心房颤动相关微小RNA1266调控钠通道蛋白

目的采用双荧光素酶报告基因分析微小RNA 1266(miR-1266)与心房颤动相关离子通道蛋白的靶向关系。方法利用TargetScan、miRanda和miRDB数据库预测出miR-1266的4种靶基因,分别为电压门控钠通道α亚基(SCN5A)、电压门控钾通道eag相关H家族2型(KCNH2)、电压门控钾通道E家族β1亚基(KCNE1)和内向整流钾通道J家族5型(KCNJ5),并针对靶基因构建3非编码区的重组荧光素酶报告质粒,4种靶基因(SCN5A、KCNH2、KCNE1和KCNJ5)分别分为miR-1266转染组、阴性对照组和空白对照组,miR-1266转染组和阴性对照组分别将SCN5A、KCNH2、KCNE1和KCNJ5的重组荧光素酶报告质粒与miR-1266及阴性对照质粒共转染于人胚肾HEK293细胞,48h后检测各组荧光素酶活性。结果 SCN5A转染中,与阴性对照组比较,miR-1266转染组相对荧光素酶活性显著降低(0.100±0.007 vs 0.209±0.011,P=0.002);而KCNH2、KCNE1和KCNJ5转染中,与空白对照组和阴性对照组比较,miR-1266转染组相对荧光素酶活性无明显变化(P=0.1269,0.0652,0.2326)。结论 SCN5A是miR-1266的直接靶基因,而KCNH2、KCNE1和KCNJ5并非miR-1266的直接靶基因,miR-1266可能通过抑制SCN5A表达参与房颤电重构,有可能成为未来干预治疗靶点。     

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.     

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.     

心房颤动患者心房肌乙酰胆碱兴奋钾电流通道基因和蛋白的表达

目的:探讨心房颤动(房颤)患者心房肌乙酰胆碱兴奋钾电流(Ik,Ach)通道基因和蛋白表达变化,以了解Ik,Ach通道在房颤发生中的可能作用机制.方法:入选行心脏瓣膜病手术患者作研究对象,其中并发房颤者9例(房颤组),窦性心律者13例(对照组).所有入选患者于开胸行心脏手术时切取右心耳组织,房颤组患者同期加取左心耳组织.用RT-PCR和Western-Blot检测不同心房肌组织中人Kir3.4基因及蛋白水平的表达.结果:房颤组患者IK,Aeh通道蛋白mRNA表达水平及蛋白含量均较对照组患者减少(P＜0.05).结论:Ik,Ach通道蛋白mRNA表达水平及蛋白含量较少可能是慢性房颤患者发病机制之一.     

微小RNA-155调节钙调磷酸酶和活化T细胞核因子4表达对心肌细胞肥大的影响

目的研究微小(microRNA,miR)-155对心肌细胞肥大的影响及对钙调磷酸酶(CaN-β)和活化T细胞核因子4(NFAT-4)表达的调控作用。方法培养大鼠心肌细胞H9C2(2-1),血管紧张素(Ang)Ⅱ诱导心肌细胞肥大,脂质体转染法将miR-155模拟物和miR-155抑制物转染入心肌细胞。分为对照组、AngⅡ组、mimics组、inhibitors组、AngⅡ+mimics组和AngⅡ+inhibitors组。实时荧光定量PCR检测心肌细胞miR-155的表达。逆转录PCR法检测心房钠尿肽(ANP)、β-肌球蛋白重链(β-MHC)和CaN-βmRNA表达水平。Western blot法检测CaN-β和NFAT-4蛋白表达水平。结果与AngⅡ组比较,AngⅡ+mimics组ANP、β-MHC、心肌细胞表面积、CaN-βmRNA和蛋白表达及NFAT-4蛋白表达明显降低,差异有统计学意义(P<0.05)。结论 CaN-β可能为miR-155的作用靶点,miR-155可通过负性调控CaN-β和NFAT-4的表达,减少心肌细胞ANP和β-MHC表达,抑制心肌细胞肥大。     

微小RNA-221和巨噬细胞迁移抑制因子及血栓调节蛋白与心房颤动并缺血性脑卒中的相关性

目的 探讨微小RNA221(miR-221)、巨噬细胞迁移抑制因子(MIF)、可溶性血栓调节蛋白(sTM)与非瓣膜性心房颤动(NVAF)合并缺血性脑卒中的相关性及其危险因素.方法 选取NVAF患者184例,根据是否合并脑卒中分为脑卒中组63例和NVAF组121例.对比2组血清miR-221、MIF、sTM水平.采用RO...     

微小RNA-126与心脑血管疾病的研究进展

<正>大量研究表明,非编码RNA在心血管疾病中起到重要作用,在非编码RNA中,微小RNA(microRNA,miRNA)是最具有代表性的分子,它是近22个核苷酸序列的RNA,通过不完全互补配对的原则与信使核糖核酸(mRNA)的3′端非翻译区特异性结合,从而使mRNA降解、抑制或者激活,调控体内大约1/3的基因表达。miRNA在生物体内细胞的生长、分化和凋亡过程中扮演着重要的角色,比如我们前期     

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].     

Tissue-specific expression of the RI and RII sodium channel subtypes.

Anti-peptide antibodies that distinguish between the rat brain sodium channel subtypes referred to as RI and RII were prepared and used to determine their relative expression in nerve and muscle tissues. Sodium channels purified from rat brain are approximately 18% RI and 80% RII. In brain, the RII subtype is preferentially expressed with RI/RII ratios ranging from 0.07 in the hippocampus to 0.17 in the cerebral cortex. The RI subtype is preferentially expressed in more caudal areas of the central nervous system with values of RI/RII of 0.98 for medulla oblongata and 2.2 for spinal cord. Expression of additional unidentified sodium channel subtype(s) is detected in midbrain, medulla, and spinal cord, and expression of unidentified sodium channel subtypes predominates over expression of RI and RII in retina and optic nerve. The RI and RII subtypes are primarily expressed in the central nervous system and are not detected in significant numbers in skeletal or cardiac muscle, sympathetic ganglia, adrenal medulla, sciatic nerve, or cauda equina. The RII subtype appears first in development of both brain and spinal cord but declines in adult spinal cord as the RI subtype increases. The strict regional expression of these two sodium channel subtypes suggests that they may have distinct functional properties or physiological roles.     

Effect of sodium balance and calcium channel blocking drugs on blood pressure responses

To study the role of calcium movements in mediating the effects of sodium chloride on the response of blood pressure to angiotensin II (ANG II), we infused ANG II before and after giving calcium channel blocking drugs (nifedipine and diltiazem) and calcium infusions to normal subjects during high and low sodium intakes. ANG II was also in nine patients with essential hypertension eating a low sodium diet. In preliminary studies, the effects of nifedipine, 20 mg p.o., on blood pressure and plasma renin activity were determined. Sensitivity to infused ANG II was calculated as the slope of the linear regression of the increase in diastolic blood pressure (DBP) expressed as a function of the ANG II infusion rate (mm Hg/ng ANG II/kg/min). During intake of a high sodium diet (Na, 200 mEq/day) both drugs significantly (p less than 0.05) reduced ANG II sensitivity, while on a low sodium diet (10 mEq Na), neither drug reduced ANG II sensitivity. There was a significant (p less than 0.001) inverse correlation between the initial ANG II-DBP sensitivity and the change in sensitivity induced by the calcium channel blocking drugs in normal subjects (r = -0.78) and in hypertensive patients (r = -0.70). Five hypertensive patients had greater than normal ANG II-DBP sensitivity that was significantly (p less than 0.05) reduced by nifedipine. Calcium infusion did not affect the ANG II-DBP sensitivity on either diet. The results suggest that in normal subjects increased DBP responses to ANG II, induced by an increase in sodium intake, are partially mediated by increased extracellular to intracellular calcium movements, since they are blocked by the structurally different calcium channel blocking drugs nifedipine and diltiazem. In hypertensive patients on a low sodium diet, increased DBP responses to ANG II infusion were blocked by nifedipine, indicating they are at least partly mediated by increased extracellular to intracellular calcium flux.     

水通道与钠通道在小鼠胸腔液体转运中的作用

目的：探讨水通道和钠通道在小鼠胸腔液体转运中的作用。方法：小鼠吸入麻醉后，胸膜腔内分别注入0．25ml高渗和等渗液体，分别予以特布他林、阿米洛利、地塞米松和氯化汞，在不同时间点回收胸腔液体，测量其渗透压、容积和示踪剂^125I－白蛋白计数。根据有腔液体滤出率和吸收率变化，研究钠通道和水通道在胸腔液体转运中的作用。结果：阿米洛利抑制高渗液体引起的胸腔液体滤出和等渗液体吸收（P＜0．05）；特布他林增加胸腔液体的滤出和吸收（P＜0．05）；地塞米松增加高渗液体引起的胸腔液体的滤出（P＜0．05），氯化汞则起抑制作用（P＜0．05），地塞米松和氯化汞对胸腔液体的吸收无影响（P＞0．05）。结论：水通道影响渗透压梯度引起的胸腔液体转运，对等渗液体的胸膜腔转运无明显影响。钠通道可影响渗透压梯度引起的胸腔液体转运和等渗液体吸收。     

Functional expression of the amiloride-sensitive sodium channel in Xenopus oocytes.

Expression of the amiloride-sensitive sodium channel was examined in Xenopus oocytes that were microinjected with A6 cell mRNA. Amiloride-inhibitable 22Na flux could be measured in intact oocytes 2-3 days after injection with 25 ng of poly(A)+ RNA isolated from aldosterone-treated A6 cells. The rate of 22Na uptake was approximately 15-fold greater in oocytes microinjected with 25 ng of poly(A)+ RNA than in water-injected control oocytes. An increase in 22Na uptake by mRNA-injected oocytes occurred whether the mRNA was isolated from A6 cells grown on a porous or nonporous support. In the presence of 4 mM NaCl, amiloride caused dose-dependent inhibition of 22Na uptake in mRNA-injected oocytes, which was half-maximal at 6 x 10(-8) M. Both 1 microM amiloride and 0.1 microM benzamil inhibited 22Na uptake in mRNA-injected oocytes by greater than 95%, whereas less than 50% inhibition occurred with 1 microM 5-(N-ethyl-N-isopropyl)amiloride. When A6 cell mRNA was size fractionated by sucrose density-gradient centrifugation, amiloride-sensitive 22Na uptake was expressed predominantly by oocytes injected with mRNA from two contiguous fractions.     

Syntrophin-dependent expression and localization of Aquaporin-4 water channel protein

The Aquaporin-4 (AQP4) water channel contributes to brain water homeostasis in perivascular astrocyte endfeet where it is concentrated. We postulated that AQP4 is tethered at this site by binding of the AQP4 C terminus to the PSD95-Discs large-ZO1 (PDZ) domain of syntrophin, a component of the dystrophin protein complex. Chemical cross-linking and coimmunoprecipitations from brain demonstrated AQP4 in association with the complex, including dystrophin, beta-dystroglycan, and syntrophin. AQP4 expression was studied in brain and skeletal muscle of mice lacking alpha-syntrophin (alpha-Syn(-/-)). The total level of AQP4 expression appears normal in brains of alpha-Syn(-/-) mice, but the polarized subcellular localization is reversed. High-resolution immunogold analyses revealed that AQP4 expression is markedly reduced in astrocyte endfeet membranes adjacent to blood vessels in cerebellum and cerebral cortex of alpha-Syn(-/-) mice, but is present at higher than normal levels in membranes facing neuropil. In contrast, AQP4 is virtually absent from skeletal muscle in alpha-Syn(-/-) mice. Deletion of the PDZ-binding consensus (Ser-Ser-Val) at the AQP4 C terminus similarly reduced expression in transfected cell lines, and pulse-chase labeling demonstrated an increased degradation rate. These results demonstrate that perivascular localization of AQP4 in brain requires alpha-Syn, and stability of AQP4 in the membrane is increased by the C-terminal PDZ-binding motif.     

电针治疗对大鼠脑缺血后电压门控型钠通道Na（v）1．6表达的影响

目的观察电针治疗对人鼠脑缺血后电压门控型钠通道Na（v）1．6表达的影响.方法将健康成年雄性SD大鼠105只,随机分为假手术组（15只）、脑缺血组（45只）和电针治疗组（45只）。线栓法制作右侧大脑中动脉永久性闭塞模型。电针治疗组在人鼠清醒后,选取内关、外关、足三里、三阴交穴位行针刺治疗,留针时间30min。1次／d：在缺血后6h,1、2、3d及7d观察大鼠神经功能缺损情况,以及纹状体区Na（v）1．6的表达（免疫荧光染色和实时荧光定量PCR法）及腑梗死体积的变化。结果①缺血组和电针治疗组火鼠神经功能缺损表现在缺血后2d最严重;相同时间点,缺血组比电针治疗组神经功能缺损稃度严重（P〈0．05）。②免疫荧光染色显示,缺血组和电针治疗组Na（v）1．6的表达均在缺血后1d达高峰,随后下调。实时荧光定量PCR显示,缺血组6h～1d表达上调,1d表达最强,2～7d表达呈下调趋势;电针治疗组从缺血6h开始至缺血7d,Na（v）1．6表达呈下调趋势。L述2种方法均昆示,相同时间点,电针治疗组较缺血组Na（v）1．6表达下调（P〈0．05）。③缺血组和电针治疗组恼梗死体积均在缺血3d最大。相同时间点电针治疗组脑梗死体积比缺血组小（P〈0．05）：结论Na（v）1．6可能参与缺血性脑损伤的发病过程。电针治疗可能通过下调Na（v）1．6表达发挥对缺血性脑损伤的保护作用：