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。     

丝裂素活化的蛋白激酶反义寡核苷酸对p42/p44,p38和JNK激酶的靶向选择性及其对血管平滑肌细胞DNA合成的抑制作用

目的:探讨丝裂素活化的蛋白激酶(MAPK)反义寡核苷酸对血清诱导的培养大鼠血管平滑肌细胞增殖的选择性及序列依赖性抑制作用。方法:用脂质体将p42-和p44-MAPK反义寡核苷酸转染入大鼠血管平滑肌细胞,设正义及随机寡核苷酸对照,20％血清刺激后,用Western Blot法测定总p44/p42-MAPK、p38 MAPK及JNKs蛋白水平及磷酸化MAPK表达。[~3H]胸腺嘧啶核苷酸掺入测定平滑肌细胞DNA合成。结果:MAPK反义寡核苷酸能明显抑制血清诱导的血管平滑肌细胞总MAPK蛋白水平及磷酸化MAPK蛋白表达,对p38 MAPK及JNKs表达无影响,并能明显抑制[~3H]胸腺嘧啶核苷酸掺入。结论:针对p42-和p44-MAPK起始部位设计的17-mer反义寡核苷酸能选择性及序列依赖性地抑制血清诱导的血管平滑肌细胞的增殖。     

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。     

黄芪抑制血管平滑肌细胞增殖及其作用机制

目的 观察黄芪(AS)对血管平滑肌细胞(VSMC)增殖的抑制作用，了解黄芪是否通过刺激VSMC产生一氧化氮(nitric oxide，NO)，使细胞周期停滞于G0／G1期，从而抑制平滑肌细胞增殖。方法 [3H]-胸腺嘧啶核苷酸([3H]-TdR)掺入测定VSMCDNA合成，流式细胞仪检测细胞周期情况，硝酸还原酶法测定细胞培养上清中NO水平。结果 黄芪以剂量依赖关系抑制血清诱导的VSMC[3H]-胸腺嘧啶核苷酸掺入，使G0／G1期细胞比例明显增多，S期细胞比例显著减少，黄芪刺激VSMC后，细胞培养上清中NO水平呈剂量依赖上升。结论黄芪能抑制VSMC增殖，使细胞周期停滞于G0／G1期，这一过程可能与黄芪刺激VSMC产生NO有关。     

卡托普利对自发性高血压大鼠血管平滑肌细胞增殖及癌基因和抑癌基因的影响

目的：观察卡托普利（CaP）对自发性高血压大鼠（SHR）血管平滑肌细胞（VSMC）增殖的作用及对原癌基因及抑癌基因的影响。方法：氚-胸腺嘧啶核苷（3H－TdR）参入，电镜，原位来交及Northernblot杂交。结果：CaP在降低SHR血压同时，能减少VSMC的线粒体，粗面内质网及3H－TdR参入量（P<0.01），并能逆转c－fos，c－myc，c－sis原癌基因mRNA表达增强（P<0.05或0.01），p53抑癌基因mRNA表达减弱（P＜0．01）。结论：Cap能抑制SHR的VSMC增殖，与癌基因调控的分子生物学机制有关。     

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.     

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

目的：研究雌二醇（17β－estradiol,E2)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.BAECHoechst 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Western blot法检测磷酸化p38和p44/42 CCDPK表达。结果：TNF－α诱导BAEC产生典型的凋亡细胞形态学变化（核浓梁,核碎裂）和DNA断片。E2（0.1pmol/L-100nmol/L)能增强TNF－α诱导的磷酸化p44/42表达,同时抑制p38 CCDPK的活化而浓度依赖性阻止TNF－α诱导的BAEC凋亡;E21nmol/L明显减弱TNF－α所致DNA断裂;p44/42 CCDPK抑制剂U0126可拮抗E2的作用。结论：雌激素通过激活p44/42 CCDPK ,同时抑制p38 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。     

黄芪抑制血管平滑肌细胞增殖及其作用机制

目的　观察黄芪(AS)对血管平滑肌细胞(VSMC)增殖的抑制作用,了解黄芪是否通过刺激VSMC产生一氧化氮(nitricoxide,NO) ,使细胞周期停滞于G₀/G₁期,从而抑制平滑肌细胞增殖。方法　[³H] 胸腺嘧啶核苷酸([³H] TdR)掺入测定VSMCDNA合成,流式细胞仪检测细胞周期情况,硝酸还原酶法测定细胞培养上清中NO水平。结果　黄芪以剂量依赖关系抑制血清诱导的VSMC[³H] 胸腺嘧啶核苷酸掺入,使G₀/G₁期细胞比例明显增多,S期细胞比例显著减少,黄芪刺激VSMC后,细胞培养上清中NO水平呈剂量依赖上升。结论　黄芪能抑制VSMC增殖,使细胞周期停滞于G₀/G₁期,这一过程可能与黄芪刺激VSMC产生NO有关     

丝裂原活化蛋白激酶参与血小板源生长因子刺激的大鼠主动脉平滑肌细胞增殖(英文)

选择大鼠主动脉平滑肌细胞作为细胞模型 ,观察丝裂原活化蛋白激酶 ( MAPK)信号通路在血小板源生长因子 ( PDGF)诱导的血管平滑肌细胞( VSMC)增殖中的作用。用 [3 H]Td R参入率作为衡量 VSMC增殖的指标 ,以 Western-印迹方法 ,用磷酸化一抗测 MAPK磷酸化的程度作为衡量 MAPK活性的指标 ,实验结果发现 ,PDGF( 0 .0 2 - 2 0μg·L-1)诱导 VSMC增殖呈剂量依赖性。 PDGF( 2μg· L-1)能持续性激活 MAPK。用 PD980 59( 1 0 -1 0 0 mol· L-1)预处理 1 5min后 ,MAPK活性较对照组均有明显差别 ( P<0 .0 5) .MAPK反义寡核苷酸能明显抑制 PDGF诱导的 MAPK的激活 ,并明显抑制 VSMC[3 H]Td R参入。上述结果表明 ,MAPK参与 PDGF促细胞增殖的信号途经 ,它的持续性激活与细胞增殖有关 ,针对 p42 /p44MAPK设计的反义寡核苷酸类能有效抑制 PDGF诱导的血管平滑肌细胞增殖。     

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.     

Effect of PKC-zeta mediating Ang II-stimulated activation of CCDPK on rat cardiac fibroblast proliferation

AIM: To investigate whether the effect of angiotensin (Ang) II or epidermal growth factor (EGF) on cardiac fibroblast proliferation involved in activation of extracellular signal-regulated kinase (ERK) 1/2 or Ca(2+)-calmodulin dependent protein kinase(CCDPK) mediated by protein kinase C (PKC)-zeta. METHODS: Relative activity of CCDPK was measured by Western blotting. DNA synthesis was assayed by [3H]thymidine incorporation. RESULTS: PDBU caused no decrease in Ang II- and 10% FCS-stimulated CCDPK activity and DNA synthesis. In contrary, 65% or 75% EGF- or tetradecanoylphorbol acetate (TDPA, formally called PMA)--stimulated CCDPK activity and 38% or 42% [3H]thymidine incorporation treated by PDBU were inhibited, respectively. Meanwhile 70% and 72% CCDPK activities induced by Ang II and EGF were inhibited by PD 98059, respectively. CONCLUSION: PKC-zeta mediated Ang II-induced activation of CCDPK and cardiac fibroblast proliferation.     

丝裂原活化蛋白激酶参与血小板源生长因子刺激的大鼠主动脉平滑肌细胞增殖(英文)

选择大鼠主动脉平滑肌细胞作为细胞模型 ,观察丝裂原活化蛋白激酶 ( MAPK)信号通路在血小板源生长因子 ( PDGF)诱导的血管平滑肌细胞( VSMC)增殖中的作用。用 [3 H]Td R参入率作为衡量 VSMC增殖的指标 ,以 Western-印迹方法 ,用磷酸化一抗测 MAPK磷酸化的程度作为衡量 MAPK活性的指标 ,实验结果发现 ,PDGF( 0 .0 2 - 2 0μg·L-1)诱导 VSMC增殖呈剂量依赖性。 PDGF( 2μg· L-1)能持续性激活 MAPK。用 PD980 59( 1 0 -1 0 0 mol· L-1)预处理 1 5min后 ,MAPK活性较对照组均有明显差别 ( P<0 .0 5) .MAPK反义寡核苷酸能明显抑制 PDGF诱导的 MAPK的激活 ,并明显抑制 VSMC[3 H]Td R参入。上述结果表明 ,MAPK参与 PDGF促细胞增殖的信号途经 ,它的持续性激活与细胞增殖有关 ,针对 p42 /p44MAPK设计的反义寡核苷酸类能有效抑制 PDGF诱导的血管平滑肌细胞增殖。     

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.     

丝裂素活化蛋白激酶反义寡核苷酸对血管紧张素Ⅱ诱导的大鼠心肌？…

AIM: To investigate the inhibitory effect of down-regulating mitogen activated protein kinase (MAPK) on c-myc gene expression and further on cardiac fibroblast proliferation. METHODS: Cultured neonatal rat cardiac fibroblasts was pretreated with a phosphorothioate-protected 17-mer antisense MAPK oligodeoxynucleotide (ODN) directed against the initiation of translation sites of the p42 and p44 MAPK isoforms by liposomal transfection. A 17-mer sense and mismatch sequence MAPK ODN were used as controls. After liposomal transfecting, cells were exposed to angiotensin II (Ang II) 10 nmol.L-1 for 5 min and then harvested in lysis buffer. MAPK activity was measured by Western blot and P-81 phosphocellulose filter paper method by using [gamma-32P]ATP and myelin basic protein as substrates. c-myc mRNA expression stimulated by Ang II for 30 min was measured by Northern blot. DNA synthesis and collagen protein synthesis induced by Ang II for 24 h were measured by [3H]thymidine incorporation and [3H]Proline incorporation, respectively. RESULTS: Antisense ODN 0.2 mumol.L-1 reduced Ang II-induced MAPK activities by 72%, MAPK protein expression by 80%, and suppressed c-myc mRNA expression by 97%, respectively. [3H]thymidine incorporation and [3H]proline incorporation in Ang II-induced cardiac fibroblast were inhibited by 59% and 58%, respectively. CONCLUSION: A 17-mer MAPK antisense oligonucleotide directed againsts the initiation of translation sites of MAPK could specifically inhibit Ang II-stimulated cultured neonatal rat cardiac fibroblast proliferation through down-regulating MAPK activity and further depleting c-myc mRNA expression.     

Cell proliferation and Ca(2+)-calmodulin dependent protein kinase activation mediated by alpha 1A- and alpha 1B-adrenergic receptor in HEK293 cells

AIM: To examine the ability of alpha 1-AR subtypes on proliferation and Ca(2+)-calmodulin dependent protein kinase (CCDPK, formerly called MAPK) activation in transfected human embryo kidney 293 (HEK293) cells. METHODS: pREP8/alpha 1A-AR, pREP4/alpha 1B-AR, and pREP9/alpha 1D-AR were transfected, respectively, into HEK293 cells by calcium phosphate precipitation. The expression of alpha 1-AR was detected by radioligand binding assays. DNA synthesis was measured by [3H]thymidine incorporation. CCDPK activity was determined by immunoprecipitation method and myelin basic protein was used as substrate. RESULTS: Three clonal HEK293 cell lines stably expressing alpha 1A- or alpha 1B- or alpha 1D-AR were chosen and characterized by radioligand binding assay with receptor densities of about 0.6 nmol.g-1. Treatment with norepinephrine (NE) in the presence of propranolol for 24 h increased DNA synthesis in HEK293/alpha 1A- or HEK293/alpha 1B-AR cells concentration-dependently, with EC50 values of 48.8 nmol.L-1 (95% confidence limits 9.7-246 nmol.L-1) and 8.4 nmol.L-1 (95% confidence limits 2.1-32.9 nmol.L-1), respectively. The increase of DNA synthesis induced by NE 10 mumol.L-1 was 201% +/- 28% and 269% +/- 44% of basal, and the activation of CCDPK was 171% +/- 84% and 292% +/- 92% of basal in HEK293/alpha 1A-AR and HEK293/alpha 1B-AR cells, respectively. Preincubation with prazosin completely abolished NE-induced CCDPK activation in HEK293/alpha 1A- and alpha 1B-AR cells. Those changes were not found in HEK293/alpha 1D-AR cells. CONCLUSION: The activation of alpha 1A- or alpha 1B-AR but not alpha 1D-AR induces cell proliferation.     

卡托普利对缺氧诱导的肺动脉平滑肌细胞增殖和胶的合成的抑制作用

AIM: To study the effect of captopril (Cap) on hypoxia-induced proliferation and collagen synthesis in vascular smooth muscle cells (VSMC). METHODS: VSMC were isolated from rabbit pulmonary artery. Cultured VSMC were evaluated by incorporation of [3H]thymidine and [3H]proline, cell number, and intracellular calcium concentration ([Ca2+]i). RESULTS: Pretreatment of pulmonary VSMC with Cap 1 mumol.L-1 blocked hypoxia-induced increase in cell number and incorporation of [3H]proline and [3H]thymidine, which were decreased 25%, 21%, and 36%, respectively, as compared with hypoxic control. It also inhibited the increase of intracellular Ca2+ concentration under hypoxic condition. Addition of nifedipine inhibited hypoxia-stimulated increase in the collagen, DNA synthesis, and [Ca2+]i. Bay-K-8644 increased cell number (35%), DNA (55%), collagen synthesis (36%), and [Ca2+]i (33%) in pulmonary VSMC, that was completely abolished by Cap 1 mumol.L-1. CONCLUSION: Cap inhibited hypoxia-induced proliferation and collagen synthesis in VSMC.     

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。     

丝裂素活化的蛋白激酶反义寡核苷酸抑制表皮生长因子诱导体外 …

目的：探讨丝裂素活化的蛋白激酶（ＮＡＰＫ）反义寡核苷（ＯＤＮ）对表皮生长因子（ＥＧＦ）诱导的培养大鼠血管平滑肌细胞增生的抑制作用。方法：用脂质体将Ｐ４２－和Ｐ４４－ＭＡＰＫ ＯＤＮ０．２μｍｏｌˉＬ＾－１转染入大鼠血管平滑肌细胞，设正义及随机ＯＤＮ为对照，用Ｗｅｓｔｅｒｎ Ｂｌｏｔ法结合Ｐ－８１滤纸法以髓磷脂碱性蛋白为底物测定ＭＡＰＫ活性。［＾３Ｈ］胸腺嘧啶核苷酸掺入测定平滑肌细胞ＤＮＡ合成。结果