NO诱导血管平滑肌细胞凋亡中Ca^2＋的作用

探讨了NO诱导血管平滑肌细胞凋亡与细胞内游离Ca^2 之间的关系,通过粘附式细胞仪和Ca^2 荧光探针Fluo-3/AM,检测分析了NO在供体SNAP的作用下,血管平滑肌细胞中游离Ca^2 浓度的变化;又通过SNAP与维拉帕米、EGTA、肝素钠、普鲁卡因共同孵育的方法,测了Ca^2 浓度变化在细胞凋亡中的作用,得出SNAP能使细胞中游离Ca^2 浓度升高,而胞外Ca^2 内流在其中起主要作用;并且阻断胞外Ca^2 内流能够抑制SNAP所诱导的血管平滑肌细胞的凋亡,提示了胞内Ca^2 浓度升高可能是SNAP诱导血管平滑机细胞凋亡的一条途径。     

NO诱导血管平滑肌细胞凋亡中Ca~(2 )的作用

探讨了ＮＯ诱导血管平滑肌细胞凋亡与细胞内游离Ｃａ２＋之间的关系．通过粘附式细胞仪和Ｃａ２＋ 荧光探针Ｆｌｕｏ－３／ＡＭ,检测分析了ＮＯ在供体ＳＮＡＰ的作用下,血管平滑肌细胞中游离Ｃａ２＋浓度的变化; 又通过ＳＮＡＰ与维拉帕米、ＥＧＴＡ、肝素钠、普鲁卡因共同孵育的方法,观测了Ｃａ２＋浓度变化在细胞凋亡中 的作用．得出ＳＮＡＰ能使细胞中游离Ｃａ２＋浓度升高,而胞外Ｃａ２＋内流在其中起主要作用;并且阻断胞外 Ｃａ２＋内流能够抑制ＳＮＡＰ所诱导的血管平滑肌细胞的凋亡．提示了胞内Ｃａ２＋浓度升高可能是ＳＮＡＰ诱导血管 平滑肌细胞凋亡的一条途径．     

血管内皮细胞与平滑肌细胞间的信息传递

血管内皮细胞和血管平滑肌细胞之间存在着肌内皮间缝隙连接，进行电和化学的信息传递，以协调血管的舒缩活动。电信号可以从内皮细胞到平滑肌细胞进行传递，相反，也可以从平滑肌细胞到内皮细胞进行传递。内皮源性超极化因子、乙酰胆碱、缓激肽、第二信使等物质亦可引起内皮细胞或，和平滑肌细胞细胞膜的超极化或去极化，参与血管内皮细胞与平滑肌细胞间的信息传递。     

雌二醇对血管平滑肌细胞增殖的影响

1材料和方法1．1体外培养小牛胸主动脉SMC[4]1．2SMC48小时增殖率的测定[5]加药情况如下：0．1ng／mlE2组,lng／mlE2组,10ng／mlE2组,100ng／mlE2组,E2溶剂（DMSO,终浓度＜0．05％）对照和培养液对照;每组两种血清浓度2％,15％,各8孔,继续培养48小时;MTT法检测增殖率。1．3SMC集落形成率的测定[6]分组：0．1ng／mlE2,1ng／mlE2,10ng／mlE2,培养液对照和溶剂对照,12天后计算集落形成率。2结果2．1体外培养SMC形态特征贴决后3－4天SMC自组织块边缘萌出（图版a,b）,原代细胞形状多样,有星形、带状三角形、梭形等…     

心脉隆胶囊对原代大鼠血管平滑肌细胞形态损伤的保护作用

目的:探讨大鼠主动脉血管平滑肌细胞(VSMCs)的原代培养方法及观察心脉隆胶囊(XMLJ)对由H2O2诱导VSMCs细胞形态损伤的保护作用。方法:采用组织贴块法培养原代大鼠VSMCs,继而用62.5μmol.L-1H2O2损伤细胞,通过HE染色观察XMLJ对VSMCs形态损伤的保护作用。结果:200μg.mL-1的XMLJ对过氧应激造成的VSMCs形态损伤,具有明显的保护作用,细胞形态接近于正常组VSMCs。结论:组织贴块法培养大鼠VSMCs方便简单,XMLJ对H2O2诱导的VSMCs细胞形态损伤具有一定的保护作用。     

缝隙连接阻断剂18α-甘草次酸对大鼠血管平滑肌细胞增殖的作用

为探讨缝隙连接是否参与抑制大鼠血管平滑肌细胞的增殖及可能的分子机制,本研究以原代及传代培养大鼠胸主动脉平滑肌细胞为模型,实验分2组：对照组、缝隙连接阻断剂18α-甘草次酸（18α-—Glycyrrhetinicacid,18Q—GA）组。MTT法及流式细胞术检测细胞的增殖活性,染料示踪分子传递法（划痕标记染料传输法）检测细胞的缝隙连接功能,Westernblotting法检测细胞中缝隙连接蛋白40（Cx40）和43（Cx43）表达。结果显示：（1）与对照组相比,18α-GA组MTT法测得的氏,。值及细胞周期S期比例均降低（P〈0．01）,细胞增殖活性减弱;（2）与对照组相比,18α-GA组罗氏黄荧光染料传递百分数显著降低,缝隙连接功能明显减弱（P〈0．01）;（3）与对照组相比,18rGA组Cx40和总Cx43蛋白表达无显著差异（P〉0．05）,磷酸化Cx43与非磷酸化Cx43的比值显著降低（P〈0．01）。由此可知,18α-GA可能主要通过下调血管平滑肌细胞磷酸化Cx43蛋白表达,引起缝隙连接通讯功能减弱,从而抑制了血管平滑肌细胞的增殖。     

水溶性壳聚糖对血管平滑肌细胞增殖的影响

通过化学方法测定了水溶性壳聚糖(water-solubility chitosan,WSC)的部分理化性质;通过原代分离培养大鼠腹主动脉平滑肌细胞和MTT实验,研究了1,10,100和1000μg/mL的WSC对血管平滑肌细胞的增殖作用.实验结果表明,WSC溶解度为125,含水量13.19%,脱乙酰度54.73%,重均相对分子质量1.17×105.在24h和48h时,大于10μg/mL的WSC可抑制原代培养的血管平滑肌细胞的增殖.其中,100和1000μg/mL的WSC在48h时对细胞的增殖的抑制作用最明显,表明在一定条件下,WSC可抑制血管平滑肌细胞增殖,减轻由血管平滑肌细胞增殖所引起的血管狭窄问题.     

兔血管平滑肌细胞和聚羟基丁酯（PHB）的细胞相容性实验研究

探索血管平滑肌细胞和新型可降解材料聚羟基西酯（PHB）的细胞相容性,为组织人工血管的构建寻找理想的支架材料。将组织法体外培养的兔血管平滑肌细胞种植在PHB膜片和PHB三维微孔支架上,在相差显微镜下观察细胞的粘附和生长情况,用MTT法测定细胞粘附率和细胞增殖指数,复合培养7d后进行扫描电镜观察并用流式细胞仪（FCM）测定细胞周期、DNA指数。结果兔血管平滑肌细胞在PHB膜片上粘附率为77％,细胞增殖符合细胞的生长曲线,在PHB三维微孔支架上生长情况良好,并被证实为二倍体细胞。结论是兔血管平滑肌细胞和聚羟基西酯（PHB）的细胞相容性较好,但细胞与材料间的粘附有待进一步改善。     

QKI对血管平滑肌细胞增殖的抑制功能

观察QKI蛋白对血管平滑肌细胞(VSMCs)增殖的影响,进一步完善血管平滑肌细胞增殖的相关机制.以大鼠的原代主动脉平滑肌细胞为研究对象,观察分别过表达QKI5、QKI6对血清促增殖作用的影响.细胞增殖采用MTT法和Western blot测定.结果血清可促进大鼠的原代主动脉平滑肌细胞增殖,过表达QKI可抑制血清刺激的血管平滑肌细胞的增殖.说明过表达QKI对血清刺激的血管平滑肌细胞增殖具有抑制作用,该作用有可能在一定程度上能够抑制高血压等病伴发的血管平滑肌增殖.     

高血压大鼠血管平滑肌细胞GRP94和CHOP的表达及替米沙坦的影响

观察替米沙坦对高血压大鼠动脉血管平滑肌细胞内质网应激相关因子葡萄糖调节蛋白94(GRP94)和C/EBP同源蛋白(CHOP)的表达,以及血管中层厚度的变化的影响。将48只成年雄性SD大鼠随机等分为假手术对照组(对照组)、手术模型组和替米沙坦组。对照组分离腹主动脉,但不行狭窄术;模型组行腹主动脉狭窄术(TAC);替米沙坦组行腹主动脉狭窄术并给予每天替米沙坦1.5 mg/kg。喂养6周用颈动脉插管法测定各组大鼠的动脉压(MAP);利用图像分析软件测量血管中层的厚度。用免疫组织化学法和western-blot法检测GRP94和CHOP的表达水平。结果:①手术模型组大鼠的MAP显著高于相应的对照组和替米沙坦组,有统计学意义(P<0.05)。②手术模型组的GRP94表达量高于对照组和替米沙坦组,有统计学意义(P<0.05)。③手术模型组的CHOP蛋白表达量高于对照组和替米沙坦组,有统计学意义(P<0.05)。④手术模型组大鼠血管平滑肌中层厚度厚于对照组和替米沙坦组,有统计学意义(P<0.05)。说明腹主动脉狭窄致高血压可引起血管平滑肌细胞内质网应激反应,导致GRP94表达及CHOP表达的增加。替米沙坦可能通过减轻内质网应激,逆转高血压所致的血管平滑肌细胞的损伤作用,对血管平滑肌细胞有保护作用。     

西洋参叶二醇组皂苷抑制血管平滑肌细胞增殖及影响原癌基因表达的研究

观察PQDS对VSMC增殖及原癌基因c-myc的mRNA表达的影响,探讨PQDS抑制VSMC的增殖作用及相关机制。方法用组织贴块法培养大鼠胸主动脉VSMC作为研究对象,用AngⅡ诱导VSMC增殖,随机分为：空白对照组、AngⅡ模型组、AngⅡ＋PQDS 25、50、100mg/L不同剂量组。用MTT法、流式细胞术、RT-PCR法观察PQDS对细胞增殖、细胞周期、增殖指数及原癌基因c-myc的mRNA表达的影响。结果PQDS可减低VSMC的增殖能力,将增殖的VSMC抑制在G0/G1期,可以下调VSMC中的原癌基因c-myc的mRNA表达。结论PQDS能抑制VSMC增殖,其机制可能与下调原癌基因c-myc的表达有关。     

清热解毒药对血管平滑肌细胞增殖、细胞周期的影响

目的 :观察清热解毒药双花、公英、虎杖、连翘对血管平滑肌细胞生长、贴壁和增殖抑制作用 ,并从对细胞周期的影响角度分析它们的作用机制。方法 :采用细胞计数、MTT法和流式细胞技术。结果 :双花、公英、虎杖、连翘对h PDGF B/B刺激下的平滑肌细胞生长和贴壁均有抑制作用。对平滑肌细胞增殖抑制率分别达 9.2 6%～ 77.35% ,7.78%～ 62 .65% ,5.88%～ 47.81 % ,3.98%～ 33.35% ,其增殖抑制作用呈量效时效关系。通过对细胞周期分析知道 ,双花、公英、虎杖均可以减少 S期 ,增加 G0 /G1期细胞数目。结论 :清热解毒药双花、公英、虎杖、连翘对平滑肌细胞生长及增殖有抑制作用 ,其作用机制可能是通过抑制 DNA合成 ,减少进入 S期细胞数目 ,使细胞增殖停留在 G0 /G1期来实现的     

The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine

Despite its very potent vasodilating action in vivo, acetylcholine (ACh) does not always produce relaxation of isolated preparations of blood vessels in vitro. For example, in the helical strip of the rabbit descending thoracic aorta, the only reported response to ACh has been graded contractions, occurring at concentrations above 0.1 muM and mediated by muscarinic receptors. Recently, we observed that in a ring preparation from the rabbit thoracic aorta, ACh produced marked relaxation at concentrations lower than those required to produce contraction (confirming an earlier report by Jelliffe). In investigating this apparent discrepancy, we discovered that the loss of relaxation of ACh in the case of the strip was the result of unintentional rubbing of its intimal surface against foreign surfaces during its preparation. If care was taken to avoid rubbing of the intimal surface during preparation, the tissue, whether ring, transverse strip or helical strip, always exhibited relaxation to ACh, and the possibility was considered that rubbing of the intimal surface had removed endothelial cells. We demonstrate here that relaxation of isolated preparations of rabbit thoracic aorta and other blood vessels by ACh requires the presence of endothelial cells, and that ACh, acting on muscarinic receptors of these cells, stimulates release of a substance(s) that causes relaxation of the vascular smooth muscle. We propose that this may be one of the principal mechanisms for ACh-induced vasodilation in vivo. Preliminary reports on some aspects of the work have been reported elsewhere.     

Action potential refractory period in ureter smooth muscle is set by Ca sparks and BK channels

In excitable tissues the refractory period is a critical control mechanism preventing hyperactivity and undesirable tetani, by preventing subsequent stimuli eliciting action potentials and Ca2+ entry. In ureteric smooth muscle, peristaltic waves that occur as invading pacemaker potentials produce long-lasting action potentials (300-800 ms) and extraordinarily long (more than 10 s) refractory periods, which prevent urine reflux and kidney damage. For smooth muscles neither the mechanisms underlying the refractory period nor the link between excitability and refractoriness are properly understood. Here we show that a negative feedback process, which depends on Ca2+ loading the sarcoplasmic reticulum (SR) during the action potential and on the subsequent activation of local releases of Ca2+ from the SR (sparks), stimulating plasmalemmal Ca2+-sensitive K+ (BK) channels, determines the refractory period of the action potential. As sparks gradually reduce the Ca2+ load in the SR, electrical inhibition is released, the refractory period is terminated and peristaltic contractions occur again. The refractory period can be manipulated, for example from 10 s to 100 s, by altering the Ca2+ content of the SR or release mechanism or by inhibiting BK channels. This insight into the control of excitability and hence function provides a focus for therapies directed at pathologies of smooth muscle.     

Urotensin Ⅱ increases endothelin production by vascular smooth muscle cells in rats

The aim of this study was to observe the effects of urotensin Ⅱ (UII) on production of endothelin (ET) in rat aortic vascular smooth muscle cells (VSMC). Cultured VSMCs incubated with various concentrations of UII were used to measure the VSMC 3H-TdR incorporation, the amount of ET mRNA and ET production in VSMCs. In this work we found that UII (10-10-10-8 mol/L) promoted VSMC 3H-TdR incorporation (47%-83%, P < 0.01) and increased the amount of ET mRNA by 17.1% (P < 0.05) to 112.8% (P < 0.01), respectively, in a concentration dependent manner compared with control. After 4 and 8 h incubation, 10-10-10-8 mol/L of UII elevated the ET synthesis and release in a concentration dependent manner. After 4 h incubation, the content of ET in medium was 4.9, 5.36 and 7.12 pg/mL (P < 0.01). After 8 h incubation, the ET content released from VSMCs was 12.6, 12.07 and 17.17 pg/mL (P < 0.01). In addition, it was found that BQ123, a specific ETA receptor antagonist, obviously decreased the VSMC DNA synthesis induced by UII. The results of this study showed that UII could stimulate the ET mRNA expression and ET production in VSMC. The effects of UII on VSMC DNA synthesis were partly mediated by ET autocrine pathway. It suggests that the interaction between UII and ET plays an important biological regulating role as endogenous active peptides.     

Inositol 1,4,5-trisphosphate activates a channel from smooth muscle sarcoplasmic reticulum

Inositol 1,4,5-trisphosphate (InsP3) can initiate calcium release into the cytoplasm in a variety of cells. From experiments using permeabilized cells, membrane vesicles, and patch-clamp techniques, it has been suggested that InsP3 acts by directly opening calcium channels. Here, we show that InsP3 induced openings of channels in planar lipid bilayers into which vesicles made from aortic muscle sarcoplasmic reticulum (SR) were incorporated. Activation of channels by InsP3 was not observed when vesicles made from SR of cardiac or skeletal muscle were incorporated into planar lipid bilayers. The present study demonstrates for the first time unique properties of an InsP3-gated calcium channel in sarcoplasmic reticulum vesicles from vascular smooth muscle. This InsP3-activated channel from aortic SR differs strikingly from the calcium-gated calcium channel of striated muscle SR in single-channel conductance and pharmacology.     

Regulation of angiotensinⅡon Gaq/11 protein of vascular smooth muscle cell and its underlying mechanism

To study the regulation of angiotensin Ⅱ(Ang Ⅱ) on Gαq/11 protein of vascular smooth muscle cell (VSMC) and its underlying mechanism, the protein synthesis was detected by [3H]-leucine incorporation. G?q/11 expression was measured by Western blot in cultured VSMC of rat aorta. The results showed that the level of G?q/11 was downregulated after stimulated by AngⅡ for 1-6 h, while it was upregulated significantly by 12-24 h stimulation (P < 0.01) in VSMC. The [3H]-leucine incorporation of VSMC was increased after 24 h Ang Ⅱ stimulation. The biphase regulation of Ang Ⅱ on G?q/11 protein was blocked by the Ang Ⅱ type Ⅰ receptor (AT1) specific antagnist losartan or PLC inhibitor U73122, while PD98059 did not have this effect. These data indicated that Ang Ⅱ contributed to VSMC hypertrophy by regulating the level of G?q/11, and this effect was mediated mainly through AT1 receptor-PLC signal transduction pathway.