VEGF通过对p38和p42／p44 CCDPK信号的共调节作用抑制TNF—α和H2O2诱导的牛主动脉内皮细胞凋亡

目的：研究血管内皮细胞生长因子（VEGF）对肿瘤坏死因子（TNF－α）和过氧化氢（H2O2）诱导主动脉内皮细胞（BAEC）凋亡的影响及信号机制。方法：BAEC培养并传代于DMEM。经TNF－α,或H2O2处理24h后,Hoechst 33258染色,荧光显微镜观察形态学变化及凋亡细胞计数。MTT法测定细胞活性,琼脂糖凝胶电泳DNA解,Western blot法检测磷酸化p38和p42/p44 CCDPK表达。结果：TNF－α5000kU/L和H2O300μmol/L均可诱导BAEC产生DNA断片。VEGF100μg/L显著增强TNF－α和H2O2诱导的磷酸化p42-p44 CCDPK表达,而明显抑制磷酸化p38 CCDPK的活化。对二者所致BAEC凋亡起明显的抑制作用。p42/p44 CCDPK抑制剂U0126可取消VEGF引起的磷酸化,p42/p44 CCDPK表达上调和其抗凋亡作用。结论：VEGF通过其共调节作用激活p42/p44 CCDPK,抑制p38 CCDPK信号途径而对TNF－α和H2O2所致凋亡产生的抑制效应,是内皮细胞存活的重要机制。     

p38和p44/42 CCDPK信号在TNF-α诱导牛主动脉内皮细胞凋亡中的相反作用(英文)

目的:研究肿瘤坏死因子(TNF-α)诱导牛主动脉内皮细胞(BAEC)凋亡及其信号途径。方法:BAEC培养并传代于DMEM。经TNF-α处理24h后,Hoechst33258染色,荧光显微镜观察形态学变化及凋亡细胞计数。MIT法测定细胞活性,琼脂糖凝胶电泳分析DNA降解,Western blot法检测磷酸化p38和p44/42CCDPK表达。结果:TNF-α诱导BAEC产生典型的凋亡细胞形态学变化(核浓染,核碎裂)和DNA断片。TNF-α(100-5000kU/L)浓度依赖性诱导BAEC凋亡,并同时刺激磷酸化p44/42和p38CCDPK的表达.p44/42CCDPK抑制剂U0126可完全阻断TNF-α诱导的p44/42CCDPK的活化,显著增强TNF-α致凋亡作用;而p38 CCDPK抑制剂SB203580可完全阻断TNF-α诱导的p38CCDPK的活化,还可增强TNF-α诱导的磷酸化p44/42CCDPK的表达,明显抑制TNF-α促凋亡作用。结论:TNF-α同时激活p38和p44/42CCDPK,这两种CCDPK信号通路在TNF-α诱导BAEC凋亡中起相反作用。     

雌激素通过对p38和p44/42 CCDPK的相反作用阻止TNF-α诱导牛主动脉内皮细胞凋亡(英文)

目的:研究雌二醇(17β-estradiol,E_2)对肿瘤坏死因子α(TNF-α)诱导牛主动脉内皮细胞(BAEC)凋亡的影响及机制.方法:BAEC培养并传代于DMEM.BAEC经Hoechst 33258染色后用荧光显微镜观察形态学变化及计数凋亡细胞;用MTT法测定细胞活性;用琼脂糖凝胶电泳分析DNA降解;用Westernblot法检测磷酸化p~(38)和p~(42) CCDPK表达.结果:TNF-α诱导BAEC产生典型的凋亡细胞形态学变化(核浓染,核碎裂)和DNA断片.E_2(0.1pmol/L-100 nmol/L)能增强TNF-α诱导的磷酸化p~(44)/42表达,同时抑制p~(38) CCDPK的活化而浓度依赖性阻止TNF-α诱导的BAEC凋亡;E_2 1nmol/L明显减弱TNF-α所致DNA断裂;p~(44)/42 CCDPK抑制剂U0126可拮抗E_2的作用.结论:雌激素通过激活p~(44)/42CCDPK,同时抑制p~(38) CCDPK而产生的抗凋亡效应,是其使内皮细胞存活的重要机制.     

p38和p42/p44 Ca~(2 )-钙调蛋白依赖性蛋白激酶信号在过氧化氢诱导牛主动脉内皮细胞凋亡中的作用

目的:研究p38和p42/p44 Ca~(2 ).钙调蛋白依赖性蛋白激酶(CCDPK)信号通路对过氧化氢(H_2O_2)诱导牛主动脉内皮细胞(BAEC)凋亡的调节作用.方法:H_2O_2处理BAEC 24 h后,荧光显微镜下观察形态学变化及凋亡细胞计数.MTT法测定细胞活性,琼脂糖凝胶电泳分析DNA降解,蛋白质印迹法检测磷酸化p38和p42/p44 CCDPK表达.结果:H_2O_2诱导BAEC产生典型的凋亡细胞形态学变化(核浓染,核碎裂)和DNA断片.H_2O_2(100-500μmol·L~(-1))浓度依赖性刺激磷酸化p42/p44和p38 CCDPK的表达.p42/p44 CCDPK抑制剂U0126显著增强H_2O_2致凋亡作用;然而p38 CCDPK抑制剂SB203580可增强H_2O_2诱导的磷酸化p42/p44 CCDPK的表达,但不影响BAEC的存活.结论:p42/p44 CCDPK对H_2O_2诱导的BAEC凋亡起保护作用,而p38 CCDPK不是介导H_2O_2所致细胞凋亡的主要信号通路.     

VEGF通过对p38和p42/p44 CCDPK信号的共调节作用抑制TNF-α和H_2O_2诱导的牛主动脉内皮细胞凋亡(英文)

目的:研究血管内皮细胞生长因子(VEGF)对肿瘤坏死因子(TNF-α)和过氧化氢(H_2O_2)诱导生主动脉内皮细胞(BAEC)凋亡的影响及信号机制.方法:BAEC培养并传代于DMEM.经TNF-α或H~2O_2处理24h后,Hoechst 33258染色,荧光显微镜观察形态学变化及凋亡细胞计数.MTT法测定细胞活性,琼脂糖凝胶电泳分析DNA降解,Western blot法检测磷酸化p~(38)和p~(42)/p~(44) CCDPK表达.结果:TNF-α5000kU/L和H_2O_2 300μmol/L均可诱导BAEC产生DNA断片.VEGF 100 μg/L显著增强TNF-α和H_2O_2诱导的磷酸化p~(42)/p~(44) CCDPK表达,而明显抑制磷酸化 p~(38) CCDPK的活化.对二者所致 BAEC凋亡起明显的抑制作用.P~(42)/p~(44) CCDPK抑制剂U0126可取消 VEGF引起的磷酸化p~(42)/p~(44) CCDPK表达上调和其抗凋亡作用.结论:VEGF通过其共调节作用激活p~(42)/p~(44) CCDPK,抑制p~(38) CCDPK信号途径而对TNF-α和H_2O_2所致凋亡产生的抑制效应,是内皮细胞存活的重要机制.     

高糖增强过氧化氢诱导牛主动脉内皮细胞凋亡(英文)

目的:研究高糖对过氧化氢(H_2O_2)诱导牛主动脉内皮细胞(BAEC)凋亡作用。方法:BAEC培养并传代于正常葡萄糖(5.5 mmol·L~(-1))和高糖(25mmol·L~(-1))中,经H_2O_2处理24 h后,Hoechst 33258染色,荧光显微镜观察形态学变化及凋亡细胞计数;琼脂糖凝胶电泳分析DNA降解,Western blot法检测磷酸化p38 CCDPK表达。结果:H_2O_2诱导BAEC产生典型的凋亡细胞形态学变化(核浓染,核碎裂)。在100-300 μmol·L~(-1)范围内,正常糖和高糖BAEC经H_2O_2处理后,浓度依赖性诱导细胞凋亡和磷酸化p38 CCDPK表达。高糖条件下诱导BAEC DNA降解浓度低于正常糖BAEC,细胞凋亡率和磷酸化p38 CCDPK表达均显著高于正常糖组(p<0.05)。结论:高糖促进H_2O_2诱导BAEC凋亡,可能与其增强磷酸化p38 CCDPK的表达相关。     

血小板源生生长因子刺激血管平滑肌细胞增殖及其分子机制

目的：探讨血小板源生生长因子（PDGF－BB）刺激血管平滑肌细胞（VSMC）增殖及其分子机制。方法：用Western Blot法测定p44/p42 CCDPK活性。[^3H]脱氧胸腺嘧啶核苷酸掺入测定VSMC DNA合成。原位杂交检测c-myc mRNA的表达。结果：PDGFBB诱导的磷酸化CCDPK蛋白表达和[^3H]脱氧胸腺嘧啶核苷酸掺入呈浓度依赖性,此作用可被PTK抑制剂Genistein,外钙络合剂依他酸和MEK抑制剂PD 98059抑制。PDGF－BB刺激可引起c-myc mRNA的明显表达,此作用可被PD 98059抑制。结论：PDGF－BB通过激活p44/p42 CCDPK,上调c-myc mRNA的表达从而促进VSMC增殖,其作用是由PTK和Ca^2 介导的。     

毛兰素诱导人白血病HL—60细胞的凋亡

目的：研究毛兰素对HL－60细胞增殖的抑制作用,探讨其诱导细胞凋亡的机制。方法：用MTT比色法测定了毛兰素对HL－60细胞增殖的抑制作用：应用荧光显微镜、透射电镜、DNA电泳及流式细胞仪观察了药物对细胞凋亡的诱导作用,并用免疫组化的方法从基因水平阐述了凋亡的发生。结果：毛兰素20－81．9nmol/L在72h内显著抑制HL－60细胞增殖,作用24h后,对HL－60细胞的IC50为38nmol/L,而阳性对照药长春新碱对HL－60细胞的IC50为101nmol/L,前者明显优于后者;形态学观察可见凋亡的特征性改变;琼脂糖电泳出现典型的DNA“ladder”;流式细胞仪结果表明细胞被阻滞于G2／M期;免疫组化可见bcl-2表达下降,bax表达升高。结论：毛兰素显著抑制HL－60细胞的生长,该抑制作用可能是通过诱导细胞凋亡和改变HL－60细胞bcl-2和bax基因的表达而实现的。     

p38 MAPK激酶抑制剂增强二烯丙基二硫化物诱导CNE2细胞凋亡

目的　研究二烯丙基二硫化物 (DADS)诱导CNE2细胞凋亡及 p38MAPK信号转导通路对此过程的作用。 方法　DADS处理CNE2细胞 2 4h后 ,荧光显微镜下观察形态学变化及凋亡细胞计数 ,MTT法测定细胞活性 ,流式细胞仪检测凋亡细胞 ,蛋白质印迹法检测磷酸化p38MAPK表达。结果　在培养的CNE2细胞中 ,DADS(50～ 1 50 μmol·L- 1 )作用 2 4h后 ,DADS诱导CNE2细胞产生典型的凋亡细胞形态学变化 (核浓染 ,核碎裂 ) ,流式细胞仪结果显示 ,随着DADS给药剂量增加 ,细胞周期中各期细胞所占百分率的变化无规律 ,细胞凋亡呈剂量依赖性 ,DADS(50～ 1 50 μmol·L- 1 )浓度依赖性刺激磷酸化p38MAPK的表达 ,p38MAPK抑制剂SB2 0 3580明显增强DADS致凋亡作用。结论　DADS诱导CNE2细胞凋亡时激活磷酸化 p38MAPK表达 ,磷酸化p38MAPK抑制剂增强DADS诱导CNE2细胞凋亡效应     

丝裂素活化的蛋白激酶反义寡脱氧核苷酸防治血管内膜增殖

目的：探讨Ca^2 -钙调蛋白依赖性蛋白激酶（丝裂素活化的蛋白激酶）（CCDPK）在生长因子诱导体外培养大鼠血管平滑肌细胞增殖中的作用及反义CCDPK寡脱氧核苷酸（ODN）对球囊损伤后大白鼠血管内膜增生的抑制作用。方法：利用脂质体转染17－mer CCDPK反义ODN进入培养的血管平滑肌细胞以抑制CCDPK活性,设正义及随机ODN作对照。用蛋白质印迹法测定CCDPK表达。[^3H]胸腺嘧啶核苷酸掺入测定平滑肌细胞DNA合成。用2F球囊导管造成大白鼠颈动脉再狭窄模型,利用多聚胶F127－ODN系统由血管外膜部位给药。于损伤后2周取样,固定及HE染色观察内膜增生情况。FITC标记的ODN观察体内外给药方法的分布及吸收情况。结果：CCDPK反义ODN能明显抑制PDGF及ET诱导的CCDPK蛋白表达及[^3H]胸腺嘧啶核苷酸掺入。在大鼠颈动脉再狭窄模型,能明显抑制血管内膜增生。结论：CCDPK介导了PDGF及ET诱导的血管平滑肌细胞增殖。针对p42-和p44-CCDPK起始部位设计的17－mer反义ODN能有效抑制生长因子诱导的血管平滑肌细胞的增殖及球囊损伤大鼠血管内膜增生。     

Opposing effect of p38 CCDPK and p44/42 CCDPK signaling on TNF-alpha-induced apoptosis in bovine aortic endothelial cells.

AIM: To investigate the pro-apoptotic role of tumor necrosis factor alpha (TNF-alpha) in cultured bovine aortic endothelial cells (BAEC) and its underlied apoptotic signaling pathways. METHODS: BAEC were cultured and passaged in Dulbecco's modified Eagle's medium (DMEM). Morphologic changes and quantification of apoptotic cells were determined under fluorescence microscope after TNF-alpha treated BAEC for 24 h with Hoechst 33258 staining. Cell viability was determined with MTT method. DNA fragmentation was visualized by agarose gel electrophoresis. The expression of phospho-p38 and phospho-p44/42 Ca2+-calmodulin dependent protein kinase (CCDPK, formerly called MAPK) was measured by Western blotting. RESULTS: TNF-alpha elicited typical apoptotic morphologic changes (chromatic condensation, nucleus fragmentation) and DNA fragmentation. At 1000-5000 kU/L, incubation with TNF-alpha for 24 h induced BAEC apoptosis and both of phospho-p38 and phospho-p44/42 CCDPK expression in a concentration-dependent manner. Interestingly, TNF-alpha-stimulated activation of p44/42 CCDPK was completely blocked, TNF-alpha-induced apoptosis was markedly increased by preincubation with U0126, a specific p44/42 CCDPK inhibitor. However, SB203580, a specific p38 CCDPK inhibitor, completely blocked TNF-alpha-stimulated activation of p38 CCDPK, and enhanced the expression of phospho-p44/42 CCDPK induced by TNF-alpha, substantially inhibited the pro-apoptotic effect of TNF-alpha. CONCLUSION: TNF-alpha simultaneously activates p38 CCDPK and p44/42 CCDPK, and these two CCDPK signaling pathways appeared to play opposing roles in TNF-alpha-induced apoptosis in BAEC.     

Effects of p38 and p42/p44 CCDPK signaling on H2O2-induced apoptosis in bovine aortic endothelial cells

AIM: To investigate the effects of p38 and p42/p44 Ca(2+)-calmodulin dependent protein kinases (CCDPK) signaling on hydroperoxide (H2O2)-induced apoptosis in cultured bovine aortic endothelial cells (BAEC). METHODS: Morphologic changes and quantification of apoptotic cells were determined under fluorescence microscope after a 24-h treatment of BAEC by H2O2. Cell viability was determined with MTT method. DNA fragmentation was visualized by agarose gel electrophoresis. The expression of phospho-p38 and phospho-p42/p44 CCDPK was measured by Western blotting. RESULTS: H2O2 elicited typical apoptotic morphologic changes (chromatic condensation, nucleus fragmentation) and DNA fragmentation. At 100-500 mumol.L-1, incubation of BAEC with H2O2 for 24 h also induced phospho-p38 and phospho-p42/p44 CCDPK expression in a concentration-dependent manner. Interestingly, H2O2-induced apoptosis was markedly increased by preincubation with U0126, a specific p42/p44 CCDPK inhibitor. However, SB203580, a specific p38 CCDPK inhibitor, enhanced the expression of phospho-p42/p44 CCDPK induced by H2O2, but had no effect on BAEC survival. CONCLUSION: p42/p44 CCDPK signaling appears to play protective roles in H2O2-induced apoptosis in BAEC, whereas p38 CCDPK is not the main signaling pathway mediating H2O2-induced cellular apoptosis.     

Biphasic effect of aspirin on apoptosis of bovine vascular endothelial cells and its molecular mechanism

AIM: To investigate the effect of aspirin on the apoptosis of cultured bovine aortic endothelial cells (BAEC) and the signal pathways involved in this process. METHODS: BAEC were cultured and passaged in Dulbecco's modified Eagle's medium culture medium. Morphologic changes and quantification of apoptotic cells were determined using fluorescence microscope after staining the cells with Hoechst 33258. Cell viability was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) method. DNA fragmentation was visualized by agarose gel electrophoresis. Phospho-p38 mitogen-activated protein kinase (MAPK) expression was detected by Western blotting. RESULTS: Aspirin at low concentrations from 1X10( -10) mol/L to 1X10( -8) mol/L decreased the apoptosis and p38 MAPK phosphorylation induced by H2O2 in BAEC, while high doses of aspirin (1X10( -7)-1X10( -4) mol/L) induced typical apoptotic changes in BAEC and stimulated the expression of phospho-p38 MAPK in a concentration-dependent manner. SB203580, a specific p38 MAPK inhibitor, blocked such effects. CONCLUSION: Aspirin exhibits a biphasic effect on the apoptosis in BAEC, reducing apoptosis at low concentration and inducing apoptosis at high concentration. p38 MAPK may be an important signal molecule mediating the effects of aspirin.     

Enhancement of diallyl disulfide-induced apoptosis by inhibitors of MAPKs in human HepG2 hepatoma cells

We examined the effects of diallyl disulfide (DADS), an oil-soluble organosulfur compound found in garlic, on human HepG2 hepatoma cells to better understand its effect on apoptosis and apoptosis-related genes. Our study has demonstrated that DADS affects cell proliferation activity and viability and elicits typical apoptotic morphologic changes (chromatic condensation and nuclear fragmentation) in human HepG2 hepatoma cells. Also, treatment with DADS induces a temporary increase in phosphorylated p38 MAPK (phospho-p38) and phosphorylated p42/44 MAPK (phospho-p42/p44) in a time- and concentration-dependent manner. Inhibition of activated/phosphorylated mitogen-activated protein kinase (MAPK) with phospho-p38 or phospho-p42/44 specific inhibitors, SB203580 or U0126, induces apoptosis without DADS treatment, indicating that at least the endogenous activated forms of p38 MAPK and p42/p44 MAPK markedly exert cytoprotective roles from cell apoptosis in the HepG2 hepatoma cells. Combined treatment with these inhibitors followed by DADS further enhances the DADS-induced apoptosis. Taken together, these results show that both DADS and the specific inhibitors of MAPKs could induce apoptosis in HepG2 hepatoma cells and that the MAPKs inhibitors further enhance the apoptotic effect in DADS-treated HepG2 hepatoma cells.     

CCDPK反义寡核苷酸抑制bFGF诱导的大鼠血管平滑肌细胞增殖(英文)

AIM: To investigate the role of Ca(2+)-calmodulin dependent protein kinase (CCDPK) on basic fibroblast growth factor (bFGF)-induced vascular smooth muscle cell (VSMC) proliferation and the inhibitory effect of antisense CCDPK oligonucleotides (ODN). METHODS: Before being exposed to bFGF, cultured rat VSMC CCDPK activity was inhibited by pretreatment with either a phosphorothioate-protected 17-mer antisense CCDPK ODN-directed against the initiation of translation sites of the p42 and p44 CCDPK isoform or with CCDPK kinase inhibitor PD98059. All ODN were introduced into cells by liposomal transfection. DNA synthesis was measured by [3H]thymidine incorporation. P44- and p42-CCDPK protein expression and phosphorylation were measured by Western blot. RESULTS: PD98059 inhibited bFGF-induced phosphorylation of CCDPK and DNA synthesis. Antisense CCDPK ODN 0.2-0.8 mumol.L-1 reduced both p44- and p42-CCDPK expression and phosphorylation of CCDPK in a concentration-dependent manner and DNA synthesis induced by bFGF. Lipofectin alone or sense and random CCDPK ODN did not affect p44- and p42-CCDPK protein expression or bFGF-induced phosphorylation of CCDPK or DNA synthesis. CONCLUSION: bFGF-stimulated rat VSMC proliferation is mediated by CCDPK. The antisense CCDPK ODN can inhibit bFGF-induced VSMC proliferation through down-regulating p44- and p42-CCDPK level.     

氧化型低密度脂蛋白诱导主动脉和心内膜内皮细胞凋亡

目的：研究氧化型低密度脂蛋白（ｏｘ－ＬＤＬ）诱导血管和心内膜内皮细胞凋亡。方法：用超速离心法分离健康人血浆低密度脂蛋白（ＬＤＬ），以ＣｕＳＯ４１０μｍｏｌ．Ｌ＾－１氧化，观察ｏｘ－ＬＤＬ对培养新生小牛主动脉内皮细胞及心内膜细胞的损伤作用，琼脂糖凝胶电泳和Ｈｏｅｃｈｓｔ３３２５８荧光密度法定性与定量分析ＤＮＡ降解，结果：ｏｘ－ＬＤＬ诱导血管内皮细胞及以内膜细胞典型凋亡形态学改变，ＤＮＡ降解呈时间和剂