采用比较蛋白质组学方法研究p38β激酶对293T细胞蛋白质表达的影响

采用比较蛋白质组学方法研究了过表达p38信号通路激酶p38β对人类肿瘤细胞293T细胞蛋白质表达的影响．利用双向电泳和质谱技术分离鉴定了13个差异表达蛋白质．这些差异蛋白的分布从细胞质膜到核．其功能涉及核酸、蛋白质、糖脂类分子的转录、合成和成熟．细胞物质跨膜运输．细胞周期调控以及细胞防御等．献分析并没有发现上述鉴定的差异表达蛋白是p38β激酶现有直接底物的证据。提示其可能为新的p38β激酶相关蛋白．这些结果表明．比较蛋白质组学方法是寻找激酶新靶标的一种有效工具．     

利用基因芯片分析人era基因表达降低后细胞周期蛋白表达的变化

为了探讨利用RNAi封闭肿瘤细胞系的era基因表达后细胞周期相关蛋白的变化,实验利用Trizol法提取总RNA,用人细胞周期相关基因cDNA芯片检测细胞周期相关蛋白的表达。生物信息学分析结果显示：封闭肿瘤细胞系的era基因表达后,有10条细胞周期相关基因表达上调,7条基因表达下调,为研究人era基因的功能提供了线索。     

p53突变蛋白对人肺腺癌细胞中E-cadherin的表达调控及细胞迁移的影响

在人肺肿瘤细胞H1299中稳定表达含R273H点突变的p53蛋白(p53-R273H),观察到细胞中E-cadherin mRNA和蛋白水平下调,同时细胞迁移能力增强.免疫荧光方法检测发现p53-R273H显著降低E-cadherin在肿瘤细胞间的表达.这些结果表明p53-R273H突变蛋白具有下调E-cadherin基因的表达和促使肿瘤细胞迁移的新功能.     

血清对人脑胶质瘤细胞株U251基因表达的影响

利用分子原位杂交和定量PCR技术分析新生小牛血清培养和无血清培养的人脑胶瘤细胞株U2 5 1的基因表达 ,发现血清对细胞周期正调控基因cdc2基因和PCNA基因的表达有激活作用 ,而对细胞周期负调控基因 p2 1和 p16基因的表达有抑制作用 .这些事实表明 ,血清刺激细胞生长时 ,一方面要激活促进细胞增殖的基因 ,另一方面要抑制或关闭抑制细胞增殖的基因     

p53突变蛋白下调调控人肺腺癌细胞中E-cadherin的表达并促进细胞迁移

摘要：p53 是最重要的抑癌基因之一。在肿瘤发生发展过程中,超过50% 的人类肿瘤组织和细胞中存在 p53 基因发生突变,而且大多数为点突变,其中包括 R273H,由此产生的点突变蛋白会失去 p53 的DNA结合和特异的转录活性。最近的研究表明p53突变蛋白还可能获得一些野生型p53蛋白不具有的新的生物学活性。在本研究中,我们在人肺肿瘤细胞 H1299 中稳定表达含R273H 点突变的p53 蛋白（p53-R273H）,观察到细胞中 E-cadherin mRNA 和蛋白水平下调,同时细胞迁移能力增强。免疫荧光方法检测发现 p53-R273H 显著降低 E-cadherin 在肿瘤细胞间的表达。这些结果表明p53-R273H 突变蛋白具有下调 E-cadherin 基因的表达和促使肿瘤细胞迁移的新功能。     

OsICK1基因植物反义表达载体的构建及遗传转化

OsICK1是水稻细胞周期蛋白依赖性激酶抑制剂基因．我们构建了水稻细胞周期蛋白依赖性激酶抑制剂基因（OsICK1）的植物反义表达载体，在农杆菌的介导下尝试使antisense OsICK1整合到水稻基因组中并初步得到验证．为进一步研究OsICK1对水稻细胞周期调控的作用奠定了基础．     

对香豆酸抑制肺癌细胞增殖、迁移并诱导其凋亡的机制研究

探究了对香豆酸（p-coumaric acid,p-CA）对人肺癌细胞细胞A549和H1299增殖、凋亡和迁移的影响及作用机制.结果显示,添加0～10 mM p-CA能显著抑制两种肺癌细胞的生长和迁移,并呈剂量和时间依赖性（P<0.001）. p-CA显著抑制了细胞周期发生,并将细胞周期阻滞在G1期,且细胞凋亡发生明显增加（P<0.05）. p-CA显著抑制了CDK2、CDK6、Cyclin D1等周期正调控因子表达,并诱导细胞周期负调控因子p21、p27等基因的表达上调;与此同时,p-CA还诱导了促凋亡因子Bax表达上升和抗凋亡因子Bcl-2表达水平的降低且caspase-3和caspase-9的表达上调,而对迁移相关基因表达的检测结果也显示p-CA抑制了β-连环蛋白（β-catenin）的表达并促进钙粘附蛋白E(E-cadherin)表达上升.上述结果表明,p-CA具有抑制肺癌细胞生长、诱导凋亡并抑制其迁移的作用,它主要是通过促进Bax、caspase-3、caspase-9、p21、p27、E-cadherin等蛋白的表达而抑制Bcl-2、Cyclin D1、β-ca...     

RNA干扰Nucleostemin基因对人结肠癌细胞株HT-29细胞增殖及细胞周期的影响

将Nucleostemin(NS)siRNA利用脂质体2000转染人结肠癌细胞株HT 29,分别利用CCK-8试剂盒和流式细胞术检测NS siRNA对HT 29细胞增殖和细胞周期的影响,进一步利用Real-time PCR和Western blotting技术检测NS基因和细胞周期相关基因p21 mRNA和蛋白的表达.结果表明,NS siRNA的转入能有效下调NS mRNA和蛋白的表达(P0.05),并能明显抑制人结肠癌细胞株HT 29的细胞增殖(P0.05),并诱导细胞周期静止在G_0/G_1期,转染NS siRNA后,p21 mRNA和蛋白的表达均明显升高,提示细胞增殖抑制和细胞周期静止与p21基因表达的升高密切相关.     

P53亚细胞定位变化对POLD1基因启动子活性的影响

POLD1基因编码DNA聚合酶δ（pol δ）的催化亚基．体外实验表明p53能够抑制POLD1基因启动子活性．文中探讨p53是否在细胞内直接与POLD1启动子结合调节POLD1基因表达. 在瞬时转染pEGFP-p53重组质粒的人乳腺癌MCF7细胞中，p53表达增强; RT-PCR结果显示POLD1基因mRNA表达受到抑制． 染色体免疫共沉淀和荧光素酶报告基因实验证明p53在细胞内与POLD1启动子直接结合抑制其启动子活性．荧光显微镜观察发现EGFPp53在G1期进入细胞核而在S期和G2期则被转运至细胞质．在稳定表达的pEGFP-p53细胞系中，细胞周期同步化后POLD1启动子活性在细胞周期各时相均处于较低水平．证明了细胞内p53高表达后通过直接与POLD1启动子结合而抑制POLD1基因表达，且这种抑制作用不受细胞周期进程的影响．     

p53基因调控网络研究进展

肿瘤抑制基因p53表达的p53蛋白是一个通用转录因子,与其上、下游功能相关基因组成了一个复杂的基因调控网络,在这个基因网络中p53基因起着关键作用;DNA损伤、缺氧、原癌基因的激活等均能刺激p53基因表达;p53表达升高后,可通过p53-MDM2反馈环路与泛素系统等对p53表达水平进行精确调节;p53通过调控多种下游/靶基因表达完成多种生物学功能,主要包括阻滞细胞周期、促进细胞凋亡、维持基因组稳定性等;认识p53基因调控网络的功能有助于理解p53及其下游/靶基因间的具体作用机制。     

刀额新对虾周期蛋白B基因的分子克隆及序列分析

周期蛋白B(cyclin B)是真核生物细胞周期的一种重要调控原件,通过调节周期蛋白依赖性蛋白激酶(cyclindependent kinase,CKD)的活性可以控制细胞周期.运用RACE(rapid-amplification of cDNA ends)技术克隆了刀额新对虾(Metapendeusensis)cyclin B基因.刀额新对虾cyclin B的cDNA全长为1681 bp,5′端非编码区为111 bp,3 ′端非编码区为355 bp,开放阅读框为1215 bp,编码404个氨基酸,平均分子质量为45767.9 u,pl为8.81.Blast比对后发现,其氧摹酸序列与斑节对虾(Penaeus monodon)的同源性达约90％.基于cyclin B序列所绘制的进化树基本上能反映出各物种间的进化关系.半定量RT-PCR结果显示,刀额新对虾的cyclinB基因在不同组织表达中具有明显的组织特异性,在鳃、卵巢和心脏中有较高的表达量,在眼柄神经节和胸神经节中表达量较低.推测该差异性主要与cyclin B在细胞周期中调控细胞分裂的功能有关.     

皮蛋模拟胃肠道消化物对HepG2细胞的影响

皮蛋是中国传统食品,其水解物具有抗氧化、抗炎和抗癌等功效,具有良好的医学前景,但目前皮蛋发挥抗癌功效的途径及作用机制尚不清楚。探究了皮蛋模拟胃肠道消化物(preserved eggs simulated gastrointestinal digests,PESD)对人肝癌HepG2细胞增殖和细胞周期的影响。将皮蛋经体外模拟胃肠道消化处理后,进行细胞实验,用CCK-8法进行细胞增殖分析,研究PESD对HepG2细胞存活率的影响,采用碘化丙啶(propidium iodide, PI)染色标记的方法,通过流式细胞术测定PESD对HepG2细胞周期的影响,用western blot法测定细胞周期相关调控蛋白的表达。研究发现:PESD以剂量依赖性方式对HepG2细胞增殖进行抑制,IC₅₀为4.17mg/mL;PESD处理显著增加了S期细胞的占比(P<0.05),使HepG2细胞阻滞于S期;4mg/mL的PESD处理HepG2细胞24h后,细胞中cyclin A、cyclin E、ATM、chk2和CDC25A的蛋白表达水平上升(P<0.05),而CDK2蛋白表达水平下降(P<0.05)。研究认为,皮蛋模拟胃肠道消化物是以剂量依赖性的方式来抑制人肝癌HepG2细胞的增殖,主要是通过ATM-chk2-CDC25A信号通路,上调cyclin A和cyclin E蛋白的表达,下调CDK2蛋白的表达来实现人肝癌HepG2细胞阻滞于S期,影响人肝癌HepG2细胞周期进程,抑制癌细胞的增殖。研究结果旨在为皮蛋抗癌作用机制的阐明提供一定的理论基础。     

Expression and significance of cyclin D1, p27kip1 protein in bronchioloalveolar carcinoma

PURPOSE: To investigate the relationship between expression of cell cycle-related protein cyclin D1, p27kip1 and the pathogenesis of bronchioloalveolar carcinoma (BAC) and the value of prediction of prognosis. METHODS: Cyclin D1 and p27kip1 protein were detected by immunohistochemical En Vision method in 43 BACs. RESULTS: The positivity of cyclin D1 in BAC was 65.1% (28/43), which was significantly higher than that in normal pulmonary tissue (0/13), P<0.01. No statistically significant association was found between cyclin D1 expression data and sex, age, tobacco-use history, histologic subtype (mucinous vs nonmucinous), stromal fibrosis, lymph node metastasis, clinical stage or postoperative survival period (P>0.05), while cyclin D1 expression was found to be negatively correlated with tumor size (P<0.05). The positivity of p27kip1 in BACs was 51.2% (22/43), significantly lower than that in normal pulmonary tissue (12/13), P<0.01. p27kip1 expression level was not associated with sex, age, tobacco-use history, tumor size or histologic subtype (P>0.05), but was negatively correlated with stromal fibrosis, lymph node metastasis and clinical stage (P<0.05); and positively associated with postoperative survival period (P<0.01). The survival rate of p27kip1 positive group was significantly higher than that of p27kip1 negative group (P<0.01). No statistically significant correlation was found between cyclin D1 and p27kip1 expression. CONCLUSIONS: Increased cyclin D1 expression and decreased p27kip1 expression are related to the pathogenesis of BAC; decreased p27kip1 expression is associated with metastasis progression; immunodetection of p27kip1 is useful for assessment of prognosis.     

Partial inhibition of CDK4 gene expression leads to the extension of G1 phase of V79-8 cells

V79-8 is an abnormal cell line which does not have detectable G1 and G2 phases in its cell cycle. This cell line is derived from V79 cell line which has Gl phase but lacks G2 phase. By using an anti-sense approach, CDK4 gene expression was partially inhibited to find whether CDK4 might contribute to the lack of Gl phase in V79-8 cells. Anti-CDK4 anti-sense plasmid was constructed and used to transfect V79-8 cells. Clones of transfected cells (V79-8-asCDK4) were examined, in comparison with V79-8 cells, to determine its growth curve, cell doubling-time (GT), the level of CDK4 gene expression and the levels of expression of some other growth related genes. V79-8-asCDK4 cells showed a slower growth rate with a doubling time 2.5-h longer than that of V79-8 cells. A flow cytometry (FCM) analysis demonstrated that the 2.5 h increase of the doubling time of V79-8-asCDK4 cells was mainly due to the appearance of Gl phase because its G2 + M phase was not significantly different from that of V79-8 cells. The decrease of CDK4 gene expression in V79-8-asCDK4 cells was shown by Northern-blot. Changes in the expression levels of the growth-related genes TGF-β, cyclin D1 and Rb were also detected in V79-8-asCDK4 cells. CDK4 functions mainly in G1 and at the transition between G1 and S phases. Expression of an anti-sense CDK4 gene fragment reduces the levels of endogenous CDK4, CDK4/cyclinD kinase activity and the phosphorylation of Rb. These events may postpone the inactivation of the check-point leading to the delay of entry into S phase and the reappearance of G1 phase in V79-8-asCDK4 cells.     

Effects of enhancing p21 waf1 on proliferation and expression of G1cyclins andCDKs in human breast cancer cells

The cyclin-dependent kinase inhibitor p21( waf1/cip1/sdil) is an important negative regulator in control of cell cycle. Its functions of inhibiting cancer cell growth and its effects on expression of G1 phase cyclins and related CDKs are a worthy topic for study. The plasmid expressing p2l with high level was transformed to human breast cancer cells, and the expression of p2l in cells was enhanced, then the cell growth rate, anchorage-independent growth and tu-morigenecity were tested, at the same time the expression levels of cyclinD1, CDK4, cyclinE and CDK2 were analyzed by Northern blot. The results showed that since the expression of p21 was enhanced in the cell, the rate of cell growth and anchorage-independent growth was inhibited, tumorigenecity was suppressed, the level of expression of cyclinE and CDK2 decreased while that of cyclinDl and CDK4 was not affected. It is suggested that the enhanced expression of p21 markedly inhibits the proliferation and lessens the tumorigenecity of breast cancer cells, and that p2l expression is not related to that of cyclinDl and CDK4, but affects the expression of cyclinE and CDK2 .     

p16与原发性肝细胞癌的研究进展

p16基因又称肿瘤抑制基因(MTS1),是近年来发现的一个重要的抑癌基因,在细胞周期中发挥重要的调节作用.现对p16基因的结构、p16蛋白的作用途径以及该基因突变与原发性肝细胞癌的关系进行综述.     

5种细胞周期调控基因在小鼠生殖细胞发育过程中的表达研究

 以成熟(10周龄以上)的昆明种正常小鼠的精巢和卵巢为材料,利用地高辛标记的基因探针进行组织切片上的DNA-mRNA分子原位杂交,研究了PCNA,cdc2,cyclin D1,p2 1和 p16 5种细胞周期调控基因在生殖细胞发育过程中的表达.结果表明:PCNA基因在睾丸组织的精原细胞和精母细胞中有强杂交信号,而在雌性生殖细胞及滤泡细胞的发育过程都没有杂交信号;cyclin D1,cdc2,p2 1,p16基因在生殖细胞的发育过程中都没有,表明这些基因并没有参与小鼠生殖细胞的生长和分化调控.这些事实表明在生殖细胞发育过程中,控制细胞增殖和增殖抑制的基因与培养细胞有不同的机制,它们可能采用了不同的调控系统.     

Phosphorylation-dependent binding of mitotic cyclins to Cdc6 contributes to DNA replication control

Cyclin-dependent kinases (CDKs) limit the activation of DNA replication origins to once per cell cycle by preventing the assembly of pre-replicative complexes (pre-RCs) during S, G2 and M phases of the cell cycle in the budding yeast Saccharomyces cerevisiae. CDKs inhibit each pre-RC component (ORC, Cdc6, Cdt1/Mcm2-7) by different mechanisms. We show here that the mitotic CDK, Clb2/Cdc28, binds tightly to an amino-terminal domain (NTD) of Cdc6, and that Cdc6 in this complex is unable to assemble pre-RCs. We present evidence indicating that this Clb2-dependent mechanism contributes to preventing re-replication in vivo. CDK interaction with the NTD of Cdc6 is mediated by the cyclin subunit Clb2, and could be reconstituted with recombinant Clb2 protein and synthetic NTD peptides. Tight Clb2 binding occurred only when the NTD was phosphorylated on CDK consensus sites. Human CDKs containing cyclins A, B and E also bound specifically to phospho-NTD peptides. We propose that direct binding of cyclins to phosphopeptide motifs may be a widespread phenomenon contributing to the targeting of CDKs to substrates.     

CDK16在食管鳞癌中的表达及其对肿瘤发生发展的影响

目的研究发现,细胞周期素依赖性激酶(cyclin dependent kinases,CDKs)家族成员与食管鳞癌的发病密切相关,但细胞周期素依赖性激酶16(CDK16)对食管鳞癌发病的影响尚不清楚.本研究旨在探讨CDK16在人食管鳞癌组织中的表达及生物学意义.方法应用组织芯片技术结合免疫组织化学技术检测45例食管鳞癌组织、45例癌旁组织中CDK16蛋白的表达情况,并使用统计分析软件研究CDK16蛋白的表达与食管鳞癌临床病理特征之间的关系.结果 CDK16在食管鳞癌组织中呈阳性表达,且表达强度与肿瘤分化程度呈负相关.另外,CDK16在食管鳞癌细胞中的表达位置与肿瘤的分化程度紧密相关.CDK16的表达与食管鳞癌的分化程度、淋巴结转移及TNM临床分期均具有显著的相关性(P<0.05),而与患者的年龄、性别以及肿瘤的部位、最大径、病理形态没有显著的相关性(P>0.05).结论 CDK16在食管鳞癌中的高表达与食管鳞癌的发生发展及转移密切相关.检测CDK16的表达有助于食管鳞癌的临床诊断和预后评估.