胞学研究

鳗鲡精巢发育可划分为6个时期,即精原细胞前增殖期,精原细胞后增殖期,精母细胞生长、成熟期,精子开始出现期,精子完全成熟期和精子退化吸收期。卵细胞的发育可划分为6个时相,即卵原细胞时相,卵母细胞单层滤泡时相,卵母细胞出现脂肪泡时相,卵母细胞卵黄充满时相,卵母细胞核极化时相和卵母细胞退化时相。以卵细胞发育6个时相在卵巢中组成的差异,也可把卵巢划分为相应的6个时期。对鳗鲡性腺发育的分期,卵黄积累方式,产卵类型等问题进行了讨论。       

Rho小G蛋白介导的细胞周期调控

Rho小G蛋白作为一个信号分子家族具有多样化的功能, 可以调节细胞骨架重排 、细胞迁移、细胞极性、基因表达、细胞周期调控等. Rho小G蛋白家族对细胞周期 调控的研究主要集中在其对于有丝分裂期细胞的调节作用,包括调节有丝分裂期前 期细胞趋圆化、后期染色体排列及收缩环的收缩作用.近期的研究显示,Rho小G蛋白及其效应分子对于细胞周期G1、S、G2期的调控主要是通过影响细胞周期的正调控因子细胞周期蛋白D1 (cyclin D1) 和负调控因子细胞周期蛋白依赖型激酶相互作用蛋白1及细胞周期蛋白依赖型激酶抑制蛋白27 (p21cip1/p27kip1) 进行的.本文总结了Rho小G蛋白及其效应分子在细胞周期调控,尤其是对G1/S期调控的研究进展,并简要阐述了Rho小G蛋白介导的细胞周期调控异常与癌症发生的关系.     

敲除 Sam68 基因导致白血病细胞系细胞生长迟缓和 G2/M 期延长

RNA 结合蛋白 Sam68 是细胞有丝分裂期 Src 酪氨酸磷酸化的靶蛋白 . 尽管确切机制尚不清楚,一些人还是认为 Sam68 可通过调控 RNA 的代谢参与细胞周期调控 . 利用基因打靶技术,在 DT40 细胞分离出 Sam68 基因缺失的细胞系 . 利用该细胞系,进行 Sam68 的功能解析 . 与野生型细胞系相比, Sam68 基因缺失细胞表现出明显的生长速度迟缓 . 通过细胞周期研究揭示 , 这些细胞生长速度延迟是由于细胞周期中的 G2/M 期延长 . 因为参与细胞周期 G2/M 期调控的周期因子 Cdc2 激酶的活性没有改变,所以提示 Sam68 不依赖于 Cdc2 激酶的活性参与细胞周期中 G2/M 期调控 .     

Skp2参与HeLa细胞G_2/M周期检查点的调控

具癌基因特性的Skp2在大多数肿瘤组织和肿瘤细胞中异常高表达,它作为SCFSkp2复合物的底物识别亚基调控p27KIP蛋白的稳定性而促进细胞G1/S期转换.为进一步明确Skp2与G2/M周期检查点的关系,在HeLa细胞中过表达Skp2以及通过反义寡核苷酸抑制Skp2表达.结果发现:Skp2能促进细胞周期运转,表现为S期细胞增多和G2/M期细胞减少,其中F-box结构域具有重要的功能意义;反义寡核苷酸抑制Skp2表达后,HeLa细胞发生显著的G2/M期阻滞;MTT检测结果表明,400nmol/L的Skp2的反义寡核苷酸能明显抑制HeLa细胞的增殖活性;Western印迹结果表明,HeLa细胞中Skp2可能通过负调控p21WAF的稳定性来参与G2/M检查点调控,这在用放线菌素D处理HeLa细胞的实验中得到验证.这些结果初步揭示了Skp2参与HeLa细胞G2/M周期检查点调控的分子机制.     

Crosstalk between elicitor-induced cell death and cell cycle regulation in tobacco BY-2 cells

The molecular links between cell cycle control and the regulation of programmed cell death are largely unknown in plants. Here we studied the relationship between the cell cycle and elicitor-induced cell death using synchronized tobacco BY-2 cells. Flow cytometry and fluorescence microscopy of nuclear DNA, and RNA gel-blot analyses of cell cycle-related genes revealed that the proteinaceous elicitor cryptogein induced cell cycle arrest at the G1 or G2 phase before the induction of cell death. Furthermore, the patterns of cell death induction and defence-related genes were different in different phases of the cell cycle. Constitutive treatment with cryptogein induced cell cycle arrest and cell death at the G1 or G2 phase. With transient treatment for 2 h, cell cycle arrest and cell death were only induced by treatment with the elicitor during the S or G1 phase. By contrast, the elicitor-induced production of reactive oxygen species was observed during all phases of the cell cycle. These results indicate that although recognition of the elicitor signal is cell cycle-independent, the induction of cell cycle arrest and cell death depends on the phase of the cell cycle.     

Changes in p34cdc2 kinase activity and cyclin A during induced differentiation of murine erythroleukemia cells.

Hexamethylene bisacetamide (HMBA)-induced murine erythroleukemia (MELC) differentiation is characterized by a prolongation of the initial G1 which follows passage through S phase in the presence of inducer. Commitment to terminal cell division is first detected in a portion of the cell population during this prolonged G1. HMBA-induced commitment is stochastic. This study has examined changes in two known cell cycle regulators, p34cdc2 and cyclin A, in cycle-synchronized MELC in the absence and presence of HMBA. Histone H1 kinase activity of p34cdc2, and the levels of CDC2Mm mRNA, 1.8-kilobase mRNA of cyclin A, and cyclin A protein changed during cell cycle progression in MELC, and all of them were suppressed during G1. The suppression of the H1 kinase activity and cyclin A expression continued through the prolonged G1 in MELC cultured with HMBA, whereas p34cdc2 protein level did not vary through the cell cycle in MELC cultured without or with inducer. Phosphorylation of p34cdc2 in uninduced MELC gradually increased as cells progressed from G1 to S. In induced MELC, an increase in phosphorylation of p34cdc2 occurred during the prolonged G1, and prior to the exit of the bulk of the cells from G1 to S. These results suggest that in HMBA-induced MELC, p34cdc2 phosphorylation per se is not a limiting factor in determining G1 to S progression. The persistent suppression of cyclin A expression and histone H1 kinase activity may play a role in HMBA-induced commitment to terminal differentiation.     

HMBA诱导人肝癌SMMC—7721细胞分化的观察

In this paper, the effects of HMBA on the differentiation of human hepatocarcinoma cell line SMMC-7721 were investigated. After treated with 5 mmol/L HMBA, the proliferation of SMMC-7721 cells was inhibited remarkably, the cell growth inhibitory rate amounted to 64.14%, the cell mitotic index was declined by 53.88%. Light microscopy and transmission electron microscopy showed that the morphology and ultrastructure of the cells treated with HMBA undergone restorational alteration. Cytochemistry and immunocytochemistry assay revealed that the activities of gamma-GT declined and the levels of AFP and PCNA downregulated while the activity of TAT increased significantly after HMBA treatment. In the meantime, flow cytometry analysis showed that HMBA could arrest the cells in G0/G1 phase. The results showed that HMBA could effectively inhibit the proliferation, reverse the malignant morphology and ultrastructure, alter the levels of enzymes and antigens, arrest the cells in G0/G1, and induce the differentiation of human hepatocarcinoma SMMC-7721 cells in vitro.     

Cell volume changes in relation to the cell cycle of differentiating erythroleukemic cells

Murine erythroleukemic cells (MELC) were synchronized by sequential exposure to thymidine and hydroxyurea. Upon removal from hydroxyurea, cells cultured with or without agents that induce erythroid differentiation, such as hexamethylene bisacetamide (HMBA) or dimethylsulfoxide (Me₂SO), proceed through S, G2 and mitosis with the same kinetics: S phase averages 5 h and G2 plus mitosis, 2 h. Cells cultured with HMBA and Me₂SO remain in the subsequent G1 for 5–7 h, compared with an average of only 3 h for cells cultured without inducer. Modal cell volume doubles as the cells proceed from G1 to G2. During the inducer-mediated prolonged G1, MELC retain a small cell volume. In cultures of non-synchronous MELC, inducers also increase the G1 fraction, as well as the proportion of small cells. An Me₂SO-resistant MELC variant (DR10), cultured with Me₂SO, shows little prolongation of G1 and little difference in the modal cell volume compared with cells without inducer. However, HMBA, which induces differentiation of DR10 cells, prolongs G1 and increases the proportion of small cells. These studies indicate that early changes in cell volume associated with induction of MELC to differentiate, in large part reflect alterations in the cell cycle. Evidence is presented which suggests that only one round of DNA synthesis in the presence of inducer may be necessary to initiate differentiation.     

HMBA诱导人肝癌SMMC-7721细胞分化的观察

本文研究HMBA对人肝癌SMMC-7721细胞的诱导分化作用.细胞生长曲线测定和细胞分裂指数观察显示HMBA可明显抑制细胞增殖,细胞生长抑制率达64.14%,分裂指数抑制率为53.88%.光镜和透射电镜观察可见HMBA能诱导人肝癌SMMG-7721细胞形态和超微结构发生恢复性改变.生化检测或免疫细胞化学方法观察显示,HM-BA处理后细胞γ.谷氨酸转肽酶(γ-GT)活性和甲胎蛋白(AFP)、增殖细胞核抗原(PCNA)表达均降低,而酪氨酸.酮戊二酸转氨酶(TAT)活性增强.流式细胞仪分析表明HMBA引起细胞发生G0/G1期阻滞.以上结果表明HMBA能有效抑制人肝癌细胞恶性增殖活性,逆转肝癌细胞恶性形态与超微结构特征,改变肝癌细胞相关酶活性和抗原表达,引发G0/G1期阻滞,从而对肝癌细胞具有明显的诱导分化作用.     

盘基网柄菌(Dictyostelium discoideum)突变型细胞allC细胞周期异常的研究

细胞计数和细胞倍增时间计算的结果表明allC细胞的倍增时间为2.36h,仅为KAx-3细胞倍增时间的1/3。为了探究allC细胞倍增时间大幅度缩短、细胞周期异常的原因我们采用流式细胞术测定两种细胞的细胞周期,并结合实时荧光定量PCR技术测定cycB1和cdk1基因的相对表达量的比值。结果表明,16h突变型allC细胞处于G2期的数目(1.51%)显著少于KAx-3细胞(16.61%)。allC细胞和KAx-3细胞的细胞周期素B1(cyclinB1)cycB1基因相对表达量分别是2.5和0.25,两者相差10倍。这些数据表明,两种类型细胞中G2期的差异十分明显,cyclinB1的相对表达量也存在显著差异。提示cyclinB1的过表达可能在一定程度上影响allC细胞的细胞周期正常的调控机制,与突变细胞的G2期异常有一定关系。     

Identification of a restriction point at the M/G1 transition during the ongoing cell cycle

Progression through the cell cycle is dependent upon numerous external factors (growth factors, extracellular matrix components) which exert their effects through the activation of signal transduction networks. During last years we have studied the regulation of progression through the ongoing CHO cell cycle. Recently, we have demonstrated that in CHO cells at least two serum dependent points exist in G1 phase that lead to different cellular responses. The first point is located immediately after mitosis and is suggested to link with apoptosis, while the second is located in late G1 phase and probably corresponds to the classical restriction point R. Because of the suggested link with apoptosis of the restriction point in early G1 phase, we have studied the possible role of PI 3-K in cell cycle progression through the ongoing G1 phase of CHO cells. In the presence of the PI 3-K inhibitors wortmannin or LY294002, cells were arrested during early G1 phase, leading to the expression of cleaved caspase-3, a central mediator of apoptosis. Addition of AP-2, an inhibitor of PKB, the downstream substrate of PI 3-K, at several time points during G1 phase demonstrated that inhibition during early G1 phase caused cell cycle arrest, while addition of the inhibitors during mid or late G1 phase had no effect on S phase entry. As for inhibition of PI 3-K, also inhibition of PKB resulted in expression of cleaved caspase-3. These results clearly demonstrate that a decision point exists in the early G1 phase of the cell cycle; in the presence of PKB activity the cells are continuing cell cycle progression, while in the absence of PKB activity the cells are induced for apoptosis.