芍药甙诱导人肝癌细胞系HepG2凋亡的研究

目的：观察芍药甙诱导人肝癌细胞系HepG2凋亡及凋亡相关基因的变化。方法：用0．5～0．2g／L芍药甙处理人肝癌HepG2细胞，光镜下观察细胞形态；MTT法检测细胞增殖率；流式细胞仪检测凋亡峰；免疫组化法检测P53，bcl-2，bax等凋亡相关基因的表达。结果：0．5～2．0g／L浓度的芍药甙对HepG2有显著抑制作用；可诱导HepG2凋亡，并能上调凋亡相关基因P53和bax表达、下调抗凋亡基因bcl-2表达。结论：0．5～2．0g／L芍药甙可诱导人肝癌细胞HepG2凋亡，其机制可能与凋亡相关基因表达增强和抗凋亡相关基因表达减弱有关。     

大黄素自身抑制血管平滑肌细胞迁移和增殖

目的探讨大黄素对血管平滑肌细胞迁移和增殖的作用以及大黄素在血管平滑肌细胞中是否存在代谢过程。方法采用Transwell迁移系统和MTT法观察大黄素对血管平滑肌细胞迁移和增殖的影响。结果大黄素能显著抑制平滑肌细胞迁移,5μg/mL大黄素抑制率为83·3%;大黄素呈浓度和时间依赖性抑制平滑肌细胞增殖。然而,血管平滑肌细胞作用24h后,上清液中的大黄素浓度并无显著降低;细胞色素p450氧化酶诱导剂或者抑制剂没有改变大黄素的细胞毒性作用或者细胞内活性氧水平。大黄素的主要代谢酶(细胞色素p450氧化酶)基因表达水平没有显著的上调。结论大黄素能够抑制血管平滑肌细胞的迁移和增殖,并且不被平滑肌细胞代谢,可能作为药物涂层支架的药物。     

p53基因增强非小细胞肺癌细胞对顺铂化学敏感性的研究

我们以具有抑制细胞增殖和促进细胞凋亡的p53基因加入肺癌细胞培养板上,观察它对DNA损伤药顺铂（cisplatin,CDDP）抑癌作用的影响,从分子水平上为提高肺癌疗效拓展新的途径。材料与方法（1）材料：非小细胞肺癌（NSCLC）细胞系LTEP-a-2购自中国科学院上海细胞生物研究所。表达型p53质粒：由美国华盛顿大学吴坡博士赠送。SA阳离子脂质体：购自中国科学院上海生物化学研究所。CDDP：锦州制药厂产,批号961201－4。(2)基因转染：收获对数生长期的LTEP-a-2细胞,继续培养24小时,取所需量的p53cDNA质粒,以每μg质粒50μl的不含小…     

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。     

野生型P53基因导入诱导血管平滑肌细胞凋亡

为观察野生型P53基因诱导血管平滑肌细胞凋亡的现象，探讨对生型P53基因导入抑制平滑肌细胞增殖的作用机理，构建了野生型P53基因的重组腺病毒载体，体外转染兔主动脉血管平滑肌细胞。应用流式细胞议分析细胞周期，氚标胸腺嘧啶脱氧核苷掺入试验检测DNA合成，琼脂糖凝胶电泳观察DNA区带图谱，末端脱氧核苷酸转移酶介导的dUTP切口末端标记技术原位检测细胞凋亡。结果发现，野生型P53基因重组腺病毒载体转梁平滑肌细胞后，细胞内DNA合成减少。流式细胞仪分析显示77％的平滑肌细胞生长停滞在G0／Gl期，约45％的细胞发生细胞凋亡。dUTP切口末端标记阳性细胞百分率为40％～50％。DNA在琼脂糖凝胶电泳中呈现阶梯状区带图谱。以上结果提示，野生型P53基因通过诱导细胞凋亡对血管平滑肌细胞增殖起抑制作用。     

金莲花黄酮对A549细胞生长及凋亡的影响

目的观察金莲花黄酮对人非小细胞肺癌A549生长及凋亡的影响及其体外抗肿瘤效应。方法体外培养A549细胞,以不同浓度的金莲花黄酮作用为实验组,并设对照组,应用CCK8法测细胞增殖抑制效应、DNA琼脂糖凝胶电泳检测细胞凋亡、流式细胞术检测A549细胞中p53和bcl-2蛋白表达情况。结果不同浓度金莲花黄酮作用A549细胞24 h增殖抑制明显(P<0.05),其抑制效应具有剂量依赖的特点并可诱导A549细胞凋亡,同时上调p53基因表达、下调bcl-2基因表达(P<0.05)。结论金莲花黄酮可剂量依赖性抑制A549细胞的生长并可诱导肿瘤细胞凋亡,其抗肿瘤效应可能与细胞凋亡相关基因表达的调控有关。     

皂荚提取物对人肝癌细胞相关癌基因表达的调控作用及端粒酶的影响

目的：研究皂荚提取物对人肝癌细胞的增殖、相关癌基因表达的调控作用及端粒酶的影响。方法：体外培养人肝癌细胞bel-7402，检测不同浓度皂荚提取物（0．05mg／ml、0．1mg/ml、0．2mg／m1）对人肝癌细胞bel-7402细胞增殖、细胞周期、肝癌相关基因（bax、bcl-2、p53）的表达及端粒酶活性的影响。结果：0．05mg／ml-0．2mg／ml浓度组皂荚提取液对人肝癌细胞bel-7402细胞增殖、调控癌基因与抑癌基因、促进癌细胞凋亡、抑制端粒酶活性差异均具有显著性意义（P〈0．05或P〈0．01）。结论：皂荚提取物能够抑制人肝癌细胞增殖，促进细胞凋亡。     

p53基因突变与人类肝癌的病因学有关联

据好医生网4月21臼报道，S．P．HUSSAIN等人对p53调控的基因转录凋亡功能和微阵列DNA损伤细胞表达进行的研究和分析，发现了新的p53靶基因。     

姜黄素对人结肠癌SW620细胞凋亡机制的影响

目的 探讨体外姜黄素对人结肠癌SW620细胞生长及凋亡的影响.方法 体外培养SW620细胞,用不同浓度的姜黄素作用为实验组,同时设对照组,以CCK8法测细胞增殖抑制作用、流式细胞仪检测细胞凋亡及p53和Bcl-2蛋白表达情况.结果 不同浓度姜黄素作用SW620细胞24 h增殖抑制显著(P<0.05),其抑制效应具有剂量依赖性,同时诱导SW620细胞凋亡、上调p53基因表达、下调Bcl-2基因表达(P<0.05).结论 姜黄素可抑制SW620细胞的生长且具有剂量依赖性,并可诱导肿瘤细胞凋亡,其抗肿瘤效应可能与细胞凋亡相关基因表达的调控有关.     

斑蝥素诱导人胰腺癌细胞凋亡的实验研究

目的 探讨斑蝥素(Cantharidin)对人胰腺癌细胞凋亡的影响及其作用机制.方法 采用MTT法观察斑蝥素对人胰腺癌细胞株SW1990细胞增殖的抑制作用.采用Hoechst 33258染色、TUNNEL染色、流式细胞术检测细胞凋亡改变,并以RT-PCR和Western blot检测凋亡调节基因p53和Bcl-2、Bax的表达.结果 5μmol/L斑蝥素能明显抑制人胰腺癌SW1990细胞的生长,呈现凋亡特征.RT-PCR和Western blot检测可见Bax、p53基因表达显著增加,而Bc1-2基因表达减少.结论 斑蝥素能诱导人胰腺癌细胞凋亡,其作用可能与上调p53、Bax基因和下调Bcl-2基因有关.     

血管平滑肌细胞细胞周期素依赖性激酶抑制因子的调节

目的 阐明有丝分裂源激活的蛋白激酶(MAPK)和磷脂酶C(PLC)通路在调节细胞周期紊依赖性激酶抑制因子(CKI)p27，p21，p57蛋白表达量中的作用及其对血管平滑肌细胞(VSMC)增殖的影响。方法 分离SD大鼠主动脉中层平滑肌，贴壁法培养平滑肌细胞，无血清培养基培养静止后，分别加入血小板源性生长因子(PDGF)(20ng／m1)、PDGF PD98059(20ng／ml 20mmol／L)、PDGF 硫酸新霉紊(20ng／ml 10mmol／L)等刺激因素，以无血清培养基培养的VSMC作对照。在刺激后90min收集细胞，用免疫沉淀法检测MAPK(p44／p42)活性的改变。在刺激后6和24h收集细胞，用Western蛋白印迹法检测p27、p57和p21蛋白表达量。结果 PDGF刺激后，MAPK活性明显升高(较对照组增加46．6％)，同时VSMC明显增殖。刺激24h后，细胞增殖程度为对照组的1．43倍，p27蛋白的表达量显著下降至对照组的71％；p21和p57蛋白表达量却明显增加；加用PD98058(MAPK抑制剂)和新霉紊(PLC抑制剂)可明显抑制PDGF引起的上述改变，MAPK活性分别较PDGF。刺激组下降了46％和37％，p27蛋白表达量则分别为PDGF刺激组的1．77倍和1．49倍，细胞增殖程度分别降为后者的68％和65％。结论 MAPK(-14／42)变化的幅度是决定CKI表达量和VSMC增殖的关键因素。PLC通路在PDGF刺激VSMC增殖信号转导中同样起关键作用。     

Carbon monoxide inhibits apoptosis in vascular smooth muscle cells

OBJECTIVE: Carbon monoxide (CO) is generated from vascular smooth muscle cells via the degradation of heme by the enzyme heme oxygenase-1. Since smooth muscle cell apoptosis is associated with numerous vascular disorders, we investigated whether CO regulates apoptosis in vascular smooth muscle. METHODS AND RESULTS: Treatment of cultured rat aortic smooth muscle cells with a combination of cytokines (interleukin-1beta, 5 ng/ml; tumor necrosis factor-alpha, 20 ng/ml; interferon-gamma, 200 U/ml) for 48 h stimulated apoptosis, as demonstrated by DNA laddering, annexin V binding, and caspase-3 activation. However, the exogenous administration of CO inhibited cytokine-mediated apoptosis. The antiapoptotic action of CO was partially dependent on the activation of soluble guanylate cyclase and was associated with the inhibition of mitochondrial cytochrome c release and with the suppression of p53 expression. Incubation of smooth muscle cells with the cytokines also resulted in a pronounced increase in heme oxygenase-1 protein after 24 h of stimulation. The addition of the heme oxygenase inhibitor, zinc protoporphyrin-IX, or the CO scavenger, hemoglobin, stimulated apoptosis following 24 h of cytokine exposure. CONCLUSIONS: These results demonstrate that CO, either administered exogenously or endogenously derived from heme oxygenase-1 activity, inhibits vascular smooth muscle cell apoptosis. The ability of CO to block smooth muscle cell apoptosis may play an important role in blocking lesion formation at sites of vascular injury.     

Inhibition of the p53 tumor suppressor gene results in growth of human aortic vascular smooth muscle cells. Potential role of p53 in regulation of vascular smooth muscle cell growth.

Loss of activity of the p53 tumor suppressor gene product has been postulated in the pathogenesis of human restenosis. Although the antioncogenes p53 and retinoblastoma (Rb) susceptibility gene have been reported to play a pivotal role in cell cycle progression in various cells, the role of p53 and Rb in the growth of human vascular smooth muscle cells (VSMC) has not yet been clarified. We used antisense strategy against p53 and Rb genes by the viral envelope-liposomal method. Transfection of antisense p53 oligodeoxynucleotides (ODN) alone resulted in an increase in DNA synthesis compared with control (P<0.01). Similarly, transfection of antisense Rb ODN alone resulted in a higher DNA synthesis rate than control (P<0.01). Moreover, increase in VSMC number was only induced by transfection of antisense p53 ODN alone or cotransfection of p53/Rb ODN (P<0.01), whereas a single transfection of antisense Rb ODN had little effect on cell number. Therefore, we hypothesized that this discrepancy is due to the induction of apoptosis mediated by p53. Interestingly, apoptotic cells were markedly increased in VSMC transfected with antisense Rb ODN alone, accompanied by the induction of p53 protein. The number of apoptotic cells was attenuated by cotransfection of antisense p53 ODN (P<0.01). We finally examined the molecular mechanisms of apoptosis induced by the absence of Rb. In VSMC transfected with antisense Rb ODN, bax, a promoter of apoptosis, was significantly increased in VSMC transfected with antisense Rb ODN (P<0.01), whereas bcl-2 and Fas did not play a pivotal role in the induction of apoptosis. Overall, these data first demonstrated that the antioncogenes p53 and Rb negatively regulated the cell cycle in VSMC, suggesting that the modulation of their activity may mediate VSMC growth such as that in restenosis and atherosclerosis. The presence of p53 plays a pivotal role in the regulation of apoptosis in human VSMC growth, probably through the bax pathway. These results provide evidence that p53 is a functional link between cell growth and apoptosis in VSMC.     

d-Alpha-tocopherol prevents the hyperglycemia induced activation of diacylglycerol (DAG)-protein kinase C (PKC) pathway in vascular smooth muscle cell by an increase of DAG kinase activity

We have reported that d-alpha-tocopherol can prevent hyperglycemia-induced activation of DAG and PKC levels in vascular tissues as well as normalizing retinal blood flow and renal hyperfiltration. The mechanism of this effect, however, is not clear. Aside from alpha-tocopherol's principal role as an antioxidant agent, it has also been shown to act as a membrane stabilizer. Another possibility is that the effect of alpha-tocopherol is focused on the activation of DAG kinase, which is a key enzyme in the metabolism of DAG. Therefore, in this study, we examined the effect of alpha-tocopherol on the DAG kinase activity in vascular smooth muscle cell. We have also examined the effect of alpha-tocopherol, its analogues, and probucol on DAG kinase activities and expression. The present study showed that d-alpha-tocopherol's inhibitory effect on DAG-PKC pathway is by increasing DAG kinase activity in rat and human vascular smooth muscle cell (VSMC). Total DAG level was increased by 40 +/- 10% (mean +/- S.E.) (P < 0.05) in human VSMC, after exposure to 22 vs 5 mM glucose. This increase was normalized by d-alpha-tocopherol treatment in a concentration-dependent manner. In parallel, DAG kinase activation by d-alpha-tocopherol was also induced in a time- and dose-dependent manner. DAG kinase activity was increased by 57 +/- 19% (P < 0.05) in human VSMC and 112 +/- 35% (P < 0.05) in rat VSMC after 24 h of incubation with d-alpha-tocopherol (100 microg/ml). Another lipophilic antioxidant, probucol, also increased DAG kinase activity by 124 +/- 34%, but other vitamin E analogues with much less antioxidant potencies were ineffective. Western blots of various DAG kinase isoforms were not changed by d-alpha-tocopherol treatment. These results provide strong and detailed evidence that d-alpha-tocopherol can prevent hyperglycemia induced DAG-PKC activation by enhancing DAG kinase activity, probably through an antioxidant effect.     

Decrease of vascular smooth muscle cell locomotion by abciximab,but not tirofiban: a possible role of different affinity to alpha v beta 3 integrins

AIM: The EPISTENT and EPIC studies demonstrated a reduction of clinically driven re-interventions after percutaneous transluminal coronary angioplasty (PTCA) and stent implantation in patients treated with abciximab, while for tirofiban no similar effects could be demonstrated. This may be explained by the different effects on the migratory and invasive potential of vascular smooth muscle cells (VSMCs) by integrin alpha v beta 3 blockade. Therefore, the objective of this study was to compare the effectiveness of abciximab and tirofiban to affect VSMC migration and invasion. METHODS: Vascular smooth muscle cells were treated with abciximab (0.1-1 microg/ml), tirofiban (0.1-1 microg/ml), and the alpha v beta 3 specific antibody LM609 (1-5 microg/ml), that was used as a positive control during the assay (treatment) over 24 h before the assay (pre-treatment), or before and during the assay (combined treatment). Sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxyy-6-nitro) benzene sulfonic acid (XTT)-assay and cell counting measured the influence of the substances on VSMC proliferation. Using a Boyden Chamber model, the capability of VSMCs for migration and invasion was tested with different chemo-attractants and barriers. RESULTS: Any influence of the platelet glycoprotein (GP) IIb/IIIa receptor (integrin alpha IIb beta 3) antagonists on VSMC proliferation could be excluded. After combined treatment, abciximab demonstrated a dose-dependent inhibition of migration (IC50 = 33 microg/ml) and invasion (IC50 = 0.5 microg/ml) of VSMCs. Administration during the assay without pre-treatment inhibited migration similarly (IC50 = 32 microg/ml) but invasion to a significant lower extent (IC50 = 44 microg/ml). Administration of tirofiban during the assay with or without pre-treatment had no inhibitory effect on VSMC migration and invasion. Pre-treatment alone with one of the substances also did not alter VSMC migration or invasion. CONCLUSION: Abciximab administration in physiological concentrations was capable of significantly inhibiting the migratory and invasive potential of VSMCs, while for tirofiban no similar effect could be demonstrated.     

内皮素促进细胞增殖相关基因的表达和血管平滑肌细胞增殖

通过３Ｈ－ＴｄＲ掺入实验和ＲＮＡ印迹分析，观察了内皮素－１对大鼠血管平滑肌细胞ＤＮＡ合成及细胞增殖相关基因表达的影响。结果表明，内皮素－１作用于血管平滑肌细胞１２ｈ，３Ｈ－ＴｄＲ掺入开始增加，２４ｈ达到峰值。在内皮素－１作用下，原癌基因ｃ－ｆｏｓ和ｃ－ｊｕｎ进行快速瞬间表达，其表达活性在３０ｍｉｎ达到峰值，３ｈ后恢复到原来水平；Ｐ３４＾激酶基因在内皮素－１刺激１２ｈ后表达活性开始增加，至２４ｈ仍维     

ACE inhibition and fibroblast growth factor in cultured human vascular smooth muscle.

The effects of the angiotensin converting enzyme (ACE) inhibitor, enalaprilat, and basic fibroblast growth factor (bFGF) on DNA synthesis and expression of ACE mRNA were examined in human vascular smooth muscle cells cultured from saphenous vein and internal mammary artery. DNA synthesis was estimated using 3H-thymidine uptake, and ACE mRNA was estimated by rt-PCR. Enalaprilat (0.125 microg/ml, 48 h) decreased 3H-thymidine uptake to 66+/-12% (SE) of the control without enalaprilat (p < 0.05). Basic FGF (10 ng/ml, 24 h) increased uptake by 41 +/- 12% (p < 0.05) while enalaprilat pretreatment (24 h) decreased uptake to 56 +/- 12% of this augmented value (p < 0.025). Basic FGF increased ACE mRNA, a process that was time dependent with an approximately 50% increase after 24 h exposure. Pre-exposure to enalaprilat (24 h) before bFGF reduced ACE mRNA to approximately 50% of that found in the presence of bFGF alone. The results indicate that ACE mRNA is present in human vascular smooth muscle cells and that exposure to an ACE inhibitor reduces DNA synthesis. Basic FGF stimulates DNA synthesis and ACE mRNA expression, and both of these effects are reduced by an ACE inhibitor. The results are consistent with the effects of bFGF being exerted through, or alternatively in concert with, angiotensin II. Further, they suggest that ACE inhibition can reduce the activity of the renin-angiotensin system by inhibiting the production of ACE, or at least the expression of ACE mRNA, in addition to producing enzyme inhibition at the ACE level.     

c-myc and skp2 coordinate p27 degradation, vascular smooth muscle proliferation, and neointima formation induced by the parathyroid hormone-related protein

Parathyroid hormone-related protein (PTHrP) contains a classical bipartite nuclear localization signal. Nuclear PTHrP induces proliferation of arterial vascular smooth muscle cells (VSMC). In the arterial wall, PTHrP is markedly up-regulated in response to angioplasty and promotes arterial restenosis. PTHrP overexpression exacerbates arterial restenosis, and knockout of the PTHrP gene results in decreased VSMC proliferation in vivo. In arterial VSMC, expression of the cell cycle inhibitor, p27, rapidly decreases after angioplasty, and replacement of p27 markedly reduces neointima development. We have shown that PTHrP overexpression in VSMC leads to p27 down-regulation, mostly through increased proteosomal degradation. Here, we determined the molecular mechanisms through which PTHrP targets p27 for degradation. S-phase kinase-associated protein 2 (skp2) and c-myc, two critical regulators of p27 expression and stability, and neointima formation were up-regulated in PTHrP overexpression in VSMC. Normalization of skp2 or c-myc using small interfering RNA restores normal cell cycle and p27 expression in PTHrP overexpression in VSMC. These data indicate that skp2 and c-myc mediate p27 loss and proliferation induced by PTHrP. c-myc promoter activity was increased, and c-myc target genes involved in p27 stability were up-regulated in PTHrP overexpression in VSMC. In primary VSMC, PTHrP overexpression led to increased c-myc and decreased p27. Conversely, knockdown of PTHrP in primary VSMC from PTHrP(flox/flox) mice led to cell cycle arrest, p27 up-regulation, with c-myc and skp2 down-regulation. Collectively, these data describe for the first time the role of PTHrP in the regulation of skp2 and c-myc in VSMC. This novel PTHrP-c-myc-skp2 pathway is a potential target for therapeutic manipulation of the arterial response to injury.     

大黄素对人血管平滑肌细胞周期蛋白D1表达的影响

目的　观察大黄素对人血管平滑肌细胞周期时相和细胞周期蛋白D1 (CyclinD1 )表达的影响 ,探讨大黄素在药物置入物 (涂层支架 )上应用的可能性。方法　取对数增长期的平滑肌细胞 ,采用四甲基偶氮唑盐 (MTT)法观察大黄素对平滑肌细胞增殖抑制的有效浓度范围 ,求出IC50 并干预细胞。然后分别用流式细胞仪和Western blot法进行细胞周期时相和CyclinD1表达的测定。结果　与对照组比较 ,IC50 时药物组G0 /G1 期细胞百分比升高 ,S期细胞百分比下降 ,CyclinD1表达高峰延迟、表达量下调 ,细胞周期受阻于G0 /G1 期。结论　大黄素可以通过下调CyclinD1表达 ,阻滞细胞周期的进程 ,是一种有效的抗平滑肌细胞增殖的药物。