Effect of vitamin K2 on β-glycerophosphate-induced calcification in rat vascular smooth muscle cells and the mechanism

Objective To explore the effect of vitamin K2 on β-glycerophosphate(β-GP)-induced rat vascular smooth muscle cells (VSMCs) calcification and and the mechanism. Methods VSMCs were obtained from rat aortic, and identified by immunocytochemistry, then randomly divided into control group, high phosphorus group, vitamin K2 group (the group was settled three subgroups according to the concentration of vitamin K2 based on the high phosphorus medium, namely 10 μmol/L, 25 μmol/L, 50 μmol/L) and noggin (bone morphogenetic protein pathway inhibitor) group. Calcification was visualized by Alizarin red staining, calcium load in cells was quantified by o-cresolphthalein complexone method and alkaline phosphatase (ALP) activity was measured after stimulating 14 days, gene expressions of bone morphogenetic protein - 2 (BMP-2), SMAD1, SMAD7 and Runx2 mRNA were detected by RT-PCR, Runx2 protein levels was detected by Western blotting after stimulating 3 days. Results Compared with the cells in control group, high phosphorus induced cell calcification, increased ALP activity, up-regulated the expression of BMP-2, SMAD1, Runx2 mRNA (P＜0.05) and down-regulated the expression of SMAD7 (P＜0.01),while compared with high phosphorus group, the calcium deposition, ALP activity and the expression of BMP-2, SMAD1, Runx2 mRNA were remarkably reduced in a dose-dependent manner by treatment with vitamin K2 (P＜0.05) and the expression of SMAD7 was increased (P＜0.01). Compared with high phosphorus group, SMAD1 and Runx2 expression in noggin group were remarkably reduced(P＜0.01). Conclusion Vitamin K2 inhibits β-glycerophosphate-induced VSMCs calcification which correlates with the suppression of the expression of osteoblast markers through the down-regulation of bone morphogenetic protein pathway.     

左心室肥厚的影响因素及机制的研究进展

生理和病理条件下,血流动力学超负荷会引起左心室肥厚。目前尚未完全阐明引起心肌肥厚的主要的刺激因素是心脏自身机械拉伸,还是神经体液因子,抑或是两者的共同作用所致。刺激因素进入细胞后,通过引起细胞内生化改变导致第二信使和第三信使的激活,进一步调节转录,激活多种心肌肥厚相关基因的表达。左心室肥厚时会出现一系列的结构改变,包括心肌细胞肥大、间质结缔组织增生以及冠状循环微血管稀少。本文参考近年来相关文献,总结左心室肥厚的影响因素及机制研究方面的发展动态,旨在为读者阐明左心室肥厚的影响因素及机制的总体情况,提供可参考的研究靶点。     

Activation of Connexin-43 Hemichannels Can Elevate [Ca]iand [Na]iin Rabbit Ventricular Myocytes During Metabolic Inhibition

ATP depletion due to ischemia or metabolic inhibition (MI) causes Na⁺and Ca²⁺accumulation in myocytes, which may be in part due to opening of connexin-43 hemichannels. Halothane (H) has been shown to reduce conductance of connexin-43 hemichannels and to protect the heart against ischemic injury. We therefore investigated the effect of halothane on [Ca²⁺]_iand [Na⁺]_iin myocytes during MI. Isolated rabbit left ventricular myocytes were loaded with 4μ m fluo-3 AM for 30 min, or with 5 μ m sodium green AM for 60 min at 37°C. After washing, the myocytes were exposed to: (1) Normal HEPES solution; (2) MI solution (2 m NaCN, 20 m 2-deoxy- -glucose and 0-glucose); or (3) MI+H (0.95 m , 4.7 m ) for 60 min. Propidium iodide (PI, 25 μ m) was added to all samples before data acquisition. The fluorescence intensity was measured by flow cytometry with 488 nm excitation and 530 nm emission for fluo-3 or sodium green, and 670 nm for PI. The [Ca²⁺]_iand [Na⁺]_iwere then calculated by calibration. In some experiments, the effect of 10 μ m tetrodotoxin (TTX) and 20 μ m nifedipine (NIF) were studied. Metabolic inhibition for 60 min caused a significant increase in [Ca²⁺]_iand [Na⁺]_iin myocytes when compared to controls, which was significantly reduced by halothane in a dose-dependent fashion. In the presence of TTX and NIF, halothane also significantly reduced the rise in the [Ca²⁺]_iand [Na⁺]_iin myocytes subjected to MI. 1-heptanol, another gap junction blocker, had similar effects. Thus, halothane reduced [Ca²⁺]_iand [Na⁺]_ioverload produced by MI in myocytes. This effect is not solely due to block of voltage-gated Na⁺and Ca²⁺channels, and is likely mediated by inhibiting the opening of connexin-43 hemichannels.     

Caspase-3在慢性心力衰竭大鼠心肌重塑中动态表达及意义

目的探讨慢性心力衰竭大鼠心肌内半胱氨酸天冬氨酸蛋白酶3（caspase-3）表达及意义。方法40只雄性Wistar大鼠随机分为四组,假手术组、心力衰竭1d组、7d组、14d组各10只。以缩窄大鼠腹主动脉,造成左心室压力负荷,制备慢性心力衰竭大鼠模型。观察和比较成模后1d、7d、14d及假手术组左心室肌重量指数,Masson染色后对比心肌胶原含量,免疫组化法测定心肌中caspase-3蛋白表达水平。结果腹主动脉缩窄术后各组大鼠均发生左室重塑和纤维化,左心室肌重量指数、胶原含量及caspase-3蛋白表达均明显高于假手术组;caspase-3于正常心肌内表达较少,而在心力衰竭后心肌内表达则明显增多,且随着心力衰竭程度的加重而表达增多。结论caspase-3可能参与了大鼠压力负荷性心肌重塑的发生和发展,其水平的高低与慢性心力衰竭的严重程度密切相关。     

罗格列酮对四氧嘧啶介导的糖尿病家兔心室肌细胞L型钙通道电流的影响

目的探讨罗格列酮对家兔心室肌细胞L型钙通道电流(I_(CaL))的影响。方法四氧嘧啶建立糖尿病家兔模型后,利用酶解法急性分离心室肌细胞,用全细胞膜片钳技术观测糖尿病和罗格列酮对心肌细胞I_(CaL)的影响,并与正常家兔进行比较。结果 (1)正常组家兔心室肌细胞I_(CaL)电流密度[(-8.83±2.31)pA/pF]大于糖尿病组[(-8.84±1.98)pA/pF],但差异不具有统计学意义。(2)给予罗格列酮后正常和糖尿病家兔组心室肌细胞I_(CaL)[正常组:基线电流密度(-8.83±2.31 pA/pF)大于5 min电流密度(-4.17±1.89 pA/pF)和10 min电流密度(-5.54±1.63)pA/pF,糖尿病组:基线电流密度(-8.84±1.98)pA/pF大于5 ml‘n电流密度(-4.97±1.15)pA/pF和10 min电流密度(-3.89±1.06)pA/pF],各时段差异均有统计学意义(各组P<0.05)。(3)空腹血糖[(7.65±1.60)mmol/L]小于建模48 h[(17.72±9.15)mmol/L]及1周后空腹血糖[(20.84±9.48)mmol/L]。各时段胰岛素水平[空腹(23.45±9.22)mmol/L,48 h后(22.69±8.94)mmol/L,1周后(21.65±11.91)mmol/L]差异均无统计学意义(各组P>0.05)。结论正常家兔组I_(CaL)电流密度与糖尿病家兔组相似,提示糖尿病对心肌细胞I_(CaL)无明显影响;罗格列酮对心脏的作用不依赖于高血糖存在与否,提示罗格列酮可能通过某些与胰岛素水平有关的特定机制作用于心肌细胞;四氧嘧啶所致糖尿病模型是一种不完全等同于1型及2型糖尿病的独特模型,但去除了胰岛素抵抗的影响。该模型可以用来作为高血糖对心肌细胞离子通道影响的研究载体。     

人骨髓间充质干细胞体外心肌样细胞诱导分化中细胞周期与凋亡的分析

目的探讨体外诱导人骨髓间充质干细胞(hMSCs)向心肌样细胞分化过程中分化与凋亡的情况,为hMSCs临床移植治疗提供数据。方法采用体外纯化、扩增后的第4代hMSCs,分3组在体外分别经0μmol/L、2.5　μmol/L、5.0μmol/L 5-杂氮胞苷(5-Aza)诱导24 h后继续培养8周,相差显微镜下观察细胞形态变化,流式细胞仪检测各组细胞周期及凋亡指数,应用RT-PCR技术分析肌球蛋白重链(β-MHC)、肌钙蛋白T(cTNT)、凋亡相关基因(Bcl-2、Bax)的mRNA在分化过程中的动态表达。结果hMSCs诱导后凋亡指数随诱导后培养时间增加,低浓度诱导组低于标准浓度诱导组,组间差异有统计学意义(P<0.05),G_0/G_1期细胞比例诱导后较对照组显著减少(P<0.05);β-MHC和CTNT在诱导后表达开始增强,在第6周时均达到高峰,第8周时表达开始衰减,Bcl-2、Bax基因表达呈时间依赖性变化。结论hMSCs在体外经纯化、扩增和诱导后可表现心肌样细胞的生物学特性,但诱导过程中hMSCs发生细胞凋亡,采用2.5μmol/L 5-Aza低浓度诱导可减少细胞凋亡发生。     

高分子修饰细菌纤维素细胞相容性的初步研究

目的筛选适于平滑肌细胞附着生长的聚乳酸－共－聚乙醇酸(PLGA)修饰的细菌纤维素材料。 方法不同比例PLGA修饰的细菌纤维素材料分为5组：单纯PLGA材料(50P组);经PLGA修饰的细菌纤维素材料组则包括聚乳酸(PLA) ∶聚乙醇酸(PGA)＝50 ∶50的50PB组、PLA ∶PGA＝75 ∶25的75PB组和PLA ∶PGA＝90 ∶10的90PB组;单纯细菌纤维素为空白对照组。将平滑肌细胞接种于不同组别的材料上培养7 d,扫描电镜观察细胞的生长状态,并用荧光染色计数活/死细胞,CCK8法测定材料上的细胞增殖情况并绘制增殖曲线。不同材料上细胞数量及增殖数据用单因素方差分析比较、两两比较用SNK检验。 结果接种后第3天的扫描电镜可见细胞均能在各组材料上成功附着并生长,但75PB组和空白对照组细胞生长形态较差。活/死细胞染色结果显示,平滑肌细胞可以在各组材料中均匀的分布以及生长,活细胞计数各组差异无统计学意义(F＝1.454,P>0.05)。细胞生长曲线检测显示,接种后3 d及5 d,平滑肌细胞在各组材料上的增殖量差异无统计学意义(3 d F＝1.672,P>0.05; 5 d F＝1.19,P>0.05);接种后7 d, 50PB组和空白对照组的平滑肌细胞增殖优于90PB组及75PB组(F＝13.328,P<0.01)。 结论PLA/PGA比例为50 ∶50的PLGA修饰细菌纤维素后的材料细胞相容性更优,可以成为用于组织修复的生物材料。     

兔骨髓间充质细胞分化为肌细胞的实验研究

目的　探讨兔骨髓间充质细胞 (BMSCs)体外分化为肌细胞的可能性。方法　穿刺法抽取成年新西兰兔双侧股骨骨髓 ,分离培养BMSCs ,5 -溴脱氧尿苷 (bromod eoxyuridine)标记 ,再经过 5一氮杂胞苷 (5 azacytidine)诱导或与乳鼠心肌细胞共培养 ,通过免疫细胞化学染色及透射电镜检查 ,研究其分化情况。结果　体外培养的BMSCs无论是在 5 aza诱导还是在与心肌细胞共培养的条件下 ,均有部分细胞表达横纹肌肌动蛋白 (α actin)。电镜检查显示分化后的细胞具有原始肌小节样结构。结论　成年兔BMSCs在 5 aza诱导或与心肌细胞共培养的条件下均可向肌细胞方向分化