姜黄素对血管平滑肌细胞增殖及cyclinD1和p21wafl/cipl蛋白表达的影响

目的 观察姜黄素(curcumin)抑制大鼠血管平滑肌细胞(VSMC)增殖和诱导细胞周期停滞的作用,以及对细胞周期蛋白cyclinDl,p21wafl/cipl表达的影响.方法 采用组织贴块法体外培养大鼠VSMC,MTT法测定姜黄素对VSMC增殖的抑制作用,流式细胞仪分析细胞周期分布,Western印迹法检测cyclinDl,p21wafl/cipl蛋白的变化.结果 MTT示不同浓度姜黄素(7.5～120 μmol/L)在24～72 h范围内,浓度和时间依赖性抑制VSMC增殖;30 μmol/L以上浓度姜黄素显著抑制增殖的VSMC细胞周期进程,使S及G2/M期减少(P<0.05),G0/G1期增加(P<0.05);30 μmol/L的姜黄素可抑制cyclinDl表达,促进p21wafl/cipl蛋白表达.结论 姜黄素具有明确的抑制VSMC增殖和细胞周期停滞的作用,其与cyclinDl,p21wafl/cipl蛋白变化有关.     

姜黄素对血管平滑肌细胞增殖及cyclinD1和p21waf1/cip1蛋白表达的影响

目的 观察姜黄素（curcumin）抑制大鼠血管平滑肌细胞(VSMC)增殖和诱导细胞周期停滞的作用,以及对细胞周期蛋白cyclinD1,p21waf1/cip1表达的影响。方法 采用组织贴块法体外培养大鼠VSMC,MTT法测定姜黄素对VSMC增殖的抑制作用,流式细胞仪分析细胞周期分布,Western印迹法检测cyclinD1,p21waf1/cip1蛋白的变化。结果 MTT示不同浓度姜黄素(7.5～120 μmol/L)在24～72 h范围内,浓度和时间依赖性抑制VSMC增殖;30 μmol/L以上浓度姜黄素显著抑制增殖的VSMC细胞周期进程,使S及G2/M期减少(P<0.05),G0/G1期增加(P<0.05);30 μmol/L的姜黄素可抑制cyclinD1表达,促进p21waf1/cip1蛋白表达。结论 姜黄素具有明确的抑制VSMC增殖和细胞周期停滞的作用,其与cyclinD1,p21waf1/cip1蛋白变化有关。     

野生型P53基因诱导血管平滑肌细胞P21基因表达

为研究野生型P53基因导入诱导血管平滑肌细胞P21基因的表达，探讨P53基因调节细胞周期进程的作用机理，体外培养了人脐动脉平滑肌细胞。将野生型P53基因导入细胞后，应用逆转录－聚合酶链反应半定量测定P21mRNA水平，以免疫组织化学法观察P21蛋白表达的变化，并用流式细胞术分析细胞周期。结果发现，正常生长的血管平滑肌细胞中P21mRNA水平较低，用免疫组织化学法检测不到P21蛋白。野生型P53基因导入并在平滑肌细胞中表达后，显著增加了P21mRNA水平，在免疫组织化学检测中呈现很强的阳性显色反应、引起平滑肌细胞停滞在G0／G1期。以上结果提示，野生型P53基因通过诱导P21基因表达调控血管平滑肌细胞周期。     

p27蛋白与血管平滑肌细胞增殖

p27蛋白是一种细胞周期蛋白依赖性激酶抑制剂,通过抑制cyclIN-CDK复合物活性使细胞停滞予G1期.p27蛋白能显著抑制血管平滑肌细胞增殖,有望成为治疗动脉粥样硬化以及PTCA后血管再狭窄的新手段.     

普罗布考抑制过氧化氢刺激大鼠主动脉平滑肌细胞增殖的机制

目的研究普罗布考抑制过氧化氢促大鼠主动脉平滑肌细胞增殖的机制。方法采用MTT3、H-胸腺嘧啶核苷掺入法、流式细胞术和逆转录聚合酶链反应观察过氧化氢刺激条件下普罗布考对血管平滑肌细胞细胞周期、DNA合成、细胞增殖和凋亡的影响。结果普罗布考抑制过氧化氢刺激血管平滑肌细胞增殖和DNA合成。与过氧化氢组比较,普罗布考 过氧化氢组细胞计数、吸光度值和3H-胸腺嘧啶核苷掺入量分别下降了46.9%、45.0%和39.5%(P<0.05)。普罗布考通过使血管平滑肌细胞生长停滞在G0/G1期抑制过氧化氢刺激细胞增殖。过氧化氢使细胞外信号调节激酶1 mRNA相对表达量增加近6倍,丝裂原活化蛋白激酶磷酯酶1 mRNA相对表达量下降了82.2%。普罗布考抑制细胞外信号调节蛋白激酶1 mRNA的表达,增强丝裂原活化蛋白激酶磷酯酶1 mRNA的表达。普罗布考诱导过氧化氢刺激条件下血管平滑肌细胞凋亡。结论普罗布考通过下调细胞外信号调节蛋白激酶1 mRNA的表达抑制细胞周期运转和诱导血管平滑肌细胞凋亡两种机制抑制过氧化氢刺激血管平滑肌细胞增殖。     

粉防己碱对大鼠静止期肝星状细胞细胞周期的影响

目的观察粉防己碱对大鼠静止期肝星状细胞（HSCs）细胞周期的影响及机制。方法分离正常大鼠HSCs,培养48 h后给予粉防己碱（1.6μmol/L）和（或）TGF-β1（5μg/L）处理3 d。流式细胞仪检测细胞周期分布;应用RT-PCR法检测Cyclin D1、Cyc-lin E、p21WAF1/CiP1和p27Kip1表达;使用Western blot法检测Cyclin D1和PPARγ蛋白水平。结果粉防己碱使G0/G1期和G2/M期细胞增多,S期细胞数显著减少,抑制TGF-β1介导的G0/G1期细胞含量下调和S期细胞含量升高,上调p21WAF1/CiP1mRNA表达,抑制Cyclin D1而维持PPARγ蛋白在一定水平。结论在静止期HSC中,粉防己碱通过抑制Cyclin D1、上调p21WAF1/CiP1和维持PPARγ表达使HSCs出现G0/G1期停滞和S期向G2/M期转换加速。     

蛋白激酶B短发夹环RNA表达载体的构建及其对血管平滑肌细胞增殖活性的影响

目的构建靶向蛋白激酶B基因的短发夹环RNA表达载体,观察其对血管平滑肌细胞增殖活性的影响。方法设计多个针对大鼠蛋白激酶B基因的短发夹环RNA序列,化学合成方法合成并经pGEM-T载体克隆后双酶切,将cDNA序列插入逆转录病毒载体pLXIN,包装后获得蛋白激酶B的逆转录表达载体,感染血管平滑肌细胞,Northern blot和Western blot法检测蛋白激酶B及其下游底物的表达变化,流式细胞仪检测细胞周期变化,MTT法检测血管平滑肌细胞增殖活性的改变。结果成功构建蛋白激酶B基因的逆转录病毒载体并包装,感染血管平滑肌细胞,证实其能显著抑制蛋白激酶B的mRNA和蛋白产物表达,下游的p70s6k表达相应减少;被感染血管平滑肌细胞的分裂、增殖过程受阻,更多细胞停滞在G0/G1期。结论成功构建蛋白激酶B基因逆转录病毒RNA干扰表达载体,感染血管平滑肌细胞能够明显抑制其分裂、分化和增殖。     

丁酸钠对人结肠癌细胞株SW480增殖及凋亡的影响及其机制

将2.85 mmol/L丁酸钠(NaB)体外作用于人结肠癌SW480细胞12、24、48 h分别用半定量RT-PCR法与Western-blot法检测p21基因mRNA及蛋白表达变化;并用FCM检测细胞周期变化.结果 p21基因mRNA 及p21蛋白表达随时间延长逐渐增加;结肠癌SW480细胞周期阻滞于G0/G1期,S期比例明显减少,细胞增殖指数下降.认为NaB可诱导结肠癌细胞凋亡,其机制可能为细胞周期阻滞及p21蛋白高表达.     

莲心碱对血管平滑肌细胞增殖及热应激蛋白70和P53表达的影响

采用培养的内皮素所致血管平滑肌细胞增殖模型。用氚-胸腺嘧啶核苷掺入法、流式细胞术、Westernblot及Northernblot分析方法，观察了莲心减对血管平滑肌细胞增殖的作用及对热应激蛋白70及其mRNA和抑癌基因P53表达的影响。结果发现，遂心碱能逆转内皮素所致的~3H－TdR掺入量增多，阻止血管平滑肌细胞由Go／G1期进入DNA合成期（S期）和有丝分裂期（G2／M期）．并能使效应激蛋白70及mRNA表达减弱，P53抑癌基因及mRNA表达增强。以上结果提示，莲心碱能抑制血管平滑肌细胞增殖，这可能与热应激蛋白70及P53的调控有关。     

肝癌组织中p57kip2基因表达缺失与甲基化的关系

p57kip2属于CIP/KIP家族成员,抑制细胞周期从G1期到S期转化,使细胞周期停滞在G1期,进而阻止细胞增殖.肝癌组织中普遍存在p57kip2 mRNA表达水平的下降,提示p57kip2与肝癌发生关系密切[1].本研究采用原位分子杂交和甲基化特异性PCR方法检测p57kip2 mRNA的表达及启动子区甲基化情况,探讨p57kip2 基因失表达机制.     

Adenosine monophosphate-activated protein kinase suppresses vascular smooth muscle cell proliferation through the inhibition of cell cycle progression

Vascular smooth muscle cell (VSMC) proliferation is a critical event in the development and progression of vascular diseases, including atherosclerosis. We investigated whether the activation of adenosine monophosphate-activated protein kinase (AMPK) could suppress VSMC proliferation and inhibit cell cycle progression. Treatment of human aortic smooth muscle cells (HASMCs) or isolated rabbit aortas with the AMPK activator 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) induced phosphorylation of AMPK and acetyl Co-A carboxylase. AICAR significantly inhibited HASMC proliferation induced by both platelet-derived growth factor-BB (PDGF-BB) and fetal calf serum (FCS). Treatment with AICAR inhibited the phosphorylation of retinoblastoma gene product (Rb) induced by PDGF-BB or FCS, and increased the expression of cyclin-dependent kinase inhibitor p21(CIP) but not that of p27(KIP). Pharmacological inhibition of AMPK or overexpression of dominant negative-AMPK inhibited both the suppressive effect of AICAR on cell proliferation and the phosphorylation of Rb, suggesting that the effect of AICAR is mediated through the activation of AMPK. Cell cycle analysis in HASMCs showed that AICAR significantly increased cell population in G0/G1-phase and reduced that in S- and G2/M-phase, suggesting AICAR induced cell cycle arrest. AICAR increased both p53 protein and Ser-15 phosphorylated p53 in HASMCs, which were blocked by inhibition of AMPK. In isolated rabbit aortas, AICAR also increased Ser-15 phosphorylation and protein expression of p53 and inhibited Rb phosphorylation induced by FCS. These data suggest for the first time that AMPK suppresses VSMC proliferation via cell cycle regulation by p53 upregulation. Therefore, AMPK activation in VSMCs may be a therapoietic target for the prevention of vascular diseases.     

Tranilast inhibits the proliferation of human coronary smooth muscle cell through the activation of p21waf1

Restenosis after percutaneous transluminal coronary angioplasty (PTCA) occurs due to vascular smooth muscle cell proliferation and migration. Recently, tranilast, an anti-allergic drug, has been used for the prevention of restenosis after PTCA. To determine the molecular mechanism involved, the effect of tranilast on the proliferation of human coronary smooth muscle cells (SMCs) was investigated. Tranilast arrested the proliferation of human coronary SMCs at the G0/G1 phase of the cell cycle. In association with this inhibitory effect, tranilast increased p21waf1 and p53 tumor suppressor factor, and decreased cyclin-dependent kinase 2 (CDK2) activity. These results suggest that tranilast inhibits the proliferation of human coronary SMCs during restenosis after PTCA via an induction of p21waf1 and p53. Tranilast may thus allow us to prevent restenosis after PTCA by interfering with this mechanism.     

凝血酶诱导CBP高表达刺激大鼠血管平滑肌细胞增殖的研究

目的:探讨凝血酶诱导大鼠CBP高表达影响大鼠血管平滑肌细胞(VSMCs)增殖的作用机制.方法:采用10-3、10-2、0.1、1、10 U/ml的凝血酶干预体外培养的VSMCs 30min.RT-PCR法、蛋白印记法分别检测rCBPmRNA和蛋白质的表达变化.流式细胞技术检测细胞周期,评价细胞增殖能力.结果:凝血酶可在...     

T-cadherin upregulation correlates with cell-cycle progression and promotes proliferation of vascular cells

OBJECTIVE: In vascular tissue, T-cadherin (T-cad) levels correlate with the progression of atherosclerosis, restenosis and tumour neovascularization. This study investigates whether T-cad influences proliferation of vascular cells. METHODS AND RESULTS: Cultures of human umbilical vein endothelial cells (HUVEC) and rat and human aortic smooth muscle cells (rSMC, hSMC) were used. T-cad was overexpressed in HUVEC and hSMC using an adenoviral expression system. In cultures released from G(1)/G(0) synchrony parallel immunoblot analysis of T-cad and cell cycle phase specific markers (p27(Kip1), cyclin D1, E2F1, PCNA, cyclin B) showed increased T-cad protein levels subsequent to entry into early S-phase with sustained elevation through S-and M-phases. T-cad was increased in G(2)/M-phase (colchicine) synchronized cultures. In FACS-sorted cell populations, expression of T-cad in S-and G(2)/M-phase was higher than G(1)/G(0)-phase. Compared with empty-and LacZ-vector infected controls, HUVEC and hSMC overexpressing T-cad exhibited increased proliferation as assessed in enumeration and DNA synthesis assays. Additionally, following release from G(1)/G(0) synchrony, HUVEC and hSMC overexpressing T-cad enter S-phase more rapidly. Flow cytometry after BrdU/propidium labelling confirmed increased cell cycle progression in T-cad overexpressing cells. CONCLUSION: In vascular cells, T-cad is dynamically regulated during the cell cycle and its expression functions in the promotion of proliferation. T-cad may facilitate progression of proliferative vascular disorders such as atherosclerosis, restenosis and tumour angiogenesis.     

The essential role of p21 in radiation-induced cell cycle arrest of vascular smooth muscle cell

The biologic mechanisms for the success and failure of intravascular radiation therapy after angioplasty have not been well studied. We investigated the molecular mechanism of radiation-induced cell cycle arrest in vascular smooth muscle cell (VSMC) and examined whether p21 knock-out is a cause of radiation failure. Using different dosages of gamma radiation, we evaluated the effect of radiation on VSMC apoptosis and cell cycle progression, and its action mechanism. Irradiation significantly retarded the growth of cultured VSMC, which was not due to induction of apoptosis but mainly due to cell cycle arrest. Radiation showed remarkable cell cycle arrest at G1 and G2 phase (G0/G1:S:G2/M phases = 61%:34%:5% with 0 Gy versus 61%:9%:30% with 16 Gy, 12 h after radiation). In immunoblot analysis and kinase assay, radiation increased the expression of p21 and decreased the expression and activity of CDK2 and 1. In contrast, radiation did not affect the expression and activity of CDK4 and 6, nor the expression of p27 and p16. When p21 was knocked out, cell cycle of VSMC was not arrested by radiation, leading to increased proliferation. These finding provide the evidence that radiation inhibits VSMC proliferation through cell cycle arrest by enhancing p21 expression and suppressing CDK1 and 2. This observation supports the key role of p21 in radiation-induced cell cycle arrest and the degree of p21 expression may be the possible mechanism of radiation failure and delayed restenosis.     

Expression of the cyclin-dependent kinase inhibitor, p21Waf-1/Cip-1/Sdi-1, in human vascular smooth muscle cells in the proliferating state

Excess proliferation of vascular smooth muscle cells (SMC) is an important aspect of atherogenesis. Cell-cycle regulatory proteins such as cyclin and cyclin-dependent kinases are vital for cell-cycle progression. To understand the role of the cyclin-dependent kinase inhibitor, p21Waf-1/Cip-1/Sdi-1 (p21Waf-1), on human atherogenesis, we tested p21Waf-1 expression in human atherosclerotic lesions and cultured SMC. Immunohistochemical staining revealed that SMC in neointimal lesions expressed p21Waf-1. No evidence of the p53 protein could be detected. By Western blotting, cultured SMC obtained from a neonate revealed that a higher level of p21Waf-1 expression correlated with a higher expression of proliferating cell nuclear antigen and a lower expression of the contractile protein than that observed in cells obtained from aged donors. When the phenotypes of SMC were changed by modification of serum concentration and cell densities, p21Waf- expression was maximal in serum-stimulated SMC at low cell densities despite the low expression of p53. Furthermore, serum stimulation transiently increased the p21Waf-1 expression of quiescent SMC, which was synchronized with the transition from the G0/G1 to the S phase as well as with cyclin D1 expression. These results may suggest that the negative regulator of cell-cycle progression also plays a role in regulating the appropriate cell-cycle progression in SMC. Growth stimuli may induce both growth-promoting and growth-inhibitory factors in SMC. The balance between these two opposing factors may play an important role in the determination of cell-cycle progression.     

曲古霉素A对人胃癌细胞的抑制作用及其机制探讨

背景：组蛋白去乙酰基酶抑制剂（HDACi）是一类新型抗肿瘤药物。曲古霉素A（TSA）是目前研究最为广泛的HDACi之一,已发现其对多种肿瘤细胞具有明显抑制作用,但关于TSA对胃癌作用的研究尚少。目的：观察TSA对人胃癌细胞增殖、凋亡、细胞周期以及相关基因表达的影响,探讨其抑制人胃癌细胞的可能作用机制。方法：以不同浓度TSA（0—1μmol／L）处理人胃癌细胞株AGS和HGC-27。CCK-8实验检测细胞增殖抑制情况,流式细胞术检测细胞凋亡和细胞周期,realtimeRT-PCR和蛋白质印迹法检测细胞凋亡、细胞周期相关基因mRNA和蛋白水平的表达。结果：TSA能剂量依赖性地抑制AGS、HGC-27细胞增殖（P=0．000）,对两者的半数致死浓度分别约为0．25μmol／L和0．5μmol／L。TSA能诱导AGS、HGC-27细胞发生G0／G1期和G2／M期阻滞,以G0／G11期阻滞更为明显。TSA对AGS细胞的诱导凋亡作用强于HGC-27细胞（P〈0．05）。TSA尚能上调p21、p53、BaxmRNA和蛋白表达,下调Bel-2、CDK2、cyelinD1mRNA和蛋白表达,作用均呈时间依赖性（P〈0．05）。结论：TSA抑制人胃癌细胞增殖、诱导细胞凋亡的作用可能是通过调节细胞凋亡、细胞周期相关分子、激活多种肿瘤相关信号通路实现的。