血管平滑肌细胞钾通道的研究进展

血管平滑肌细胞(vascular smooth muscle cells ,VSMCs)上分布着多种钾通道,它们参与了细胞膜电位的复极与超极化,影响细胞膜的兴奋性,调节VSMCs的舒缩活动,对血管紧张度的调控具有十分重要的作用.

BMPR-Ⅱ信号转导功能异常与肺动脉平滑肌细胞增殖/凋亡的关系

近年来,转化生长因子-β(transforming growth fac-tor-β,TGF-β)超家族已经成为医学领域研究热点.尤其是骨形态构建蛋白(bone morphogenetic proteins,BMPs)做为TGF-β超家族的一员,不仅在骨及软骨的生成、分化、发育中具有重要作用,在其它重要组织器官(如心、肺、脑等)也有表达,对多种细胞(间充质细胞、成纤维母细胞、角质化细胞、星型细胞、肾脏皮质细胞、内皮细胞以及肿瘤细等)的增殖、分化、迁移、凋亡具有调控作用[1～2].骨形态构建蛋白Ⅱ型受体(bone morphogenetic proteins typeⅡreceptor,BMPR-Ⅱ)做为BMPs信号转导通路上重要成分,完成细胞内、外信息的传递.然而,在某些疾病(如原发性肺动脉高压)中及环境因素刺激下,正常的BMPR-Ⅱ表达下降或功能缺陷,导致信号转导异常,引起肺血管细胞生长状态改变并诱发肺血管重构.本文对BMPR-Ⅱ异常与肺血管细胞增殖/凋亡的关系作一综述.

肺动脉平滑肌细胞氧敏感钾通道与低氧性肺血管收缩

Lopez Barnc于1988年采用全细胞膜片钳技术发现了大鼠颈动脉体Ⅰ型细胞膜上对低氧敏感的钾(K+)通道,称为"氧敏感K+通道"(O2-sensitive K+channels).在肺动脉平滑肌细胞(PASMCs)中亦存在此种离子通道.低氧时PASMCs的K+外向电流发生抑制,膜发生除极化,继而L-型电压门控Ca2+通道开放,Ca2+内流使PASMCs内[Ca2+]升高,平滑肌细胞收缩,终导致低氧性肺血管收缩(hypoxic pulmonary vasoconstriction,HPV).

氧化低密度脂蛋白诱导血管平滑肌细胞凋亡的泡沫化

目的:动脉粥样硬化(atherosclerosis, As)发生的细胞学基础是内皮细胞的受损以及血液单核-巨噬细胞、血管平滑肌细胞(vascular smooth muscle cells, VSMC)的迁移和增殖.

低氧上调大鼠PASMCs钙激活性氯离子通道表达的ERK1／2通路机制

目的：探讨钙激活性氯离子通道2（ calcium－activated chloride channel 2， CLCA2）在大鼠低氧性肺动脉平滑肌细胞（ pulmo－nary artery smooth muscle cells，PASMCs）中mRNA和蛋白表达的变化及其与ERK1／2信号通路的关系。方法：取14只雄性SD大鼠，使用酶消化法进行PASMCs原代培养，应用小鼠抗大鼠SM α－actin免疫荧光细胞化学法进行细胞鉴定；经培养鉴定PASMCs随机分为常氧组（N组，n＝6）、低氧组（H组，n＝6）、DMSO对照组（D组，n＝6）、U0126干预组（U组，n＝6）和stau－rosporine aglycone干预组（ SA组，n＝6）；采用免疫印迹法检测CLCA2蛋白的表达，选用RT－PCR技术测定CLCA2 mRNA水平的表达。结果：与N组比较，H组PASMCs中CLCA2 mRNA和蛋白的表达量均显著上调（均P＜0．01）；与D组比较， U组CLCA2 mRNA和蛋白的表达均明显上调（均P＜0．01），SA组mRNA的表达显著下调（P＜0．01），蛋白的表达轻微下调（但P＞0．05）。结论：低氧可上调大鼠PASMCs中CLCA2 mRNA和蛋白的表达；ERK1／2通路抑制剂U0126能上调PASMCs中CLCA2 mRNA和蛋白的表达；ERK1／2通路激活剂staurosporine aglycone可下调PASMCs中CLCA2 mRNA和蛋白的表达。

It is very difficult for doctor to treat patient with persistent hypotension in the late stage of shock. The aim of present study was to elucidate the reason for lower vasoreactivity in severe shock. Irreversible hemorrhagic shock of rat was reproduced and the vasoreactivity of arteriole in spinotrapezius muscle to norepinephrine (NE) was measured. The resting membrane potential of isolated arterial strips was detected with a microelectrode. The effect of NO on the membrane potential and intracellular ［Ca2＋］i level in isolated arteriolar smooth muscle cells (ASMCs) was determined with fluorescent probes under confocal microscope. KATP channel of ASMCs was measured with patch clamp method. It was shown that membrane hyperpolarization appeared in arteroles 2 h post hemorrhage, while the resting potential was increased from (-36.9±6.3) mV of control value to (-51.0±9.1) mV with the NE threshold increased to 15 times more than preshock value. The hyperpolarization of ASMCs was closely related to vascular hyporeactivity (correlation coefficient 0.96, P＜0.01). The hyperpolarization was enhanced by lack of ATP, increase in H＋，and OONO- in ASMCs. Single KATP channel conducatance, mean open time and open probability was increased in ASMCs, and the increased ［Ca2＋］i level of ASMCs stimulated by NE was reduced to 50% of normal value. The vasoreactivity, blood pressure and survival rate could be improved by the treatment of glybenclamide and NaHCO3. The study indicates that hyperpolarization of ASMCs is a major reason for lower vasoreactivity in severe shock, since it inhibits the voltage dependent Ca2＋ channel (POC) with the reduction of NE stimulated ［Ca2＋］i increase. The decrease in ATP and increase in H+, and OONO- in ASMCs involves in the activation of KATP channel, leading to the ASMCs hyperpolarization.

Wild type fragment of erythropoietin(Epo)3'-enhancer (W18) and its mutant type fragment (M18) were synthesized. Primary cultures of endothelial cells of different sources (ECs) and human umbilical venous endothelial cell line (HUVEC) as well as the primary culture of pulmonary artery smooth muscle cells (PASMC) were transfected with either W18 or M18. RT-PCR was performed to detect mRNA and investigate the effect of Epo3'-enhancer fragment on the hypoxia- induced gene expression in ECs. Electrophoretic mobility shift assay(EMSA) was used to test DNA-binding activity of extracted nuclear protein. ［3H］-TdR incorporation, MTT test and flow-cytometry were used to determine the proliferation of PASMC and the effect of Epo3'-enhancer fragment on it. The results showed as follows:(1)The OD value of COX-2 mRNA expressed in hypoxic rat aortic EC was 2.34±0.32, the OD values of COX-2 and VEGF mRNAs expressed in hypoxic EC of pulmonary microvasculature were 1.78±0.21 and 4.71±0.52, the OD value of TXS and ET-1 mRNAs in hypoxic HUVEC were 13.01±4.27 and 1.01±0.05, they were all higher than their counterpart normoxic group (P＜0.05). If the cells were pretransfected with W18, the OD values in these hypoxic groups became 1.28±0.25,0.77±0.09, 2.29±0.41, 4.88±1.05. and 0.51±0.15, respectively, just as low as those of the relevant normoxic groups; (2)The conditioned medium of hypoxic pulmonary artery endothelial cell (PAEC) caused proliferation of PASMC, the cpm values of ［3H］-TdR incorporation was 917.00±527.11, higher than that of normoxic control (P＜0.05). In addition, it was found by using flow-cytometry an increase in the percentage of cells of phases S and G2/M in PASMC incubated in hypoxic EC conditioned medium in comparison with normoxia group and group pretransfected with W18; (3)Hypoxia could induce proliferation of PASMC directly, transfection of W18 could decrease its effect. Their cpm values were 829.50±228.10 and 497.00±52.45, respectively(P＜0.05).The results of MTT test was similar; (4)The inducer of HIF-1, CoCl2 could increase the expression of COX-2 and TXS mRNA in HUVEC, which could also be inhibited by W18 but not by M18;(5)The HIF-1 DNA binding activity was found in hypoxic HUVEC in EMSA with　32　　　　P labeled W18, but not found with 　32　　　　P M18. These results suggest that there might be a common pathway in hypoxic responses of different cells, i.e. regulating the transcription of genes by binding of HIF-1 to a sequence similar to Epo3'-enhancer.

AIM:R-spondin 2 (Rspo2), one member of R-spondin family which contains four secreted proteins , plays an important role in skeletal muscle development .However, the impact of Rspo2 on vascular smooth muscle cell ( SMC) differentiation is little known . This study aims at revealing the role and mechanism of Rspo 2 on SMC differentiation from embryonic stem cells (ESCs).METHODS:A well-established model for studying SMC differentiation from ESCs were used , in which mouse embryonic stem cells ( ES-D3) were seeded on collagen IV-coated flasks and cultured in differentiation medium (DM) for 2, 4, 6 and 8 days.Smooth muscle specific markers, includingα-smooth muscle actin (α-SMA), SM22 and smooth muscle myosin heavy chain (SM-MHC), were detected to in-sure the successful model by qRT-PCR and Western blot .After 3-day pre-differentiation, ESCs were treated with recombinant Rspo 2 protein, overexpression plasmid or shRNA plasmid for 96 h, and the mRNA and protein expression of smooth muscle markers was detected.To explore the role of Rspo2 on SMC differentiation in vivo, 3-day predifferentiated ESCs (106 in 50μLα-MEM) incubated with Rspo2-overexpression plasmid were mixed with 50 μL of Matrigel ( Becton Dickinson Labware ) and then subcutaneously injected into C57BL/6J mice.After 12 days, mice were sacrificed and the implants were harvested for immunofluorescence staining , qRT-PCR and Western blot.Furthermore, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation assay (ChIP) and lucif-erase reporter assay were performed to investigate the transcriptional activity of SMC differentiation related transcription factors , inclu-ding serum response factor (SRF), myocardin (MYO), myocyte-specific enhancer factor 2C (MEF-2C).Involvement of Rspo2 re-ceptor, leucine-rich repeat-containing, G-protein-coupled receptors (Lgr)4,5,6, and β-catenin pathway during Rspo2-induced MSC differentiation were also uncovered by overexpression or inhibition of the respective protein .RESULTS:Our results showed that Rspo 2 mRNA and protein expression was significantly and consistently increased during ESC differentiation towards SMCs .Recombinant Rs-po2 protein and enforced Rspo 2 expression in ESCs resulted in up-regulation of smooth muscle markers and transcription factors , while knockdown decreased the expression of these genes .Expectedly , Rspo2 overexpression also promotes SMC differentiation in vivo.
Mechanistically , our data showed that Rspo 2 could promote SRF binding to SM22 promoter region .Evidence also revealed that one of three Rspo2 receptors, LGR5, was up-regulated while the other two , LGR4 and LGR6, was down-regulated.Silencing of LGR5 inhibi-ted Rspo2-induced SMC differentiation, whereas knockdown of LGR4 had no impact.Finally, activation or inhibition of β-catenin could promote or inhibit SMC differentiation , respectively .CONCLUSION: Our findings demonstrate for the first time that Rspo 2 plays a positive role during smooth muscle cell differentiation from embryonic stem cells .We confirmed that Rspo 2 can up-regulate smooth muscle markers at transcription level .We also revealed Rspo promote smooth muscle cell differentiation through activation of LGR 5 re-ceptor and Wnt/β-catenin pathway .

张应变诱导分泌型miR－27 a调控GRK6在高血压内皮细胞异常增殖中的机制研究

目的：本研究旨在揭示高血压条件下血管平滑肌细胞（ vascular smooth muscle cells ， VSMCs ）响应高周期性张应变后调控血管内皮细胞（ endothelial cells ， ECs）异常增殖的可能机制。方法：在体条件下，构建腹主动脉缩窄型高血压大鼠模型；体外条件下，用FX－4000T张应变加载系统对VSMCs施加5％和15％的周期性张应变。结果：与正常组相比，高血压组大鼠胸主动脉ECs中GRK6的表达水平显著降低，ECs增殖水平显著上升；体内和体外条件均存在VSMCs 源性MPs （ VSMC－MPs）；miR－27a存在于 VSMC－MPs 中，并可靶向调控GRK6；15％周期张应变条件下产生的 VSMC－MPs 中miR－27a 的含量显著高于5％组，作用于ECs 后，15％组ECs 中miR－27a的水平显著高于5％组， GRK6的表达水平显著低于5％组， ECs 的增殖能力显著高于5％组；用从转染生物素连接的miR－27a （B－miR－27a）的VSMCs培养液中分离得到的MPs作用于ECs，在ECs中可以检测到B－miR－27a的存在；miR－27a正向调控ECs 的增殖，GRK6负向调控ECs 的增殖。结论：在高血压条件下，高周期性张应变促进VSMCs 分泌miR－27a，其可通过VSMC－MPs 转移到ECs 中，抑制GRK6表达，并终诱导ECs 异常增殖。

张应变调控的核骨架蛋白在高血压血管平滑肌细胞增殖中的作用

目的：高张应变诱导的血管平滑肌细胞（vascular smooth muscle cells， VSMCs）异常增殖在高血压血管重建发生发展中起重要作用。本研究探讨细胞核骨架（ nuclear envelope ，NE）在其中的作用及其机制。方法：应用腹主动脉缩窄构建高血压大鼠动物模型；应用FX4000张应变加载系统对体外培养大鼠胸主动脉VSMCs分别施加5％（正常生理状态）和15％（模拟高血压状态）幅度的周期性张应变；Western blot检测NE蛋白emerin和lamin A蛋白表达水平；染色质免疫共沉淀、芯片（ CHIP－on－chip）结合MOTIF生物信息学分析检测与emerin和lamin A结合的DNA序列及其特性；protein／DNA array检测抑制emerin或lamin A表达后转录因子活性变化。结果：高血压大鼠颈总动脉emerin和lamin A表达水平明显降低，中膜VSMCs增殖明显增加；体外加载15％周期性张应变模拟高血压病理条件下VSMCs受到的张应变力学刺激，VSMCs的emerin和lamin A表达明显降低，细胞增殖明显增加，这一作用可被emerin或lamin A的高表达载体转染所部分逆转。 emerin和lamin A能够分别与包含多种转录因子启动子结合位点的DNA片段结合，进而调控多种与VSMCs增殖相关的转录因子活性。结论：NE蛋白emerin和lamin A能够响应力学刺激，并通过调控与特异性转录因子启动子区域的结合调控转录因子活性，参与VSMCs增殖功能调控和高血压血管重建。

微小 RNA 与肺动脉高压形成关系的研究进展

肺动脉高压（ pulmonary arterial hypertension ，PAH）是一类以肺血管病变、肺血管阻力进行性增加和肺动脉压力升高为特征的致命性疾病，严重影响患者的生活质量并带来沉重的经济负担。其特点主要是增加了肺血管阻力，从而介导肺血管内皮功能紊乱、血栓形成、炎症及血管结构重构，其发生发展是复杂多因素的病理过程。虽然其病理机制尚不完全清楚，但研究发现肺动脉平滑肌细胞（ pulmonary arterial smooth muscle cells ， PASMCs ）及内皮细胞（ pulmonary artery endothelial cells ）异常增生、去分化和迁移对于PAH的形成和发展起到关键作用［1］。而肺血管成纤维细胞（ pulmonary vascular fibroblast ）过度增殖、迁移及炎症激活等在PAH的形成和发展中同样发挥重要的作用［2］。通过对其进行研究，将对PAH的诊断及治疗提供更多帮助。微小 RNA （ microR－NA，miRNA）是一类进化上高度保守的单链非编码小分子RNA，典型的miRNA由18～25个核苷酸组成，大部分由内含子或编码多个miRNA的复式内含子经RNA聚合酶Ⅱ转录而来［3］。 miRNA可通过转录后翻译抑制或裂解靶mRNA，而参与调节细胞增殖、分化、凋亡、发育和肿瘤、免疫性疾病等生物学过程。研究发现， miRNA与血管平滑肌细胞（ vas－cular smooth muscle cells ，VSMCs）、内皮细胞及肺血管成纤维细胞的增殖、迁移等有着极为密切的关系。而近期的研究表明，miRNA表达于以上这3种细胞中并参与调节其增殖、迁移和分化等。有些miRNA的表达则促进PAH的发生，有些则抑制PAH的发生，通过对这些miRNA的研究，可为PAH提供新的治疗干预靶点。本文就miRNA与PAH的关系进行综述。

哮喘气道平滑肌细胞表型转化调控的研究进展

气道平滑肌细胞（ airway smooth muscle cells ， ASMCs）表型转化是ASMCs的一个重要特征，决定着ASMCs的作用特点和影响方向。现已发现，哮喘发病过程中ASMCs异常表型转化是导致哮喘气道炎症加重、气道高反应性、气道重塑发生的关键环节，其调控机制已成为哮喘研究的热点。现综述如下。

血管平滑肌细胞增殖相关microRNA的研究进展

microRNAs (miRNAs)是1993年Ambro教授在研究线虫发育时序时先发现的.miRNAs与其靶mRNA分子组成一个复杂的调控网络,参与机体发育、干细胞分化、心血管疾病、肿瘤、病毒感染等生理或病理过程[1].多种miRNAs在心血管系统的血管平滑肌细胞 (vascular smooth muscle cells,VSMCs)中高度表达,影响VSMCs的转化,增殖和迁移等过程[2].miRNAs在VSMCs异常增生所引起的增生性血管疾病的发生和发展中,起着非常重要的调控作用.本文着重介绍在VSMCs 中特异性表达的miR-21、miR-143、miR-145、miR-221、miR-222、Let-7miRNA、miR-26a、miR-31、miR-146a对VSMCs增殖的影响.

冠状动脉血管平滑肌KATP通道的特性和作用

Noma于1983年首次在心脏中发现KATP通道,随后相继在血管平滑肌细胞(VSMC)、 骨骼肌细胞、神经细胞等组织中发现有这些通道的存在[1].此通道的共同特征是可 以被二磷酸核苷酸(NDPs)例如二磷酸腺苷(ADP)、尿苷-5'-二磷酸(UDP)、二磷酸鸟苷(GD P)等和钾离子通道开放剂(KCOs)所激活,被磺酰腺(SU)类药物所抑制,例如优降糖和三磷酸 腺苷(ATP).由于VSMC膜KATP通道的研究较其他类型的组织细胞困难,人们对于冠脉V SMC的KATP通道的认识较局限.随着分子克隆技术和电生理技术的发展,目前对于冠 脉VSMC的KATP通道的结构、生理特性、通道的调节、在正常生理及病理情况下对冠脉血流的调节有了进一步的认识,本文就此作一论述.