p27^Kipl反义寡核苷酸对B淋巴瘤细胞WEHI—231细胞周期的影响

目的 探讨p27^Kipl在抗原受体介导的B淋巴瘤细胞WEHI-231细胞周期停止信号中的作用。方法 用抗IgM抗体诱导WEHI-231细胞细胞周期停止，用合成的p27^Kipl反义寡核苷酸抑制p27^Kipl基因的表达，采用流式细胞仪，分析细胞核的DNA含量和细胞周期的变化，用体外激酶实验检测Cdk2的活性，Western blot检测Rb蛋白的磷酸化水平。结果 合成的p27^Kipl反义寡核苷酸能阻断抗IgM抗体诱导的p27^Kipl基因表达的上调，用p27^Kipl反义寡核苷酸处理WEHI-231细胞，可恢复抗原受体交联引起的周期素依赖性激酶Cdk2活性的降低，以及Rb蛋白磷酸化水平的下降，并使细胞周期恢复运转。结论 p27^Kipl可能在抗原受体信号介导的WEHI-231细胞的细胞周期停止中发挥主要作用。     

p27Kip1与肿瘤

真核生物的细胞周期进程是由一系列调控因子有序的聚合和激活来调节控制的,其正常与否和细胞以及个体的生长、分化、衰老和癌变密切相关。参与细胞周期调控的主要分子有：细胞周期蛋白（ｃｙｃｌｉｎ）,细胞周期蛋白依赖性激酶（ｃｙｃｌｉｎｄｅｐｅｎｄｅｎｔｋｉｎａ...     

SIRT1上调大鼠血管平滑肌细胞P27Kip1的表达

目的 研究III类去乙酰化酶SIRT1在血管平滑肌中对p27Kip1表达的影响及其可能的机制。方法 用H2O2和oxLDL刺激大鼠平滑肌细胞A7r5,Western blot检测平滑肌细胞内源性SIRT1的表达变化;在动物血管损伤模型中,Western blot检测血管损伤处平滑肌的SIRT1表达水平。用SIRT1重组腺病毒感染A7r5细胞和原代大鼠平滑肌细胞,Western blot观察SIRT1过表达引起的p27表达的改变;用SIRT1抑制剂NAM处理平滑肌细胞并观察p27的表达改变;用免疫共沉淀技术探寻SIRT1上调p27基因表达可能的分子机制。 结果 在H2O2和oxLDL氧化应激刺激的A7r5细胞中,内源性SIRT1的表达随作用时间逐渐增加;动物血管损伤模型中,内源性SIRT1的表达明显上调;过表达SIRT1能够在10%血清刺激的早期上调p27表达,而SIRT1抑制剂NAM则能够减少p27表达;在原代平滑肌细胞中,腺病毒介导的SIRT1过表达同样能够显著上调p27的表达。在血管平滑肌细胞中检测到SIRT1能够与FOXO3a相互作用。结论 SIRT1在血管平滑肌细胞中上调p27的表达。     

脂质体转染细胞周期素B1反义脱氧寡核苷酸对HL60细胞增殖调控的影响

目的:探讨脂质体转染细胞周期素B1(cyclinB1)反义脱氧寡核苷酸(ASON)对HL60细胞增殖调控的作用。方法:用针对cyclinB1mRNA5’端编码区起始密码子(ATG/AUG)的ASON,通过脂质体导入HL60细胞共培养后,用流式细胞术(FCM)和RT-PCR分别检测cyclinB1蛋白和mRNA的表达水平,电镜和原位细胞凋亡检测法(POD)、FCM及DNA凝胶电泳法检测细胞凋亡。结果:CyclinB1ASON组与SON及空白对照组相比,ASON能特异地抑制cyclinB1蛋白及mRNA水平的表达,当ASON的浓度达到一定程度时,HL60细胞的增殖及集落形成率均明显受抑制,出现细胞凋亡,并且此作用随ASON浓度的升高而增强。结论:CyclinB1的特异ASON能封闭其蛋白及mRNA的表达水平,可剂量依赖性地抑制白血病细胞增殖,诱导细胞凋亡。     

核因子-κB p65反义寡核苷酸对溃疡性结肠炎肠黏膜单个核细胞细胞因子表达的影响

探讨 NF- κB p6 5反义寡核苷酸对溃疡性结肠炎肠黏膜单个核细胞 NF- κB p6 5的表达以及对细胞因子释放的影响。3例溃疡性结肠炎患者 (未用过任何与溃疡性结肠炎治疗相关的药物 ,符合 1993年太原会议溃疡性结肠炎诊断标准 )的活检组织被用于固有层单个核细胞 (L PMC)的分离和培养 ,并用 NF- κB p6 5反义寡核苷酸 (A-SON:5 '- GGAACAGTTCGTCCATGG- 3')及错义寡核苷酸 (MSON:5 '- GGAACAGTTCGTCTATGG- 3')和地塞米松 (EDX)进行干预。采用 :1Western蛋白印迹分析检测 NF-κB p6 5蛋白表达情况 ;2逆转录聚合酶链反应 (RT-PCR)检测 IL- 1βm RNA和 IL- 8m RNA的表达 ;3酶联免疫吸附试验 (EL ISA)测定 IL- 1β,IL- 8的分泌水平。结果显示 ,NF-κB p6 5反义寡核苷酸可明显减少 L PMC的 NF-κB p6 5表达 ,阻断 L PS刺激引起的 IL - 1βm RNA和 IL -8m RNA的表达的增强以及两种细胞因子分泌水平均的增高 ,其作用明显强于地塞米松 (P<0 .0 5 )。结论认为 :NF-κB p6 5反义寡核苷酸有可能作为治疗溃疡性结肠炎的新基因药物 ,为治疗溃疡性结肠炎开辟了一条新途径     

细胞周期蛋白依赖激酶抑制剂——p27kip1研究进展

ｐ２７ｋｉｐ１是一个广谱的周期蛋白依赖激酶抑制剂，在细胞周期的调控中起重要的作用，本综述ｐ２７ｋｉｐ１的发现，ｐ２７ｋｉｐ１的功能、作用机制以及ｐ２７ｋｉｐ１与肿瘤的关系。     

细胞周期素D1与套细胞淋巴瘤

肿瘤发生过程中原癌基因的激活是重要因素之一。原癌基因活化的机制有基因扩增、点突变、染色体易位或缺失等〔1〕。在Ｂ细胞淋巴瘤中 ,染色体重排可导致原癌基因的激活而产生过量的癌蛋白 ,使正常的细胞发生癌变〔2〕。现将原癌基因ｂｃｌ 1及其蛋白产物细胞周期素 (ｃｙｃｌｉｎ)Ｄ1与套细胞淋巴瘤的关系综述如下。1　ｃｙｃｌｉｎＤ1结构和功能细胞周期由三类调节因子进行调控 ,它们分别是周期素依赖性激酶 (ｃｙｃｌｉｎ ｄｅｐｅｎｄｅｎｔｋｉｎａｓｅｓ,ＣＤＫｓ)、周期素 (ｃｙ ｃｌｉｎｓ)和周期素依赖性激酶抑制因子 (ｃｙｃｌｉ…     

反义p38 MAPK寡核苷酸对AT1R介导的血管收缩的影响及其机制

目的研究反义p38 MAPK寡核苷酸在血管紧张素Ⅱ1型受体（AT1R）介导的血管平滑肌收缩反应中的作用及其机制。方法采用反义寡核苷酸（ASODN）基因封闭技术来特异性调控p38 MAPK的表达,观察p38 MAPK在AT1R介导的大鼠主动脉血管平滑肌收缩中的作用及其与血管平滑肌肌球蛋白轻链激酶（MLCK）的关系。结果 AngⅡ诱导大鼠主动脉产生浓度依赖性的收缩,AT1R拮抗剂losartan和p38 MAPK-ASODN处理均可拮抗AngⅡ诱导的血管收缩反应。同时,AngⅡ处理可明显升高血管MLCK的活性,AT1R拮抗剂和p38 MAPK-ASODN也明显抑制了AngⅡ的这一作用。结论 p38 MAPK参与了AT1R介导的血管收缩反应的调节,其机制与MLCK调节途径有关。     

小鼠p27Kip1基因的克隆及与外周B细胞凋亡和生存的关系

目的：克隆小鼠的p27^kip1基因，并以此为探针分析p27^kip1在外周血B细胞中的表达，以及其在抗原受体和CD40介导的抑制和促进增殖信号中的作用。方法：从培养的B细胞中提取总RNA，用RT-PCR扩增并克隆p27^kip1基因，用流式细胞仪检测B细胞凋亡，用Northern blot观察p27^kip1mRNA的表达水平。结果：成功地克隆了小鼠的p27^kip1基因，发现抗原受体交联在引起外周血B细胞凋亡之前，出现p27^kip1表达上调，若同时激活CD40则使p27^kip1表达降低，B细胞也免于凋亡用针对p27^kip1的反义寡核苷酸处理，可阻断抗原受体交联引起的外周血B细胞凋亡。结论：p27^kip1可能在抗原受体信号介导的B细胞凋亡和CD40信号介导的B细胞生存中发挥一定的作用。     

p27~(Kip1)在永生化人神经前体细胞分化中的作用研究

目的:利用全反式视黄酸(all-trans retinoic acid,RA)诱导永生化的人神经前体细胞(immortalized human neural progenitor cells,hSN12W-TERTcells)分化,研究分化过程中细胞周期调节蛋白p27Kip1,p21Cip1,细胞周期蛋白激酶2(cyclin-dependent kinase2,cdk2)及细胞周期蛋白E(cyclinE)的变化,探讨永生化人神经前体细胞分化的相关分子机制。方法:取本课题组已经建立的永生化人神经前体细胞系hSN12W-TERT细胞(第12代)培养并给予1μmol/L RA诱导。在RA诱导的第3,7d观察细胞形态变化,用RT-PCR方法检测RA诱导前后hSN12W-TERT细胞p27Kip1,p21Cip1,cdk2及cyclinE mRNA的变化,用免疫细胞化学染色方法比较RA诱导前后p27Kip1蛋白表达的变化。结果:RA诱导第3d,hSN12W-TERT细胞比未经RA诱导的正常hSN12W-TERT细胞生长缓慢且形态发生改变,表现为胞体变小,突起延长增多。至RA诱导第7d时,hSN12W-TERT细胞形态变化更加明显,接近成熟神经元形态。RT-PCR结果表明,hSN12W-TERT细胞中p27Kip1mRNA的表达在RA诱导后明显增加,而p21Cip1mRNA的表达在RA诱导后略呈下降趋势。hSN12W-TERT细胞中cdk2、cyclinE的mRNA水平在RA诱导前后没有明显变化。免疫细胞化学染色结果显示,RA诱导第3d,hSN12W-TERT细胞p27Kip1的表达比未经RA诱导的正常hSN12W-TERT细胞明显增加(P0.05),RA诱导第7d,p27Kip1的表达进一步增加(P0.05)。结论:p27Kip1参与RA诱导的永生化人神经前体细胞生长阻滞和分化过程,p27Kip1可能在永生化人神经前体细胞的神经元分化过程中发挥重要作用;且RA诱导后p27Kip1蛋白含量的增加是通过转录水平调节的。     

Differential modulation of cyclin-dependent kinase inhibitor p27Kip1 by negative signaling via the antigen receptor of B cells and positive signaling via CD40

The cross-linking of surface immunoglobulins (sIg) of B cells can transmit a negative signal, resulting in cell cycle arrest, apoptosis or both. Signaling via the B cell antigen CD40 reverses the sIg-mediated negative signaling and induces activation and proliferation of B cells. We investigated the molecular mechanisms for cell cycle regulation by negative and positive signaling via sIg and CD40, respectively, by using the B cell line WEHI-231. Cross-linking of sIg almost completely reduced the activity of cyclin-dependent kinase (Cdk) 2, essential for cell cycle progression in the late G1 phase, although the level of Cdk2 was not reduced. Among the factors that regulate Cdk2 activation, the activity of the Cdk-activating kinase (CAK) appeared intact and cyclin E was reduced only partially in sIg-cross-linked WEHI-231. In contrast, sIg cross-linking induced a significant Cdk inhibitor (CKI) activity. Since a 27-kDa protein was co-precipitated with Cdk2 in anti-Ig-treated, but not untreated WEHI-231, and the CKI activity in anti-Ig-treated WEHI-231 was neutralized by anti-p27^Kip1, antibodies, it is most likely that p27^Kip1 is responsible for the CKI activity induced by sIg cross-linking. p27^Kip1 may thus play a role in growth inhibition of B cells by negative signaling via sIg. In contrast, CD40 signaling enhanced Cdk2 activity and reduced the p27^Kip1 level in anti-Ig-treated WEHI-231, suggesting that the reduction of p27^Kip1 plays an important role in the abrogation of sIg-mediated growth arrest by CD40 signaling. Taken together, p27^Kip1 is likely to be a crucial target molecule of the negative signaling via sIg and the positive signaling via CD40 essential for T cell-dependent immune responses.     

Suppressor activity of anergic T cells induced by IL-10-treated human dendritic cells: association with IL-2- and CTLA-4-dependent G1 arrest of the cell cycle regulated by p27Kip1

We have previously shown that human IL-10-treated dendritic cells (DC) induce an antigen-specific anergy in CD4+ T lymphocytes. These anergic T cells are characterized by an inhibited proliferation, a reduced production of IL-2, and additionally display antigen-specific suppressor activity. In this study we investigated the mechanisms underlying the anergic state and regulatory function of these T cells. We did not observe enhanced rates of programmed cell death of anergic CD4+ suppressor T cells compared to T cells stimulated with mature DC. Cell cycle analysis by DNA staining and Western blot experiments revealed an arrest of anergic CD4+ T suppressor cells in the G1 phase. High levels of the IL-2-dependent cyclin-dependent kinase (cdk) inhibitor p27Kip1 were found in anergic CD4+ suppressor T cells resulting in an inhibited activation of retinoblastoma protein and an arrest of cell cycle progression in the G1 phase. Addition of IL-2, but not blocking of the CTLA-4 pathway restored the proliferation of the suppressor T cells. In contrast, both treatments induced a down-regulation of p27Kip1 and acomplete inhibition of the antigen-specific regulatory function as demonstrated by high proliferation and enhanced IFN-gamma production of co-cultured T cells. Further experiments demonstrated that p27Kip-expressing regulatory CD4+CD25+ T cells did not contribute to induction of T cell anergy in this model. Our data show that regulatory function of anergic CD4+ suppressor T cells is associated with an arrest in the G1 phase of the cell cycle mediated by increased levels of the IL-2- and CTLA-4-dependent cdk inhibitor p27Kip1.     

碱性成纤维细胞生长因子对人肝癌Bel-7402细胞Skp2和p27^Kip表达的影响

目的:研究碱性成纤维细胞生长因子(bFGF)对肝癌Bel-7402细胞Skp2、p27Kip表达的影响,探讨bFGF调控肝癌细胞增殖的信号转导机制.方法:分为对照组、bFGF组、bFGF+Wortmannin组,MTT法分析细胞增殖程度;流式细胞术分析细胞周期;RT-PCR法检测Skp2的mRNA表达;免疫印迹法检测Skp2、p27Kip蛋白的表达.结果:bFGF组细胞增殖比显著增加;bFGF可诱导细胞由G1期进入S期,而且上调Skp2的mRNA和蛋白表达、下调p27Kip蛋白表达;PI3K的抑制剂Wortmannin可部分抑制bFGF的上述作用.结论:bFGF可能通过激活PI3K/PKB途径调节Skp2、p27Kip表达,促进肝癌Bel-7402细胞增殖.     

Cytoplasmic p27Kip1 promotes tumorigenesis via suppression of RhoB activity

The cell cycle inhibitor p27^Kip1 is a tumor suppressor via the inhibition of CDK complexes in the nucleus. However, p27 also plays other functions in the cell and may acquire oncogenic roles when located in the cytoplasm. Activation of oncogenic pathways such as Ras or PI3K/AKT causes the relocalization of p27 in the cytoplasm, where it can promote tumorigenesis by unclear mechanisms. Here, we investigated how cytoplasmic p27 participates in the development of non-small cell lung carcinomas. We provide molecular and genetic evidence that the oncogenic role of p27 is mediated, at least in part, by binding to and inhibiting the GTPase RhoB, which normally acts as a tumor suppressor in the lung. Genetically modified mice revealed that RhoB expression is preferentially lost in tumors in which p27 is absent and maintained in tumors expressing wild-type p27 or p27^CK–, a mutant that cannot inhibit CDKs. Moreover, although the absence of RhoB promoted tumorigenesis in p27^−/− animals, it had no effect in p27^CK– knock-in mice, suggesting that cytoplasmic p27 may act as an oncogene, at least in part, by inhibiting the activity of RhoB. Finally, in a cohort of lung cancer patients, we identified a subset of tumors harboring cytoplasmic p27 in which RhoB expression is maintained and these characteristics were strongly associated with decreased patient survival. Thus, monitoring p27 localization and RhoB levels in non-small cell lung carcinoma patients appears to be a powerful prognostic marker for these tumors. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Prognostic implications of cyclin B1, p34cdc2, p27(Kip1) and p53 expression in gastric cancer

PURPOSE: Cell cycle progression is regulated by interactions of specific cyclins and cyclin dependent kinases (CDKs) at the G1-S and G2-M checkpoints and cell cycle deregulation plays a major role in carcinogenesis of human cancers. PATIENTS AND METHODS: To investigate the role of cell cycle regulators in the pathogenesis and progression of human gastric cancers, 23 cases of gastric carcinomas were examined for the expression of cyclin B1, p34cdc2, p27(Kip1) and p53 by immunohistochemical methods, and gene expression was correlated with various clinicopathologic findings. RESULTS: Out of 23 cases studied, cyclin B1 was diffusely expressed in 20 cases (87.0%), p34cdc2 in 14 cases (60.9%) and p53 in 12 cases (52.2%), whereas in normal gastric tissues, cyclin B1 and p34cdc2 were weakly expressed and p53 was not expressed. In contrast, p27(Kip1) was expressed in only 8.7% of gastric carcinomas compared with 78.3% of normal gastric tissues. There was correlation between the expression of cyclin B1 and expression of p34cdc2 (p=0.002), between the expression of cyclin B1 and loss of p27(Kip1) (p=0.025), and between the expression of p34cdc2 and loss of p27(Kip1) (p=0.065). In addition, expression of cyclin B1 was correlated with regional lymph node metastasis (p=0.032). CONCLUSION: Our results indicate that cyclin B1 and p34cdc2 are involved in the genesis or progression of gastric cancers. Furthermore, overexpression of cyclin B1 may play an important role in lymph node metastatic potential of gastric cancer. Thus, abnormal expression of cyclin B1 and CDKs, overexpression of p53 and loss of p27(Kip1) expression may play important roles in human gastric carcinogenesis.     

Inverse expression of Jun activation domain binding protein 1 and cell cycle inhibitor p27Kip1: influence on proliferation in hepatocellular carcinoma

Recently, the functional role of Jun activation domain binding protein 1 (Jab1) as a putative novel oncogene in hepatocellular carcinoma (HCC) has been postulated. We show that expression of p27(Kip1), a negative cell cycle regulator, correlates inversely with Jab1 expression in HCC (P = .014). We observed nuclear Jab1 expression in 57% (55/97) and p27(Kip1) expression in 32% (31/97) of HCCs. Neither Jab1 nor p27(Kip1) nor inverse Jab1 and p27(Kip1) expression correlated with clinicopathological parameters. However, HCCs lacking p27(Kip1) with increased proliferative activity were frequently found to express Jab1 (P = .048). Normal liver tissue, cirrhosis, and tumor-like lesions (focal nodular hyperplasia, dysplastic nodules in cirrhotic liver) showed no significant Jab1 expression. In transfection studies in the hepatoma cell line Huh 7, Jab1 overexpression resulted in reduced p27(Kip1) protein levels. We conclude that Jab1 expression may lead to down-regulation of the negative cell cycle regulator p27(Kip1), pointing to a possible mechanism that promotes hepatocarcinogenesis.     

p27(Kip1) expression and grading of breast cancer diagnosed on cytological samples

The progressive reduction in p27(Kip1) (p27) protein immunohistochemical staining with increasing histological grading is a well-established finding occurring in breast cancer, and its role as diagnostic complement and prognostic marker has been thoroughly evaluated. To clarify whether this test may be applied to breast cytopathology, we performed p27 immunostaining on fresh fine-needle cytology (FNC) samples from 10 benign and 40 malignant breast lesions. On average, p27 immunostaining was significantly lower in carcinomas than in benign lesions (P < 0.005). In particular, among carcinomas, p27 immunostaining progressively reduced from well-to poorly differentiated lesions (G1 vs. G2, P < 0.05; G1 vs. G3, P < 0.001; G2 vs. G3; P < 0.001). A similar trend was noted in a subgroup of 20 matched FNCs and histological samples of breast carcinomas, when p27 immunostaining on FNCs was stratified according to the histological grading (G1 vs. G2, P = 0.18; G1 vs. G3, P < 0.05; G2 vs. G3, P < 0.05). In addition, p27 immunostaining on FNCs showed a good positive correlation with that on histology (Spearman R = 0.58; P < 0.01), with a diagnostic concordance between samples of 85%, by using the standard 50% positive cell cutoff. Taken in concert, our data suggest that p27 immunostaining is a reliable marker of tumor cell differentiation in breast cytopathology as well as in histopathology. Accordingly, staining FNCs for p27 may be an useful complement in addition to cytological grading in the preoperative assessment of breast cancer.     

p27~(Kip1)调控永生化人神经前体细胞分化的机制

目的研究p27Kip1在永生化人神经前体细胞分化中的作用,探讨神经前体细胞的分化机制。方法取本课题组已建立的永生化人神经前体细胞系hSN12W-TERT细胞(第12代)进行培养,在细胞进入对数生长期时给予1μmol/L全反式视黄酸(RA)诱导,重复诱导3次,每次均在诱导的第3、5、7天收集细胞,用流式细胞仪分析RA诱导第3天细胞周期的变化,用免疫印迹法检测RA诱导第3、5、7天p27Kip1、p21cip1细胞周期蛋白依赖激酶2(cdk2)及S期激酶相关蛋白2(skp2)的变化。结果流式细胞术结果显示,hSN12W-TERT细胞经1μmol/LRA诱导3d后,G0/G1期细胞的比例由77.25%上升至85.68%,而S期的比例由9.38%下降到8.57%。免疫印迹法结果显示,RA诱导第3天,hSN12W-TERT细胞p27Kip1蛋白表达比未经RA诱导的细胞增加,并在RA诱导第5天达到高峰(P0.05)。未经RA诱导的正常hSN12W-TERT细胞p21Cip1蛋白表达弱,RA诱导后p21Cip1蛋白的表达略呈下降趋势。RA诱导前后hSN12W-TERT细胞cdk2蛋白的表达变化不明显,但反映cdk2活性的磷酸化cdk2(p-cdk2)的表达在RA诱导后明显下降,同时,参与p27Kip1泛素降解途径的重要因子skp2的表达在RA诱导后明显下降。结论在RA诱导hSN12W-TERT细胞分化的过程中,p27Kip1通过抑制cdk2的活性而发挥促细胞分化的作用,且RA诱导后p27Kip1蛋白含量增加与其泛素降解途径被抑制密切相关。     

p27Kip1蛋白在子宫内膜癌中的表达及其意义

目的：探讨子宫内膜癌中ｐ２７＾Ｋｉｐ１蛋白的表达情况及临床病理意义。方法：采用免疫组化Ｓ－Ｐ法检测１０５例子宫内膜癌ｐ２７＾Ｋｉｐ１蛋白的表达,并运用流式细胞术检测４６例子宫内膜癌组织的ＤＮＡ含量。结果：子宫内膜癌ｐ２７＾Ｋｉｐ１蛋白阳性表达率为６９％（７２／１０５例）,明显低于正常增生期子宫内膜及单纯增生、复合增生子宫内膜总的阳性率（９１％,Ｐ〈０．０１）,而与不典型增生子宫内膜的ｐ２７＾Ｋｉｐ