大鼠平滑肌细胞增殖与西洛他唑抑制作用的体内实验

目的:以球囊损伤SD大鼠模型颈动脉内膜增生为基础,比较西洛他唑给药组和生理盐水对照组大鼠血管内膜增生强度和平滑肌细胞增殖能力方面的差异。方法:①实验于2004-01/12在解放军沈阳军区总医院全军心血管病研究所完成。实验选用健康雄性SD大鼠,体质量300～500g。②建立动物模型:腹腔麻醉36只大鼠后,暴露左颈总动脉及颈内、外动脉,自颈外动脉向近心端插入2FFogarty球囊导管至颈总动脉起始部,球囊充水0.10～0.15mL,慢速回拉至颈内外动脉分叉处,反复3次,完成后结扎颈外动脉。③36只SD大鼠随机分为2组,每组18只,分别为生理盐水组,西洛他唑给药组(7.5×10-7mol/L)。给药方式:灌肠给药,5mL/次,术后即刻开始,持续给药28d。④观察并比较两组大鼠损伤血管壁各成分结构变化图像定量分析结果。应用免疫组化分析大鼠动脉血管壁增殖情况,并应用反转录聚合酶链式反应分析两组损伤的动脉半胱天冬酶3在mRNA水平表达变化。结果:大鼠36只均进入结果分析。①大鼠动脉血管壁增殖情况免疫组化分析结果:西洛他唑给药组Ki67免疫组化染色阴性,细胞增殖能力下降;生理盐水组Ki67免疫组化染色阳性,细胞增殖能力旺盛。②损伤血管壁各成分结构变化图像定量分析结果:西洛他唑给药组大鼠颈动脉血管壁损伤后28d内膜增生面积明显小于生理盐水组犤(0.015±0.002),(0.197±0.004)mm2,t=299.06,P<0.01犦;管腔面积明显大于生理盐水组犤(0.193±0.008),(0.011±0.008)mm2,t=118.21,P<0.01犦。两组大鼠损伤动脉中膜增生面积差异不明显(P>0.05)。③两组损伤的动脉半胱天冬酶3在mRNA水平表达反转录聚合酶链式反应分析结果:西洛他唑给药组平滑肌细胞中半胱天冬酶3表达明显高于生理盐水对照组。结论:①通过在体大鼠颈动脉内膜增生模型建立,有效地模拟了西洛他唑抑制体内血管平滑肌细胞增殖的发生过程。②西洛他唑能够有效抑制体内血管平滑肌细胞增殖并诱导其凋亡,抑制血管损伤后新生内膜的形成,为临床防治增生性血管疾病的提供理论依据。     

大鼠血管平滑肌细胞迁移与西洛他唑的干预效应

目的:探讨西洛他唑对原代培养大鼠血管平滑肌细胞迁移能力的影响及其可能的调控作用。方法:实验于2003-03/12在解放军沈阳军区总医院全军心血管病研究所完成。使用胶原酶消化法处理健康雄性SD大鼠,获得原代培养的鼠血管平滑肌细胞,加入终浓度为0.5μmol/L西洛他唑培养72h为西洛他唑组,对照组为加入等量的Dulbecco改良的Eagle培养液培养的血管平滑肌细胞。应用细胞刮伤实验和基质金属蛋白酶活性测定分析西洛他唑对原代培养血管平滑肌细胞迁移能力的影响。应用蛋白质印记杂交方法分析给药前后血管平滑肌细胞内基质金属蛋白酶2,9和细胞外信号调节激酶蛋白的表达情况。结果:①细胞刮伤实验显示,与对照组比较,西洛他唑组血管平滑肌细胞迁移能力明显受抑,迁移距离明显缩短。明胶酶活性分析发现,西洛他唑组细胞基质金属蛋白酶活性明显低于对照组。②蛋白质印迹杂交分析发现,西洛他唑可引起原代培养血管平滑肌细胞迁移能力下降。西洛他唑组细胞内基质金属蛋白酶2,9蛋白表达明显低于对照组(14.34±0.70,12.65±0.98;93.67±1.90,83.67±1.05,t=67.86,85.65,P<0.05)。③蛋白质印迹杂交分析检测显示,西洛他唑组与对照组血管平滑肌细胞内信号调节激酶1,2蛋白表达无明显差异(P>0.05);细胞内磷酸化信号调节激酶蛋白表     

白藜芦醇对自发性高血压大鼠血管平滑肌细胞增殖、迁移的影响及机制研究

【目的】探讨白藜芦醇（resveratrol,Res）对自发性高血压大鼠（SHR）血管平滑肌细胞（VSMCs）增殖及迁移的影响及其机制。【方法】分别培养SHR及其对照鼠（WKY）VSMCs,对SHR组VSMCs进行不同浓度Res药物处理。噻唑蓝（MTT）检测各组细胞增殖能力差异,细胞划痕检测细胞迁移能力变化,流式细胞术检测各组细胞周期情况,ELISA检测细胞上清液中白介素‐6（IL‐6）分泌情况,Real‐timePCR检测细胞中miR‐21及IL‐6mRNA表达水平,WesternBlot检测细胞信号传导与转录激活因子（STAT3）及磷酸化STAT3（p‐STAT3）表达情况。【结果】与WKY组相比,SHR组大鼠VSMCs增殖及迁移能力显著升高,细胞周期检测结果提示细胞G1期比例降低、S期及G2／M期比例升高,细胞上清液中IL‐6分泌增强,miR‐21及IL‐6mRNA表达上升,STAT3磷酸化程度增强（P＜0．05）;经过Res干预后,VSMCs细胞增殖及迁移能力减弱,细胞周期阻滞在G1期,细胞上清液中IL‐6分泌减弱,miR‐21及IL‐6mRNA表达降低,STAT3磷酸化程度减弱,并且呈一定浓度依赖关系（P＜0．05）。【结论】Res可能通过抑制IL‐6／STAT3／miR‐21途径影响自发性高血压大鼠VSMCs增殖及迁移。     

卡维地洛抑制大鼠心脏成纤维细胞增殖及胶原合成的实验研究

【目的】探讨卡维地洛对的实验性大鼠（Wistar）心脏成纤维细胞（CFs）增殖及胶原合成的影响。【方法】以12周龄Wistar健康大鼠为研究对象。采用胰酶消化法培养CFs,采用改良的MTT比色法观察CFs的增殖状况,采用3H-胸腺嘧啶核苷（3H-TdR）掺入法测定CFs的DNA合成功能,3H-脯氨酸（3H-Proline）掺入法测定胶原合成。【结果]CFsMTT吸光值随卡维地洛浓度增加和作用时间延长而减低,并呈浓度和时间依赖性;0．01～10．00μmol／L卡维地洛分别干预72h时,Wistar大鼠CFs 3H-TdR及3H-Proline掺入率均较空白对照组明显减低（分别P〈0．05,P〈0．01）。随着卡维地洛干预浓度的增加,其抑制作用逐渐增强,呈浓度依赖性。【结论】卡维地洛可显著抑制CFs的DNA合成及细胞增殖,并进一步抑制胶原沉积及逆转左室肥厚。     

卡维地洛对自发性高血压大鼠心脏成纤维细胞增殖的影响

【目的】研究卡维地洛对自发性高血压大鼠(SHR)和实验性大鼠(Wistar)心脏成纤维细胞(CFs)一氧化氮(NO)含量的影响,探讨卡维地洛抑制CFs增殖的作用机制。【方法】以12周龄SHR10只为对象,培养CFs,分为空白对照组和卡维地洛干预组;另以Wistar健康大鼠10只的CFs为正常对照组。采用消化法培养CFs,用硝酸还原酶法检测不同浓度卡维地洛干预下CFs培养液中的NO含量,用改良的MTT比色法观察CFs的增殖状况。【结果】①在基础状态下培养72h,SHR空白对照组的NO含量明显低于正常大鼠空白对照组(P<0.01);SHR空白对照组的OD值明显高于正常大鼠空白对照组(P<0.05)。②卡维地洛以浓度依赖的方式促进CFs的NO含量增加。③卡维地洛以浓度依赖的方式抑制CFs的OD值。④在不同浓度卡维地洛干预下,SHRCFs的OD值随NO含量的增高而减低,二者呈显著负相关(r=-0.949,P<0.01)。【结论】卡维地洛能促进CFs的NO合成,直接影响了CFs的增殖,进一步抑制高血压的左室重构。     

西洛他唑对缺血性卒中进行二级预防探讨

目的：探讨西洛他唑对缺血性卒中进行二级预防的安全性和有效性。方法：选取缺血性卒中患者80例,随机分为肠溶阿司匹林组和西洛他唑组,在常规治疗的基础上,肠溶阿司匹林组给予肠溶阿司匹林100mg,1次/d,西洛他唑组给予西洛他唑50 m g,2次/d,用药1 a后进行随访。结果：西洛他唑组卒中复发2例,有效率为95%;肠溶阿司匹林组卒中复发8例,有效率为80%。两组差异有统计学意义。结论：与肠溶阿司匹林相比,西洛他唑可能更为有效。     

潘托拉唑对大鼠胃黏膜保护作用的研究

目的：探讨潘托拉唑对大鼠胃黏膜保护作用.方法：在乙醇诱导大鼠胃黏膜损伤前,预先给予潘托拉唑、L-硝基-精氨酸甲酯（L-NAME）静脉注射.测定胃黏膜血流量（GMBF）、胃液pH和胃黏膜NO2-/NO3-含量,并观察了胃黏膜损伤指数（Ulcer index,UI）、溃疡坏死组织和中性粒细胞浸润严重程度的变化.结果：与模型损伤组比,潘托拉唑组大鼠UI明显降低（P〈0.01）,溃疡坏死组织和中性粒细胞浸润程度明显减轻（P〈0.01）.预先用L-NAME处理后,潘托拉唑保护胃黏膜损伤作用明显减弱.静脉注射潘托拉唑,可增加胃黏膜血流和胃黏膜NO2-/NO3-,L-NAME可逆转这种作用,但对潘托拉唑抑制酸分泌作用无明显影响.结论：潘托拉唑对大鼠胃黏膜具有重要的保护作用,一氧化氮（Nitric oxide,NO）介导了这种作用.     

大鼠血管平滑肌细胞迁移与西洛他唑的干预效应

目的：探讨西洛他唑对原代培养大鼠血管平滑肌细胞迁移能力的影响及其可能的调控作用。方法：实验于2003—03／12在解放军沈阳军区总医院全军心血管病研究所完成。使用胶原酶消化法处理健康雄性SD大鼠，获得原代培养的鼠血管平滑肌细胞，加入终浓度为0．5μmol／L西洛他唑培养72h为西洛他唑组，对照组为加入等量的Dulbecco改良的Eagle培养液培养的血管平滑肌细胞。应用细胞刮伤实验和基质金属蛋白酶活性测定分析西洛他唑对原代培养血管平滑肌细胞迁移能力的影响。应用蛋白质印记杂交方法分析给药前后血管平滑肌细胞内基质金属蛋白酶2，9和细胞外信号调节激酶蛋白的表达情况。结果：①细胞刮伤实验显示，与对照组比较，西洛他唑组血管平滑肌细胞迁移能力明显受抑，迁移距离明显缩短。明胶酶活性分析发现，西洛他唑组细胞基质金属蛋白酶活性明显低于对照组。②蛋白质印迹杂交分析发现，西洛他唑可引起原代培养血管平滑肌细胞迁移能力下降。西洛他唑组细胞内基质金属蛋白酶2，9蛋白表达明显低于对照组(14，34&;#177;0．70，12．65&;#177;0，98；93，67&;#177;1．90，83．67&;#177;1．05．1=67．86，85，65，P&;lt;0．05)。③蛋白质印迹杂交分析检测显示，西洛他唑组与对照组血管平滑肌细胞内信号调节激酶1，2蛋白表达无明显差异(P&;gt;0．05)；细胞内磷酸化信号调节激酶蛋白表达明显低于对照组(20．44&;#177;0．76，83．67&;#177;1．28，t=73，67，P&;lt;0．05)。结论：西洛他唑可以有效抑制血管平滑肌细胞迁移，这种抑制作用可能与其对细胞内信号调节激酶活性的影响有关。     

gax基因与血管平滑肌细胞增殖

gax基因是一个主要存在于心血管系统的同源盒基因,能显著抑制血管平滑肌细胞增殖,有望成为防治动脉粥样硬化和经皮血管介入治疗后再狭窄的靶基因。     

血管平滑肌细胞增殖与转化生长因子β/smads的表达

目的:观察增殖和分化两种血管平滑肌细胞表型改变与转化生长因子β/smads信号传递的关系。方法:实验于2003-09/10哈尔滨医科大学医学遗传学研究室完成。取体质量200g左右的健康雄性SD大鼠,常规胶原酶消化法原代培养大鼠血管平滑肌细胞。用体积分数为0.2和0.05(去血清培养)胎牛血清和的Dulbecco改良的Eagle培养基,37℃,体积分数0.05CO2进行培养,2.5g/L胰酶消化传代。血管平滑肌细胞经锥虫蓝染色,存活细胞数在98%以上。形态学上出现典型的“峰和谷”样生长状态,抗平滑肌特异α-肌动蛋白免疫组织化学染色阳性。取3～8代细胞用于实验。流式细胞分析检测不同表型血管平滑肌细胞增殖能力,蛋白质印迹分析方法检测血管平滑肌细胞分化相关基因平滑肌特异性肌动蛋白表达变化,反转录聚合酶链反应和细胞免疫荧光方法检测转化生长因子βI型受体,smad2,smad3,smad4和smad7的表达变化。结果:①流式细胞分析及蛋白印迹分析方法检测发现,去血清培养后,原代培养大鼠血管平滑肌细胞增殖能力下降;DNA合成前期细胞明显增加犤(52.42±2.35)%犦,细胞分化基因平滑肌特异性α-肌动蛋白明显表达。体积分数为0.2胎牛血清培养后,大鼠血管平滑肌细胞增殖旺盛,DNA合成前期细胞数相对较少犤(17.23±1.58)%犦,细胞分化相关基因平滑肌特异性肌动蛋白表达明显下调。②反转录聚合酶链反应和细胞免疫荧光方法检测发现,去血清培养后的分化表型平滑肌细胞中,转化生长因子βI型受体表达增加,smad2和smad3表达无明显变化,smad4表达增加,smad7表达下降。而高血清培养诱导平滑肌细胞转化为增殖表型后,转化生长因子βI型受体表达下降,smad4表达下降,抑制型smad7表达增加,smad2和smad3表达无明显变化。结论:转化生长因子β/smads表达与血管平滑肌细胞表型状态密切相关。血管平滑肌细胞分化时,可能通过转化生长因子βI型受体/smad4调控细胞分化功能;而血管平滑肌细胞增殖时,细胞则可能通过smad7信号传导通路发挥作用。提示转化生长因子β/smads在血管平滑肌细胞由合成表型逆转为分化表型的分子调控中发挥作用。     

3H-thymidine渗入法测定Urocortin对大鼠血管平滑肌细胞增殖的影响

目的探讨Urocortin(UCN)对大鼠血管平滑肌细胞增殖的影响。方法分离、培养大鼠血管平滑肌细胞,分别加入不同浓度的UCN以及UCN+Astressin,UCN+Glybenclamide培养,采用3H-thymidine掺入法,观察不同浓度UCN对血管平滑肌细胞增殖的影响,及其相关作用机制。结果随着Urocortin浓度的增高,VSMC对3H-thymidine摄取率呈下降趋势,而该作用不受Astressin或Glybenclamide的影响。结论Urocortin具有抑制大鼠血管平滑肌细胞增殖的作用,其作用不是通过CRF受体或KATP通道。     

15脱氧前列腺素J2对同型半胱氨酸诱导大鼠血管平滑肌细胞增殖的抑制作用

目的：探讨15脱氧前列腺素J2（15d-PGJ2）对同型半胱氨酸（Hcy）诱导大鼠血管平滑肌细胞（VSMCs）增殖的抑制作用和对细胞外信号调节蛋白激酶（ERK1/2）的作用。方法：用不同浓度的Hcy和15d-PGh对大鼠VSMCs进行刺激，以^3H-TdR掺入方法观察15d．PGJ2对Hcy诱导大鼠VSMCs增殖的抑制作用，以Western印迹方法分析ERK1/2蛋白表达和ERK1/2磷酸化水平。结果：15d-PGh可明显抑制Hcy诱导的大鼠VSMCs增殖，但对Hcy诱导的ERK1/2磷酸化无明显作用。结论：15d-PGJ2可能通过MAPK／ERK1/2信号途径的下游步骤抑制Hcy诱导的大鼠VSMCs增殖。     

Pressure promotes DNA synthesis in rat cultured vascular smooth muscle cells.

High blood pressure is one of the major risk factors for atherosclerosis. In this study, we examined the effects of pressure on cell proliferation and DNA synthesis in cultured rat vascular smooth muscle cells. Pressure without shear stress and stretch promotes cell proliferation and DNA synthesis in a pressure-dependent manner. Pressure-induced DNA synthesis was inhibited significantly by the phospholipase C (PLC) inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate, the protein kinase C inhibitor H-7, 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine, staurosporine, and the tyrosine kinase inhibitor ([3,4,5-trihydroxyphenyl]methylene)propanedinitrile. To clarify whether activation of PLC and calcium mobilization are involved in pressure-induced DNA synthesis, production of 1,4,5-inositol trisphosphate (IP3) and intracellular Ca2+ was measured. Pure pressure increased IP3 and intracellular Ca2+ in a pressure-dependent manner. The increases in both IP3 and intracellular Ca2+ were inhibited significantly by 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate. This study demonstrates a novel cellular mechanism whereby pressure regulates DNA synthesis in vascular smooth muscle cells, possibly via activation of PLC and protein kinase C.     

Cyclic GMP accumulation by atriopeptins in cultured rat renal and vascular smooth muscle cells

To evaluate the role of cyclic guanosine monophosphate (cGMP) in the vascular and renal action of atrial natriuretic peptide (ANP), we compared the effects of atriopeptins (APs) on cGMP accumulation in cultured cells from rat mesenteric vascular smooth muscle (VSM), glomerular mesangium (GM) and renal papillary collecting tubule (RPCT), and also evaluated the relationship between renal sodium or water excretion and urinary cGMP in AP-infused rats. Both AP I and AP III increased intracellular cGMP levels dose-dependently in all types of cells, while they did not affect intracellular cAMP levels or prostaglandin synthesis. AP III was 100 times more potent than AP I. The magnitude change in cGMP levels was largest in GM cells. The sensitivity of VSM and GM cells to AP III were greater than that of RPCT cells. There were significant positive relationships between urinary excretion of sodium or water and that of cGMP levels in AP-infused rats. These results may suggest that GM and VSM cells are the principal targets for ANP to stimulate cGMP synthesis and, possibly, to exert the renal sodium and water excretion, and also support the hypothesis that cGMP mediates the cellular action of ANP.     

Insulin attenuates agonist-mediated calcium mobilization in cultured rat vascular smooth muscle cells.

Insulin has been shown to attenuate pressor-induced vascular contraction, but the mechanism for this vasodilatory action is unknown. This study examines the effect of insulin on angiotensin II (ANG II)-induced increments in cytosolic calcium in cultured rat vascular smooth muscle cells (VSMC). 20-min incubations with insulin (10 microU/ml to 100 mU/ml) did not alter basal intracellular calcium concentration ([Ca2+]i), but inhibited the response to 100 nM ANG II in a dose-dependent manner (ANG II alone, 721 +/- 54 vs. ANG II + 100 mU/ml insulin, 315 +/- 35 nM, P < 0.01). A similar effect of insulin on ANG II action was observed in calcium poor buffer. Moreover, insulin did not effect calcium influx. ANG II receptor density and affinity were not affected by 24-h incubation with insulin. To further clarify the mechanisms of these observations, we measured ANG II-induced production of inositol 1,4,5-triphosphate (IP3), and IP3-releasable 45Ca. Insulin treatment did not alter ANG II-stimulated IP3 production. However, IP3-stimulated release of 45Ca in digitonin permeabilized cells was significantly reduced after 5-min incubations with 100 mU/ml insulin. Thapsigargin induced release of calcium stores was also blocked by insulin. Thus, insulin attenuates ANG II-stimulated [Ca2+]i primarily by altering IP3-releasable calcium stores. Insulin effects on ANG II-induced [Ca2+]i were mimicked by preincubation of VSMC with either sodium nitroprusside or 8-bromo-cGMP. As elevations in cGMP in vascular tissue lower [Ca2+]i, it is possible that insulin affects IP3 release of calcium by a cGMP-dependent mechanism that would contribute to its vasodilatory effects.     

Angiotensin II modulates frizzled-2 receptor expression in rat vascular smooth muscle cells

Ang II (angiotensin II) has multiple effects on vascular smooth muscle cells through the modulation of different classes of genes. Using the mRNA differential-display method to investigate gene expression in rat aortic smooth muscle cells in culture in response to 3 h of Ang II stimulation, we observed that Ang II down-regulated the expression of a member of the family of transmembrane receptors for Wnt proteins that was identified as Fzd2 [Fzd (frizzled)-2 receptor]. Fzds are a class of highly conserved genes playing a fundamental role in the developmental processes. In vitro, time course experiments demonstrated that Ang II induced a significant increase (P<0.05) in Fzd2 expression after 30 min, whereas it caused a significant decrease (P<0.05) in Fzd2 expression at 3 h. A similar rapid up-regulation after Ang II stimulation for 30 min was evident for TGFbeta1 (transforming growth factor beta1; P<0.05). To investigate whether Ang II also modulated Fzd2 expression in vivo, exogenous Ang II was administered to Sprague-Dawley rats (200 ng.kg(-1) of body weight.min(-1); subcutaneously) for 1 and 4 weeks. Control rats received normal saline. After treatment, systolic blood pressure was significantly higher (P<0.01), whereas plasma renin activity was suppressed (P<0.01) in Ang II- compared with the saline-treated rats. Ang II administration for 1 week did not modify Fzd2 expression in aorta of Ang II-treated rats, whereas Ang II administration for 4 weeks increased Fzd2 mRNA expression (P<0.05) in the tunica media of the aorta, resulting in a positive immunostaining for fibronectin at this time point. In conclusion, our data demonstrate that Ang II modulates Fzd2 expression in aortic smooth muscle cells both in vitro and in vivo.     

Inhibition of rat vascular smooth muscle proliferation in vitro and in vivo by bone morphogenetic protein-2.

Vascular proliferative disorders are characterized by the proliferation of vascular smooth muscle cells (SMCs) and excessive extracellular matrix synthesis. We found that bone morphogenetic protein-2 (BMP-2) inhibited serum-stimulated increases in DNA synthesis and cell number of cultured rat arterial SMCs in a fashion quite different from that in the case of transforming growth factor-beta1 (TGF-beta1). In addition, TGF-beta1 stimulated collagen synthesis in SMCs, whereas BMP-2 did not. In an in vivo rat carotid artery balloon injury model, the adenovirus-mediated transfer of the BMP-2 gene inhibited injury-induced intimal hyperplasia. These results indicate that BMP-2 has the ability to inhibit SMC proliferation without stimulating extracellular matrix synthesis, and suggest the possibility of therapeutic application of BMP-2 for the prevention of vascular proliferative disorders.