miR-664b-3p调控Caspase-1介导细胞焦亡在深静脉血栓形成中的作用

目的 观察深静脉血栓形成(DVT)患者外周血微小RNA-664b-3p(miR-664b-3p)、半胱氨酸天冬氨酸蛋白酶1(Caspase-1)水平变化,分析二者在细胞焦亡中的调控关系.方法 选择健康志愿者(健康对照组)和DVT患者(DVT组)各30例,空腹抽取外周静脉血,分离血清及单个核细胞(PBMC).Wester...     

微小RNA-144表达对大鼠心肌细胞凋亡的影响

目的 探讨微小RNA-144(miR-144)对心肌细胞凋亡的影响.方法运用转染的方法,使大鼠H9C2胚胎心肌细胞中高表达miR-144(miR-144组),实时定量PCR确定miR-144表达上调后,通过细胞计数试剂盒-8(CCK-8)、半胱天冬酶(Caspase)-3、流式细胞术等方法测定细胞凋亡水平,观察miR-144对细胞凋亡的影响.空白脂质体及随机微小RNA片段作为空白对照及阴性对照.结果实时定量PCR结果显示,miR-144组miR-144的表达(2178.84±838.52)明显高于空白对照组(1.00±0.00)和阴性对照组(2.06±0.73)(P均＜0.01).与阴性对照组和空白对照组比较,miR144组细胞增殖明显低、Caspase-3活性明显高、细胞凋亡率明显高,而阴性对照组与空白对照组以上数值比较差异均无统计学意义.结论合成的miR-144片段(miR-144 mimics)能选择性上调miR144的表达,并促进心肌细胞凋亡、抑制细胞增殖.

Abstract：

Objective To investigate the effects of microRNA-144 (miR-144) expression on H9C2(2-1) myocytes. Methods MiR-144 was up-regulated in primary cultured H9C2 (2-1) myocytes through transfection. Cells transfected with LipofectamineTM 2000 and its mixture with miRNA synthesized randomly as blank control and negative control respectively. The up-regulation of miR-144 was confirmed by real-time PCR. Cell apoptosis was evaluated by means of CCK-8, Caspase-3 and flow cytometry. Results Real-time PCR results showed that the miR-144 expression was obviously increased in miR-144 up-regulation group (2178.84±838.52) compared with negative (2.06±0.73) and blank (1.00±0.00) control group ( all P ＜0. 01 ). The proliferation was lower, the activity of Caspase-3 was elevated and the apoptosis rates were significantly increased in miR-144 up-regulation group compared with negative and blank control group,while no significant difference was found between the latter 2 groups. Conclusion MiR-144 mimics may selectively up-regulate the expression of miR-144 in myocardial cells and consequently promote apoptosis and inhibit proliferation in myocardial cells.     

微小RNA-144表达对大鼠心肌细胞凋亡的影响

Objective To investigate the effects of microRNA-144 (miR-144) expression on H9C2(2-1) myocytes. Methods MiR-144 was up-regulated in primary cultured H9C2 (2-1) myocytes through transfection. Cells transfected with LipofectamineTM 2000 and its mixture with miRNA synthesized randomly as blank control and negative control respectively. The up-regulation of miR-144 was confirmed by real-time PCR. Cell apoptosis was evaluated by means of CCK-8, Caspase-3 and flow cytometry. Results Real-time PCR results showed that the miR-144 expression was obviously increased in miR-144 up-regulation group (2178.84±838.52) compared with negative (2.06±0.73) and blank (1.00±0.00) control group ( all P ＜0. 01 ). The proliferation was lower, the activity of Caspase-3 was elevated and the apoptosis rates were significantly increased in miR-144 up-regulation group compared with negative and blank control group,while no significant difference was found between the latter 2 groups. Conclusion MiR-144 mimics may selectively up-regulate the expression of miR-144 in myocardial cells and consequently promote apoptosis and inhibit proliferation in myocardial cells.     

微小RNA-144表达对大鼠心肌细胞凋亡的影响

Objective To investigate the effects of microRNA-144 (miR-144) expression on H9C2(2-1) myocytes. Methods MiR-144 was up-regulated in primary cultured H9C2 (2-1) myocytes through transfection. Cells transfected with LipofectamineTM 2000 and its mixture with miRNA synthesized randomly as blank control and negative control respectively. The up-regulation of miR-144 was confirmed by real-time PCR. Cell apoptosis was evaluated by means of CCK-8, Caspase-3 and flow cytometry. Results Real-time PCR results showed that the miR-144 expression was obviously increased in miR-144 up-regulation group (2178.84±838.52) compared with negative (2.06±0.73) and blank (1.00±0.00) control group ( all P ＜0. 01 ). The proliferation was lower, the activity of Caspase-3 was elevated and the apoptosis rates were significantly increased in miR-144 up-regulation group compared with negative and blank control group,while no significant difference was found between the latter 2 groups. Conclusion MiR-144 mimics may selectively up-regulate the expression of miR-144 in myocardial cells and consequently promote apoptosis and inhibit proliferation in myocardial cells.     

细胞焦亡与心房颤动心房结构重构关系的研究进展

<正>心房颤动(AF)是临床工作中最常见的心律失常,发病率随着年龄的增长而增加。目前,全球约有3350万例AF患者,其中中国约占800万例,AF已成为世界性的公共健康问题,为社会和家庭都带来了沉重的经济负担^[1]。相关研究表明,AF的发生机制与炎症过程密切相关,炎性因子可引起心肌细胞溶解、变形、纤维化等,进一步导致心肌细胞电生理特性的改变,促进折返的形成^[2]。     

细胞焦亡在心肌缺血再灌注损伤中的研究进展

心肌缺血再灌注损伤(MIRI)是导致心肌梗死患者经积极血运重建后仍发生较高死亡率的主要原因之一,寻找减轻MIRI的有效干预靶点并探索其保护机制具有重要的意义。细胞焦亡是细胞坏死和凋亡之外的一种炎性程序性细胞死亡方式。近年来,研究发现细胞焦亡与MIRI的发生发展具有密切联系。本文对细胞焦亡及其在MIRI中的作用机制进行综述,并讨论小分子药物、天然药物以及临床常用药物影响细胞焦亡并应用于MIRI防治的研究新进展。     

细胞焦亡在痛风炎症机制中的研究

细胞焦亡是一种程序性死亡方式, 能引起细胞肿胀及细胞膜完整性破坏, 造成细胞穿孔, 导致细胞代谢物和炎性因子释放。痛风作为炎性疾病的一种, 近来研究发现细胞焦亡在其发生发展过程中扮演着重要角色, 主要是通过激活的核苷酸结合寡聚化结构域样受体蛋白3炎性小体活化半胱氨酸天冬氨酸蛋白酶-1, 切割下游消皮素D, 引起细胞膜成孔, 并释放IL-1β诱发痛风发作。因此, 探索细胞焦亡在痛风炎症机制中的作用有望进一步揭示痛风炎症的发病机制, 本文从细胞焦亡发生机制阐述细胞焦亡与痛风的关系, 加深对该疾病发病机制的认识, 并为痛风的治疗提供新的方向。     

细胞焦亡在人体寄生虫病中的研究进展

细胞焦亡是近年发现的一种依赖半胱氨酸天冬氨酸蛋白酶（caspase）的新型细胞死亡方式,活化后的caspase以剪切消皮素（GSDM）蛋白家族为途径,使细胞膜发生破裂,同时释放炎性细胞因子及其内容物,最终导致细胞死亡,其发生、形态学特征及调控机制与凋亡、坏死、自噬、铁死亡等细胞死亡方式有明显不同。细胞焦亡信号通路可分为经典途径、非经典途径和其他途径。目前大量研究证实,细胞焦亡参与寄生虫病的发展历程。本文就细胞焦亡的定义、不同途径机制及其在多种人体寄生虫病中发挥的作用作一综述,以期为开拓细胞焦亡与寄生虫疾病之间的作用提供依据。     

微小RNA-21对肝纤维化影响的初步研究

成熟的微小RNAs(miRNAs)在细胞质内与靶mRNA序列3'末端非翻译区互补配对,阻止其蛋白质翻译过程,对该基因形成转录后抑制[1～2].肝纤维化主要依靠肝星状细胞(HSC)活化,产生大量胶原纤维[主要是Ⅰ型胶原蛋白(ColⅠ)]及细胞外基质[3],HSC的活化通过转化生长因子(TGF)β Smad信号传导通路完成,Smad-7对该通路有强烈的抑制作用[4].     

微小RNA-137调控帕金森致病基因参与癫痫发生的作用机制

目的 初步探究微小RNA-137(miR-137)调控帕金森致病基因(Parkin)发挥抗癫痫机制.方法 选择SD健康清洁级雄性大鼠60只建立癫痫模型后随机分为模型组、低表达组、阴性组、3-MA组、联合组(n=12),分别给予生理盐水、miR-137低表达溶液、miR-137阴性对照溶液、自噬抑制剂3-MA溶液、miR...     

过表达miR-29a对大鼠心肌细胞凋亡的影响

目的 探讨microRNA-29a (miR-29a)对大鼠心肌细胞凋亡的调控作用.方法 体外培养新生SD大鼠心肌细胞,合成人miR-29a的拟似物(mimic).用Lipofectamine RNAiMAX转染miR-29a的mimic进入心肌细胞,转染48 h后用荧光定量PCR方法检测心肌细胞miR-29a的表达变化,流式细胞仪检测细胞凋亡水平变化,Western blot法检测凋亡相关蛋白Caspase-3和Caspase-9前体的表达变化.结果 心肌细胞转染miR-29a的mimic 48 h后,心肌细胞中miR-29a的表达水平较对照组明显升高(P＜0.05);心肌细胞的凋亡水平也明显升高,凋亡相关蛋白Caspase-3和Caspase-9前体的含量则明显下降(P＜0.05).结论 在大鼠心肌细胞中过表达miR-29a能促进心肌细胞凋亡,其机制可能是通过Caspase-3和Caspase-9途径起作用.     

慢性间歇性低氧对OSAHS大鼠心肌细胞焦亡的影响

目的 观察慢性间歇性低氧（CIH）对阻塞性睡眠暂停低通气综合征（OSAHS）大鼠心肌细胞焦亡的影响。方法 将SD大鼠随机分为对照组、OSAHS组各8只。将OSAHS组置于间歇性低氧舱,建立OSAHS模型;对照组置于常氧舱内,提供常氧空气。8周后将大鼠处死,开胸取心脏。用扫描电子显微镜观察心肌细胞的微观结构,分别用实时荧光定量PCR法、Western blotting法检测心肌组织中焦亡关键蛋白（Caspase-1、GSDMD、NLRP3）mRNA、蛋白表达。结果 对照组心肌细胞膜完整、结构正常;OSAHS组心肌细胞膜完整性被破坏,表现为细胞膜表面欠光滑,形成裂隙、凹陷和大小不等的凸起,细胞膜表面有纤维组织附着,能够看到明显的泡状突起和焦亡小体流出。与对照组比较,OSAHS组Caspase-1、NLRP3 mRNA相对表达量高（P均<0.05）,两组GDMDS mRNA相对表达量比较差异无统计学意义（P>0.05）;与对照组比较,OSAHS组Caspase-1、GSDMD、GSDMD-N、NLRP3蛋白相对表达量高（P均<0.05）。结论 CIH可促进OSAHS大鼠心肌...     

乔松素对缺氧/复氧诱导大鼠心肌细胞焦亡的预防作用及其机制

目的 观察乔松素（PIN）对缺氧/复氧（H/R）诱导的大鼠心肌细胞焦亡的预防作用,并探讨其预防作用机制。方法 将大鼠心肌细胞H9c2分为PIN-H+740-Y-P组、PIN-H组、PIN-L组、模型组、对照组。PIN-H+740-Y-P组用加入25μmol/L PIN+50μg/mL 740-Y-P的培养基、PIN-H组用加入25μmol/L PIN的培养基、PIN-L组用加入12.5μmol/L PIN的培养基、模型组用空白培养基培养1 h之后进行H/R处理。对照组仅用空白培养基培养。取上述各组细胞继续培养24、48、72 h,采用CCK-8法测定细胞活力;取上述各组细胞,采用碘化吡啶（PI）染色法测算各组心肌细胞焦亡比例,ELISA法测定细胞中LDH,Western blotting法检测细胞中磷脂酰肌醇3-激酶（PI3K）、蛋白激酶B(AKT)、核因子-κB(NF-κB)蛋白和细胞焦亡相关蛋白（Caspase-1、ASC和NLRP3蛋白）,荧光定量PCR法测定细胞中PI3K、AKT、NF-κB mRNA。结果 与对照组相比,模型组细胞活力（48 h、72 h）下降（P均<0...     

细胞焦亡及其在心肌缺血再灌注损伤中作用机制

急性心肌梗死是世界范围内发病率和病死率的主要原因,缺血心肌血流恢复可改善心肌功能,然而血流再通可能会导致严重心肌损伤,即心肌缺血再灌注损伤(MIRI).细胞焦亡是一种高度促炎的细胞程序性死亡.活化的半胱天冬氨酸蛋白酶(caspases)裂解造孔蛋白Gasdermin D(GSDMD)引起质膜破裂,导致促炎因子白细胞介素...     

微小RNA-199a对大鼠心肌肥厚的影响

目的 探讨微小RNA(miRNA)-199a在心肌肥厚中的作用.方法 (1)Sprague-Dawley (SD)大鼠12只,分为腹主动脉缩窄(abdominal aortic constriction,AAC)组(AAC组,n=6)和假手术组(n=6).AAC组通过腹主动脉缩窄术建立大鼠心肌肥厚模型.实时定量聚合酶链反应(qRT-PCR)检测心肌中部分miRNA表达的变化.(2)SD乳鼠心肌细胞分为两组,即miRNA-199a重组腺病毒(Ad-miRNA-199a)组(n=8)和腺病毒载体(Ad-vector)组(n=8),分别转染SD乳鼠心肌细胞48 h后qRT-PCR检测心肌细胞中miRNA-199a以及心肌肥厚标志分子α肌球蛋白重链(α-myosin heavy chain,αMHC)、β肌球蛋白重链(β-myosin heavy chain,βMHC)、心房钠尿肽(atrial natriuretic peptide,ANP)编码基因myh6、myh7、Nppa的表达变化,并利用免疫荧光分析检测细胞表面积的变化.(3)SD乳鼠心肌细胞分为两组,即miRNA-199a的反义寡核苷酸(As-miRNA-199a)组(n=8)和混杂寡核苷酸(As-ctl)组(n=8),分别转染SD乳鼠心肌细胞48 h后qRT-PCR检测心肌细胞中miRNA-199a的表达变化.(4)SD乳鼠心肌细胞分为4组,即空白对照组(n=8)、苯肾上腺素组(phenylephrine,PE)(n=8)、PE+As-cd组(n=8)和PE+As-miRNA-199a绀(n=8),分别转染SD乳鼠心肌细胞48 h,qRT-PCR检测心肌肥厚标志分子编码基因的表达变化,免疫荧光检测细胞表面积的变化.结果 (1)qRT-PCR结果显示,AAC组大鼠造模后1周miRNA-1、miRNA-133、miRNA-181a及miRNA-499的表达均显著低于假手术组,而miRNA-199a表达显著高于假手术组.(2)qRT-PCR结果显示,AdmiRNA-199a组SD乳鼠心肌细胞中miRNA-199a的表达显著高于Ad-vector组,myh7的表达亦显著高于Ad-vector组,而myh6的表达低于Ad-vector组.免疫荧光显示Ad-miRNA-199a组SD乳鼠心肌细胞的表面积大于Ad-vector组,P<0.05.(3)qRT-PCR结果显示,As-miRNA-199a组SD乳鼠心肌细胞中miRNA-199a的表达显著低于As-ctl组,P<0.05.(4)qRT-PCR结果显示,PE组Nppa及myh7的表达量显著高于空白对照组,而myh6的表达量低于空白对照组,P<0.05.免疫荧光检测显示,PE+As-miRNA-199a组SD乳鼠心肌细胞的表面积显著小于PE+As-ctl组,P<0.05.结论 miRNA-199a在心肌肥厚过程中可能发挥着调节作用.

Abstract：

Objective To investigate the role of miRNA-199a on cardiac hypertrophy.Methods (1)Male Sprague-Dawley rats were subjected to pressure overload induced by abdominal aortic constriction (AAC,n=6)and quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the change of microRNAs(miRNAs).(2)Neonatal rat ventricular myocytes were isolated from 2-day old Sprague-Dawley rats.The myocytes were divided into two groups:adenovirus miRNA-199a(Ad-miRNA199a) or adenovirus vector(Ad-vector).They were transfected in cardiomyocytes for 48 h using Lipofectamine 2000.qRT-PCR was used to detect the change of myocardial hypertrophy markers α-myosin heavy chain(αMHC,myh6),β-myosin heavy chain(βMHC,myh7)and atrial natriuretic peptide(ANP,Nppa).Software Axio Vision was used to detect the change of cardiomyocytes surface areas.(3)Neonatal rat ventricular myocytes were divided into two groups:antisense oligonucleotide-miRNA-199a(As-miRNA199a) and scramble oligonucleotides (As-ctl). They were transfected to cardiomyocytes respectively for 48 h. qRT-PCR was used to detect the change of miRNA-199a. (4) Neonatal rat ventricular myocytes were divided into four groups : A : control ( ctl), B : phenylephrine ( PE), C : PE + As-ctl, D : PE + As-miRNA199a. qRT-PCR was used to detect the change of myh6 ,myh7 and Nppa. Software Axio Vision was used to detect the change of cardiomyocytes surface areas. Results ( 1 ) qRT-PCR results showed that miRNA-1,miRNA-133, miRNA-181a and miRNA-499 were significantly decreased, while the miRNA-199a was significantly increased at 1 week post AAC hearts compared with the sham group. (2) qRT-PCR results showed that miRNA-199a and myh7 were increased and myh6 was decreased significantly in Ad-miRNA199a group compared with Ad-vector group. The cardiomyocytes surface area was increased in Ad-miRNA199a group detected by immunofluorescence. (3) qRT-PCR results showed that miRNA-199a was significantly decreased in As-miRNA-199a group compared with Ad-vector group. (4) The Nppa and myh7were significantly increased and myh6 was decreased in cardiomyocytes stimulated by PE for 48 h. The cardiomyocytes surface area determined by immunofluorescence was increased in PE + As-miRNA-199a groups compared with PE + As-ctl groups. Conclusion miRNA-199a may play a regulatory role in cardiac hypertrophy.     

小胶质细胞焦亡与缺血性脑卒中的关系研究进展

<正>缺血性脑卒中是我国居民主要的死亡和致残原因,其中涉及到的病理生理过程复杂,主要包括氧化应激、兴奋性毒性、炎性反应等~([1-2])。近年来,越来越多的研究发现,由小胶质细胞焦亡介导的炎性反应可能是加剧脑缺血性损伤的重要因素。本研究对小胶质细胞焦亡与缺血性脑卒中的关系作一综述。1小胶质细胞功能与缺血性脑卒中小胶质细胞是中枢神经系统中负责免疫监视和吞噬功能的常驻免疫细胞,其介导的炎性反应是缺血性脑卒中重要的病理机制~([3-4])。在脑缺血性损伤早期,小胶质细胞迅速增殖活化,活化的小胶质细胞在形态和功能上与巨噬细胞相     

细胞焦亡相关分子在原发性痛风性关节炎中的表达及其临床意义

目的探讨原发性痛风性关节炎患者PBMCs中细胞焦亡的分子机制, 为治疗痛风提供新思路。方法采集30例急性期痛风(AG)患者(AG组)、30例间歇期痛风(IG)患者(IG组)和40名健康患者(HC组)的外周血样本及临床资料;实时荧光定量检测细胞焦亡相关分子, 包括核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、半胱氨酸天冬氨酸蛋白酶-1/4/5(caspase-1/4/5)、消皮素A/B/C/D/E(GSDMA/B/C/D/E)的mRNA表达水平。蛋白质免疫印迹法检测了NLRP3、前体caspase-1(pro-caspase-1)、剪切的caspase-1(caspase-1+p10)、GSDMD、N段GSDMD(GSDMD-N)、前体IL-1β(pro-IL-1β)、成熟IL-1β(cleced IL-1β)。正态或近似正态分布的计量资料采用独立样本t检验或单因素方差分析(ANOVA), 非正态分布的计量资料采用Mann-WhitneyU检验或Kruskal-WallisH检验, 计数资料使用χ2检验进行比较。正态分布的连续性变量间采用Pearson相关分析, 非正态分布的连续性变...     

微小RNA-199a-3p靶向非受体酪氨酸激酶信号转导和转录激活因子3对心力衰竭的影响

目的 探究微小RNA(miR)-199a-3p调控非受体酪氨酸激酶(JAK)信号转导和转录激活因子3(STAT3)通路对心力衰竭(HF)大鼠模型心脏功能的影响和机制.方法 30只建模成功的大鼠随机分为HF+miR-199a-3p组和HF组(n=15),选取15只正常大鼠作为对照组.HF+miR-199a-3p组尾静脉注...     

黄芩苷对脂多糖诱导心肌细胞炎症反应和焦亡的抑制作用及其机制

目的 观察黄芩苷对脂多糖（LPS）诱导心肌细胞焦亡、炎症反应的抑制作用并分析其机制。方法 体外培养大鼠心肌细胞H9C2,取对数生长期细胞分为对照组、LPS组、黄芩苷组,对照组不做干预,LPS组以LPS刺激H9C2细胞构建体外细胞损伤模型,黄芩苷组在LPS诱导后分别加入10、20、40、80μmol/L黄芩苷,CCK-8法检测各组细胞活力,取细胞活力最高的黄芩苷浓度作为最佳作用浓度进行后续实验。而后取对数期H9C2细胞分为对照组、LPS组、黄芩苷组、抑制剂组、黄芩苷+抑制剂组及黄芩苷+激活剂组,除不做干预的对照组外均给予LPS刺激构建细胞损伤模型;黄芩苷组在此基础上给予黄芩苷处理,抑制剂组给予磷脂酰肌醇3激酶（PI3K）/丝苏氨酸蛋白激酶（AKT）通路抑制剂LY294002处理,黄芩苷+抑制剂组给予黄芩苷及LY294002处理,黄芩苷+激活剂组给予黄芩苷及PI3K/AKT通路激活剂胰岛素样生长因子1(IGF-1)处理。采用ELISA法检测细胞上清液炎症因子白细胞介素（IL）-1β、IL-6,Western blotting法检测细胞焦亡相关蛋白焦孔素D(GSDMD)、核苷酸寡聚化结构域样...