霉酚酸对内皮素-1诱导的肺动脉平滑肌细胞增殖迁移收缩的影响

目的 研究霉酚酸对内皮素-1诱导的大鼠肺动脉平滑肌细胞(PASMCs)的增殖、收缩和迁移的影响,探讨霉酚酸酯对肺动脉高压的作用机制.方法 采用四甲基偶氮唑蓝(MTT)、划痕实验、Millicell培养小室及测微尺计量的方法观察霉酚酸对内皮素-1诱导PASMCs增殖、迁移和收缩的影响,并了解鸟嘌呤对霉酚酸抑制PASMCs增殖的逆转作用.采用配对t检验进行统计分析.结果 与内皮素-1组相比,低浓度霉酚酸组平滑肌细胞增殖降低[吸光度(A)值:0.348±0.036与0.447±0.013,t=6.357,P=0.000];高浓度MPA组进一步降低.与低浓度霉酚酸组相比,霉酚酸加鸟嘌呤组平滑肌细胞增殖有所增加(A值:0.390±0.018与0.348±0.036,t=2.573,P=0.028).霉酚酸组细胞平均迁移距离较内皮素-1组缩短,霉酚酸组平均迁移细胞数较内皮素-1组减少,霉酚酸组平均细胞长度长于内皮素-1组.结论 霉酚酸具有抑制内皮素-1诱导的PASMCs增殖、迁移和收缩的作用,外源性鸟嘌呤可部分逆转霉酚酸抑制PASMCs增殖的作用.     

低氧条件下共培养体系中肺动脉内皮细胞经Notch1/Jagged1信号通路调控肺动脉平滑肌细胞的增殖

目的探讨低氧条件下细胞共培养体系中肺动脉内皮细胞经由Notch1/Jagged1信号通路调控肺动脉平滑肌细胞的增殖。方法建立Transwell共培养体系,将肺动脉内皮细胞(PAEC)和平滑肌细胞(PASMC)分别接种于下室和上室,PAEC经DAPT(Notch信号阻断剂)或(和)S iR-NA-Jagged1预处理后,与PASMC共同置入自动常压缺氧孵箱进行低氧处理。根据PAEC是否行DAPT、Jagged1小RNA干扰预处理进行实验分组:常氧时PAEC与PASMC共培养对照组(N)、低氧时PAEC与PASMC共培养组(H1)、低氧时DAPT(终浓度10μmol/L)预处理PAEC与PASMC共培养组(H2)、低氧时S iRNA-Jagged1预处理PAEC与PASMC共培养组(H3)、低氧时DAPT加S iRNA-Jagged1预处理PAEC与PASMC共培养组(H4)。用3H-TdR掺入法检测PAEC和PASMC增殖情况,RT-PCR检测PAEC中Notch1、Jagged1 mRNA表达水平。结果低氧时各组PAEC和PASMC的3H-TdR掺入量明显高于常氧共培养对照组(均P<0.01);与H1组比较,...     

Intermedin modulates hypoxic pulmonary vascular remodeling by inhibiting pulmonary artery smooth muscle cell proliferation

BackgroundHypoxic pulmonary arterial hypertension (PAH) is a disabling disease with limited treatment options. Hypoxic pulmonary vascular remodeling is a major cause of hypoxic PAH. Pharmacological agents that can inhibit the remodeling process may have great therapeutic value.ObjectiveTo examine the effect of intermedin (IMD), a new calcitonin gene-related peptide family of peptide, on hypoxic pulmonary vascular remodeling.MethodsRats were exposed to normoxia or hypoxia (∼10% O₂), or exposed to hypoxia and treated with IMD, administered by an implanted mini-osmotic pump (6.5 μg/rat/day), for 4 weeks. The effects of IMD infusion on the development of hypoxic PAH and right ventricle (RV) hypertrophy, on pulmonary vascular remodeling, on pulmonary artery smooth muscle cell (PASMC) proliferation and apoptosis, and on the activations of l-arginine nitric oxide (NO) pathway and endoplasmic reticulum stress apoptotic pathway were examined.ResultsRats exposed to hypoxia developed PAH and RV hypertrophy. IMD treatment alleviated PAH and prevented RV hypertrophy. IMD inhibited hypoxic pulmonary vascular remodeling as indicated by reduced wall thickness and increased lumen diameter of pulmonary arterioles, and decreased muscularization of distal pulmonary vasculature in hypoxia-exposed rats. IMD treatment inhibited PASMC proliferation and promoted PASMC apoptosis. IMD treatment increased tissue level of constitutive NO synthase activity and tissue NO content in lungs, and enhanced l-arginine uptake into pulmonary vascular tissues. IMD treatment increased cellular levels of glucose-regulated protein (GRP) 78 and GRP94, two major markers of endoplasmic reticulum (ER) stress, and increased caspase-12 expression, the ER stress-specific caspase, in lungs and cultured PASMCs.ConclusionsThese results demonstrate that IMD treatment attenuates hypoxic pulmonary vascular remodeling, and thereby hypoxic PAH mainly by inhibiting PASMC proliferation. Promotion of PASMC apoptosis may also contribute to the inhibitory effect of IMD. Activations l-arginine–NO pathway and of ER stress-specific apoptosis pathway could be the mechanisms mediating the anti-proliferative and pro-apoptotic effects of IMD.     

肺动脉平滑肌细胞:肺动脉高压的关键治疗靶点

肺动脉高压(PAH)是一种进展快、预后欠佳、死亡率高的心血管疾病。研究表明,肺血管重构是PAH发生发展的重要病理基础,而肺动脉平滑肌细胞的增殖和肥大是PAH肺血管重构的主要病理改变。在PAH时,肺血管平滑肌细胞由收缩表型向增殖状态的合成表型转化,主要表现为肺血管平滑肌细胞的增殖和肥大。上述病理改变最终导致肺血管管腔狭窄,管壁僵硬,进而促进PAH的发生发展。本文对肺动脉平滑肌细胞在PAH中的关键作用及作用机制进行阐述,为临床防治PAH提供新靶点和新策略。     

Antiproliferative effect of sildenafil on human pulmonary artery smooth muscle cells

Pulmonary arterial hypertension (PAH) is characterized by vasoconstriction and by obstructive changes of the pulmonary vasculature including smooth muscle cell proliferation which leads to medial hypertrophy and subsequent luminal narrowing. Sildenafil, an orally active inhibitor of cGMP phosphodiesterase–type–5, exerts pulmonary vasodilator activity in PAH patients. We evaluated the effects of sildenafil on growth of cultured human pulmonary artery smooth muscle cells (PASMC). The results indicate that sildenafil reduced DNA synthesis stimulated by PDGF and dose dependently inhibited PASMC proliferation. These effects were paralleled by a progressive increase in cGMP content, followed by an accumulation of cAMP. The treatment with 8–bromo–cGMP or dibutyryl–cAMP mimicked all the effects of sildenafil. On the other hand, treatment of PASMC with inhibitors of cGMP–dependent protein kinase (PKG) or cAMP–dependent protein kinase (PKA) reversed the antiproliferative effect of sildenafil. In addition, sildenafil inhibited the phosphorylation of ERK, a converging point for several pathways leading to cell proliferation. This effect was partially reduced by PKG inhibition and completely abolished by PKA inhibition.We conclude that sildenafil exerts an antiproliferative effect on human PASMC that is mediated by an interaction between the cGMP–PKG and the cAMP–PKA activated pathways, leading to inhibition of PDGF–mediated activation of the ERK.     

Melatonin attenuates hypoxic pulmonary hypertension by inhibiting the inflammation and the proliferation of pulmonary arterial smooth muscle cells

Hypoxia‐induced inflammation and excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) play important roles in the pathological process of hypoxic pulmonary hypertension (HPH). Melatonin possesses anti‐inflammatory and antiproliferative properties. However, the effect of melatonin on HPH remains unclear. In this study, adult Sprague–Dawley rats were exposed to intermittent chronic hypoxia for 4 wk to mimic a severe HPH condition. Hemodynamic and pulmonary pathomorphology data showed that chronic hypoxia significantly increased right ventricular systolic pressures (RVSP), weight of the right ventricle/left ventricle plus septum (RV/LV+S) ratio, and median width of pulmonary arterioles. Melatonin attenuated the elevation of RVSP, RV/LV+S, and mitigated the pulmonary vascular structure remodeling. Melatonin also suppressed the hypoxia‐induced high expression of proliferating cell nuclear antigen (PCNA), hypoxia‐inducible factor‐1α (HIF‐1α), and nuclear factor‐κB (NF‐κB). In vitro, melatonin concentration‐dependently inhibited the proliferation of PASMCs and the levels of phosphorylation of Akt and extracellular signal‐regulated kinases1/2 (ERK1/2) caused by hypoxia. These results suggested that melatonin might potentially prevent HPH via anti‐inflammatory and antiproliferative mechanisms.     

S100A4/Mts1在低氧诱导肺动脉平滑肌增殖中的作用机制

目的探讨S100A4/Mtsl在低氧诱导肺动脉平滑肌增殖中的作用机制。方法大鼠肺动脉平滑肌细胞(PASMCs)分为对低氧刺激组及AG490干预组;免疫荧光分别检测常氧组、低氧刺激组及AG490干预组RPASMCs细胞S100A4/Mtsl的表达情况,Westernblot分别检测三组细胞的S100A4/Mtsl、p-JAK2、p-STAT3蛋白表达。结果低氧0hS100A4/Mtsl在胞浆中极少表达,低氧刺激后胞浆及胞核中明显表达;S100A4/Mtsl、p-JAK2、p-STAT3蛋白在低氧刺激8h比低氧刺激0h表达明显升高(P<0.05),AG490干预低氧刺激8h组三种蛋白的表达较低氧刺激8h组显著下降(P<0.05)。结论低氧刺激下S100A4/Mtsl基因在PAMSCs增殖中发挥重要作用,并且可被AG490抑制。     

低氧致大鼠肺动脉平滑肌细胞中PTEN/Akt1表达的变化与细胞增殖的关系

目的 通过观察低氧致大鼠肺动脉平滑肌细胞(PASMC)与细胞张力蛋白同源在10号染色体有缺失的磷酸酶(PTEN)和丝氨酸/苏氨酸蛋白激酶1(Akt1)mRNA及其蛋白表达水平的变化与低氧PASMC增殖的关系,探讨PTEN/Akt1信号途径在低氧肺血管重建中的可能调控作用.方法 用组织块法培养PASMC.采用半定量逆转录-PCR技术检测常氧组、低氧2、8、12、24 h组PTEN、Akt1基因mRNA的表达水平,采用Western blot技术检测相应的蛋白表达水平.采用噻唑蓝比色法和氚-胸腺嘧啶脱氧核苷(3H-TdR)掺入法检测PASMC的增殖改变.数据用x±s表示,采用Excel 2003软件进行t检验,P<0.05为差异有统计学意义.结果低氧刺激PASMC不断增殖,3H-TdR法检测的吸光度值低氧12 h组(0.70±0.10)比常氧组(0.37±0.06)明显增高(t=14.29,P<0.01);噻唑蓝法检测的吸光度值低氧24 h组(11 208±679)比常氧组(8374±545)明显增高(t=19.56,P<0.01).各组均检测出PTEN、Akt1基因mRNA及蛋白表达的变化.常氧组Akt1的mRNA、总蛋白、磷酸化蛋白的吸光度值分别为0.76±0.09、25±6和48±8,随着低氧培养时间延长,吸光度值逐渐升高,低氧8 h组达到高峰,分别为1.05±0.09、41±7和79±14,与常氧组比较,差异均有统计学意义(t值为8.31～168.00,P<0.05和P<0.01),随后开始下降,低氧24 h组恢复至常氧组水平.常氧组PTEN的mRNA、磷酸化蛋白的吸光度值分别为0.25±0.06和98±8,并随着低氧培养时间延长而逐渐升高,低氧24 h组达到高峰,分别为0.38±0.05和232±12,与常氧组比较,差异均有统计学意义(t值分别为22.04和50.46,均P<0.01).结论 PTEN/Akt1的转录和激活与低氧肺血管重建的PASMC增殖密切相关.     

SOCS 3基因对缺氧肺动脉平滑肌细胞增殖及原癌基因mRNA表达的影响

目的探讨细胞因子信号负调控因子(SOCS)3基因对缺氧大鼠肺动脉平滑肌细胞(PASMCs)原癌基因c-fos、c-jun mRNA表达及细胞增殖的影响.方法实验分pEFSOCS3和pSV2neo共转染组(A组)、未转染组(B组)、pEF和pSV2neo共转染组(C组).以脂质体为载体将pEFSOCS3和pSV2neo 共转染至体外培养的PASMCs中,用免疫细胞化学法测转染前后细胞中SOCS3蛋白表达;用半定量RT-PCR检测转染前后常氧、缺氧2、4、6、8、12 h细胞c-fos、c-jun mRNA表达水平;流式细胞仪测转染前后上述各时相点细胞周期的变化.结果免疫细胞化学法证实转染后细胞中有SOCS3稳定表达.半定量RT-PCR结果,B、C组缺氧2 h c-fos mRNA表达水平达高峰,4 h下降, 6 h降至正常,8 h再次达高峰,12 h 下降;与B组同时相点相比,A组缺氧2、8 h c-fos mRNA表达水平低(P<0.01).B、C组缺氧2 h c-jun mRNA表达水平升高,6 h达高峰;与B组同时相点相比,A组缺氧2、4、6、8 h c-jun表达水平低(P<0.01).B、C组缺氧6 h即出现G0/G1期细胞比例减少,S+G2/M期细胞比例增加(P<0.01);与B组同时相点相比,A组G0/G1期细胞比例增多,S+G2/M期细胞比例减少(P<0.01).结论缺氧可能通过诱导PASMCs c-fos、c-jun表达而促进PASMCs增殖;SOCS3蛋白可能通过下调c-fos、c-jun基因表达从而抑制缺氧诱导的PASMCs增殖.     

肺动脉高压相关因子研究进展

肺动脉高压以肺血管阻力升高为特征,由于小肺动脉血管平滑肌细胞增殖,肺动脉血管阻力升高,导致右心室衰竭直至死亡。肺动脉高压的发生不能以单一的病理生理理论来解释,而是涉及多种分子介质、遗传因素和平滑肌细胞、内皮细胞等多种细胞异常。最近的研究表明多种因子通过不同的途径参与了肺动脉高压的发病机制,包括骨形成蛋白Ⅱ型受体、Not...     

KLF4在低氧诱导肺动脉平滑肌细胞增殖及表型转化中的作用初步研究

目的：本实验通过观察低氧诱导大鼠原代肺动脉平滑肌细胞(pulmonary artery smooth muscle cell,PASMC)增殖与分化标志改变过程中,krǖppel 样因子4(krǖppel-like factor 4, KLF4)表达的变化,并检测 KLF4过表达对该过程的影响,探讨 KLF4是否在其中担当一定作用。方法采用贴片法培养原代 PASMC,低氧(5%O 2)培养24 h、48 h、72 h 后,以正常氧浓度培养细胞为对照,采用实时荧光聚合酶链反应(real-time PCR)、Western blot 及免疫共沉淀方法,观察低氧24 h、48 h、72 h,PASMC 中 KLF4乙酰化水平及表达水平的变化。cck-8检测细胞增殖情况,碘化丙啶(PI)染色检测细胞周期,提取细胞总 RNA 和蛋白,采用 real-time PCR、Western blot 方法检测细胞中增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的表达水平,综合分析低氧对细胞增殖的影响,同时采用 real-time PCR、 Western blot 方法检测 PASMC 分化标志物平滑肌肌动蛋白α(smooth muscleα-action,SMα-actin)的表达水平。通过转染 KLF4表达质粒,提高 PASMC 中 KLF4的表达水平,检测细胞周期、增殖活力及 PCNA、SM α-actin 的变化,观察低氧下 KLF4过表达对PASMC 增殖、分化的影响。结果低氧24 h,PASMC 中 KLF4表达水平均较常氧培养细胞明显增高,期间 SMα-actin 表达较常氧培养细胞明显减少,PASMC 增殖不明显。随后 KLF4表达水平逐渐降低,至72 h 降低至正常水平,而 SMα-actin 逐渐增高,同时 PASMC 增殖活力明显增强,S 期细胞逐渐增多。KLF4过表达后,低氧刺激 PASMC 增殖活力明显降低,SMα-actin 表达水平也降低,细胞阻滞于 G1期。结论低氧诱导 PASMC 发生去分化与增殖不同步;KLF4参与调节低氧诱导的PASMC 表型转化与增殖过程,该作用涉及 KLF4乙酰化水平改变。     

肺动脉平滑肌细胞钾电流记录方法的建立

目的 在肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells,PASMCs)上建立钙离子激活钾通道(calcium-activated potassium channel,KCa)电流、电压门控钾通道(voltage-gated potassium channel,Ky)电流和内向整流钾通道(Inward rectifier channel,Ku)电流的记录方法 .方法 用急性酶解的方法 分离出单个大鼠PASMC,利用全细胞膜片钳方法 记录钾电流.结果 ①急性酶解分离得到高质量的单个大鼠PASMC,呈梭形,边界清楚,胞浆均匀透亮.②Kv电流可以被5 mmol/L4-AP明显抑制,使电流-电压关系曲线明显下移,在55 mV时,Kv电流密度从(133.86±7.36)pA/pF减少到(59.09±3.35)pA/pF(n=6,P<0.05).③1 mmol/L TEA对KCa电流有明显抑制作用,使电流-电压关系曲线明显下移,在55 mV时,KCa电流密度从(9.03±1.42)pA/pF减少到(2.12±0.52)pA/pF(n=7,P<0.05).④KATP电流可以被10 μmol/L尼可地尔激活,使电流-电压关系曲线明显上移,在50 mV时,Kv电流密度从(29.08±5.90)pA/pF增加到(88.90±7.98)pA/pF(n=10,P<0.05).结论 在肺动脉平滑肌细胞上成功地建立了KCa、Kv和Kir电流的记录方法 ,可以为肺动脉相关疾病的发病机制和治疗方案的探索,提供有效的细胞模型基础.     

钾通道与肺动脉平滑肌细胞凋亡

钾通道与肺动脉平滑肌细胞的生长、增殖和凋亡密切相关.在肺动脉高压的发生、发展中起着重要作用.肺动脉平滑肌细胞膜钾通道活性降低或表达下降不仅导致细胞浆游离Ca2+浓度升高,肺动脉平滑肌细胞收缩、增殖和肺血管重构,而且也与肺动脉平滑肌细胞凋亡减少有关.凋亡性容积减少是细胞凋亡的必要前提和早期标志.钾通道参与凋亡性容积减少调节、细胞色素C释放、Caspase激活和DNA降解等凋亡事件以及抗凋亡蛋白Bcl-2、survivin的调节.因此.钾通道有望成为诱导肺动脉平滑肌细胞凋亡药物的靶点,为未来攻克肺动脉高压带来新希望.     

Grape seed procyanidin suppresses inflammation in cigarette smoke-exposed pulmonary arterial hypertension rats by the PPAR-γ/COX-2 pathway

Background and aimPulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling, which is mainly caused by inflammation. Inhibiting inflammation can relieve PAH. Grape seed procyanidin (GSP) possesses remarkable anti-inflammatory property and vascular protective function. In this experiment, we verified the anti-inflammatory property of GSP in cigarette smoke-exposed PAH rats and revealed its molecular mechanism.Methods and resultsIn vivo, 45 Sprague Dawley (SD) rats were divided into 5 groups randomly, treated with normoxia/cigarette smoke (CS)/GSP + CS/CS + solvent/GSP. After GSP + CS administration, a decrease in mPAP, PVR, RVHI, WT%, and WA% was detected in the rats as compared to those treated with CS. In vitro, the proliferation of pulmonary arterial smooth muscle cells (PASMCs) caused by cigarette smoke extract (CSE) was effectively attenuated with GSP + CSE administration. Furthermore, GSP significantly increased the expression of peroxisome proliferator-activated receptor γ (PPAR-γ) together with the lowered expression level of cyclooxygenase 2 (COX-2) in PASMCs co-incubated with CSE.ConclusionThese findings indicate that GSP ameliorates inflammation by the PPAR-γ/COX-2 pathway and finally inhibits the proliferation of PASMCs, which leads to pulmonary vascular remodeling.     

Safety and Feasibility of Performing Pericardiocentesis on Patients with Significant Pulmonary Hypertension

Background/purposePericardial effusion (PE) is a complication of pulmonary hypertension (PHT) and, specifically, pulmonary arterial hypertension (PAH), that confers a worse prognosis. The safety of performing pericardiocentesis in patients with PHT has not been established. We aimed to assess the safety and feasibility of performing pericardiocentesis in patients with significant PHT.Methods/materialsWe performed a retrospective analysis from August 2013 to December 2018 at our tertiary-care center of patients who underwent a pericardiocentesis procedure. Patients, procedure, echocardiographic findings, any major intraprocedural complications, and post-procedural related complications up to 30 days were recorded. Specifically, we studied patients with significant PHT.ResultsThe cohort included 170 patients, with an average age of 62.6 years and an even distribution of gender and co-morbidities. The etiology for the PE varied. Major complications were rare (1.7%) and only 10 patients (5.9%) required re-intervention for reaccumulation of fluid. There were 27 patients (15.9%) with significant PHT, 5 with World Health Organization (WHO) Group I PAH (2.94%). In the entire cohort, there were only 3 major complications (1.7%), none among PHT patients.ConclusionsPericardiocentesis is a safe procedure, including in patients with significant PHT, including those with WHO Group I PAH. We advocate the use of invasive hemodynamic monitoring in patients with significant PHT.SummaryPericardiocentesis tends to be a safe procedure. However, the safety of performing pericardiocentesis in patients with significant pulmonary hypertension has not been well established. We aimed to assess the safety and feasibility of performing pericardiocentesis, and specifically in patients with significant PHT out our tertiary center by performing a retrospective analysis.     

肺动脉平滑肌细胞合成和分泌功能及相关调控机制

肺血管重塑以纤维母细胞、平滑肌细胞和内皮细胞增殖为最大特征,并导致管腔闭塞。而肺动脉平滑肌细胞(pulmonary artery smooth muscle cells,PASMCs)是肺血管重塑的主要效应细胞。了解 PASMCs 在刺激因素诱导下的合成和分泌功能的改变及其调控机制对预防和治疗以肺血管重塑为主要病理基础的 COPD 患者具有重要意义。现就影响 PASMCs 的合成分泌功能的因素和相关调控机制作一综述。     

Ad-Gax转染对缺氧性大鼠肺动脉平滑肌细胞凋亡及其相关基因表达的影响

目的观察Gax基因转染对缺氧性肺动脉平滑肌细胞（PASMCs）凋亡及其相关基因表达的影响。方法腺病毒介导Gax转染体外原代培养的PASMCs。透射电镜观察细胞凋亡形态;原位细胞凋亡检测法观察Ad-Gax转染前后在常氧和缺氧处理2h,6h、12h、24h、48h大鼠PASMCs凋亡情况;免疫细胞化学法测PASMCsBcl-2、Bax蛋白表达。结果透射电镜观察到Ad-Gax转染后PASMCs产生凋亡现象。未转染缺氧刺激前后,均无或可见极少量的阳性细胞;Ad-Gax转染后,如无缺氧刺激,也无或仅可见少量的阳性细胞,予缺氧刺激后,阳性细胞显著增多,尤其在转染后24～48h更明显。转染组常氧、缺氧2h,6h、12h、24h、48h细胞凋亡百分率均显著高于未转染组（P〈0．01）。Ad-Gax转染前,与常氧时比较,缺氧刺激PASMCs后,Bax蛋白表达略为升高但无统计学意义;而Bcl-2蛋白表达显著升高,差异有统计学意义（P〈0．05）。Ad-Gax转染PASMCs后,缺氧刺激使Bcl-2蛋白表达显著降低（P〈0．01）,Bax蛋白表达显著增高（P〈0．01）。转染组细胞凋亡率与Bcl-2／Bax比值呈负相关（r=-0．53,P〈0．01）。结论Ad-Gax转染可诱导缺氧性PASMCs凋亡,其机制可能是通过上调Bax蛋白和下调Bcl-2蛋白的表达,尤其是通过降低Bcl-2／Bax比值实现的。     

钌红、丁卡因对肺动脉高压模型大鼠肺动脉平滑肌细胞[Ca2+]i释放功能的影响

目的:分析肺动脉高压时肺动脉平滑肌细胞雷诺定(Ryanodine)受体[Ca2+]i释放功能的改变及钌红(Rutheniumred)、丁卡因对肺动脉高压模型大鼠肺动脉平滑肌细胞[Ca2+]i释放功能的影响.方法:清洁级Wister大白鼠25只,腹腔注射野百合碱,建立大鼠肺动脉高压模型,死亡2只,最终23只;原代培养肺动脉平滑肌细胞;Fura-2/AM(钙离于荧光指示剂)负载培养细胞;荧光测钙技术比较雷诺定诱导的野百合碱组(n=15)及对照组(n=8)[Ca2+]i数值,观测钌红、丁卡因对雷诺定受体激动剂雷诺定诱导的[Ca2+]i变化的影响.结果:10 nmol/L雷诺定使对照组[Ca2+]i平均增加(93.31±12.41)nmoL/L,使野百合碱组[Ca2+]i平均增加(141.71±13,59) nmol/L;两组样本[Ca2+]i增加的数值比较差异有统计学意义(P＜0.01).钌红浓度为o.5～ 10 μmol/L、丁卡因浓度为2～40 μnoL/L时,可使雷诺定受体激动剂雷诺定诱导的[Ca2+]i值最大下降(115.32±7.47) nmol/L和(107.97±7.11) nmol/L.结论:肺动脉高压大鼠肺动脉平滑肌细胞对雷诺定受体激动剂雷诺定的敏感性增强;钌红、丁卡因对雷诺定诱导的肺动脉高压大鼠肺动脉平滑肌细胞[Ca2+]i上升有明显的抑制作用.     

DAPT suppresses the proliferation of human glioma cell line SHG-44

Objective:To explore the suppressing effect ofγ-secretase inhibitor DAPT on proliferation of human glioma cell line SHG-44 in vitro and its mechanism.Methods:The SHG-44 cell was treated by DAPT with different concentration.The proliferation of cells was detected by MTT assay;cell cycle and TSC of CD133~+were determined by flow cytometry analysis technique;the key factor in Notch signaling pathway(Notch-1,Delta-1,Hes-1)was measured by reverse transcrip tase-polymerase chain reaction and western blotting.Results:DAPT inhibited the growth and proliferation of SHG-44 cells significantly(P0.05).And the inhibiting effect on SHG-44 cells produced by DAPT showed a dose-dependent manner.DAPT increased the rate of cells in G_0/G_1 phase of SHG-44 cells,while it decreased the rate of cells in S phase.TSC of CD133~+was significantly reduced after DAPT treated SHC-44 cells.The expression of protein and mRNA of Notch-1,Delta-1 and Hes-1 were gradually downregulated with the increase of DAPT doses.Conclusions:DAPT can downregulate these key factor in Notch signaling pathway,reduce the TSC of CD133+and inhibit the proliferation of SHC-44 cells.