新生鼠心室成纤维细胞条件培养液增加心肌细胞搏动频 …

目的和方法：利用细胞培养和ＴＲ－ＰＣＲ技术研究新生大鼠心脏成纤维细胞增加心肌细胞搏动频率的作用。结果：成纤维细胞条件培养液在４８ｈ内能明显增加心肌细胞的搏动频率,并具有时间依赖性和剂量依赖性,内皮素－１（ＥＴ－１）ＥＴＡ受体拮抗剂ＢＱ１２３能部分阴断成纤维细胞条件培养液增加心肌细胞搏动频率作用,而血管紧张素ⅡＡＴ１受体拮抗剂ＣＶ１１９７４和α－肾上腺素受体拮抗剂Ｒｅｇｉｔｉｎ却无此效果：ＲＴ－ＰＣ     

心室成纤维细胞条件培养液促进成纤维细胞胶原合成和增殖

采用心室成纤维细胞条件培养液培养心室成纤维细胞,通过测定[^3H]－脯氨酸（[^3H]－proline）的掺入率来了解心室成纤维细胞总胶原合成速率,通过测定[^3H]－胸腺嘧啶核苷（[^3H]－TdR）的掺入率以及c-fos基因的表达丰度来了解心室成纤维细胞的增殖速率。结果显示：心室成纤维细胞条件培养液（FCGM）能增加细胞自身的[^3H]－proline的掺入率和[^3H]－TdR的掺入率,并具有剂量依赖性;FCGM也能促进细胞自身c-fos基因的表达,刺激后1h达高峰。ETA受体拮抗剂BQ123能部分阻断FCGM增加成纤维细胞胶原合成的增殖作用,而AT1受体拮抗剂CV11974和α肾上腺素受体拮抗剂regitin无此效果。结果提示：心室成纤维细胞具有自分泌功能,能分泌内皮素等生物活性物质,促进成纤维细胞胶原的合成和增殖。     

成纤维细胞直接重编程为心肌细胞的研究进展

最近有研究通过直接重编程的方法,采用心脏特异的转录因子和miRNA的不同组合形式,成功地将人和小鼠成纤维细胞转化为心肌样细胞,这些细胞具有与心肌相似的基因表达模式和肌节结构,甚至有少量可以跳动的细胞。直接心肌细胞重编程可将心脏原位成纤维细胞转换成有功能的心肌细胞,成为心血管再生医学的一个全新的方法。该文综述了小鼠和人类成纤维细胞在体内、外直接重编程为心肌细胞研究的发展和现状,对其研究价值、重编程方法、研究过程中的教训、存在的问题及采用这些方法获得的细胞的启示与不足进行了比较分析。     

大鼠骨髓间充质干细胞条件培养液促进心肌细胞肥大

体外模拟心肌缺血微环境,研究骨髓间充质干细胞(MSCs)的旁分泌作用对心肌细胞的影响。以大鼠MSCs各时间点的条件培养液刺激心肌细胞,观察心肌细胞蛋白含量、[3H]-Leu掺入、ANF-荧光素酶(luciferase)表达和心肌细胞面积的变化。MSCs条件培养液处理心肌细胞后,与对照组相比较6h及9h时间点的条件培养液可明显增加心肌细胞蛋白含量、[3H]-Leu掺入、ANF-荧光素酶表达以及心肌细胞面积,其中以6h时间点条件培养液的作用最为显著(P<0.01)。MSCs条件培养液能够通过旁分泌作用刺激心肌细胞肥大,此现象提示移植入心肌缺血区MSCs可能通过旁分泌作用影响心肌细胞,从而参与细胞移植后心功能的改善。     

成纤维细胞直接重编程为心肌细胞研究进展

心肌细胞的再生疗法作为心脏疾病的新型疗法受到人们的广泛关注。细胞直接重编程技术为诱导获得心肌细胞提供了新的方法,它可以绕过多潜能的阶段,将一种终末分化的细胞直接重编程为心肌细胞,为将来细胞移植提供更为安全的细胞来源。文中对体内外直接重编程成纤维细胞为心肌细胞的研究方法及其存在的问题进行了总结,并对心肌细胞直接重编程的未来发展进行展望。     

成纤维细胞直接转换为心肌细胞

既然成纤维细胞可以通过导入转录因子而转变为诱导多能干细胞,研究人员认为终末分化的体细胞也可以通过同样的方法直接转变成为其它系谱的体细胞而不经过干细胞这一环节。美国加州大学的研究人员     

活性氧介导内皮素-1诱导的培养新生大鼠心肌细胞肥大

实验在原代培养的新生大鼠心肌细胞中进行,检测内皮素-1(endothelin-1,ET-1)及其他药物对心肌细胞活性氧(reactiveoxygen species,ROS)产生和心肌细胞肥大的作用,以探讨ROS在ET-1诱导的心肌细胞肥大信号通路中的作用及ROS与蛋白激酶C(protein kinase C,PKC)活化的关系。细胞内ROS水平用ROS敏感的荧光探针2,7-dichlorofluorescin dictate(DCF-DA)反映,心肌细胞肥大通过细胞内RNA含量、细胞内蛋白质含量、细胞表面积大小来确定。实验结果如下:单独使用ET-1后,心肌细胞内反应ROS含量的DCF-DA荧光值比对照组增加77%,反应心肌肥大的PI荧光值、细胞内蛋白质含量、细胞表面积也分别比对照组增加128%、87%和151%。ET-1合用内皮素受体A亚型(ET_A)受体拮抗剂ABT-627、PKC抑制剂CC或过氧化氢酶后,DCF-DA的增加分别减弱62%、60%和51%,同时心肌细胞肥大也被抑制,单独使用PKC激动剂佛波醇脂(PMA)也能使DCF-DA的产生比对照组增加74%。因此,在ET-1诱导心肌细胞肥大的过程中,ET-1能够使心肌细胞产生ROS和诱导ROS依赖的心肌细胞肥大,这一作用依赖于ET_A受体的激活和PKC的活化,·ROS在ET-1诱导心肌细胞肥大中起信号传递的作用。     

碱性成纤维细胞生长因子对乳鼠心肌细胞缺氧复氧损伤的影响

本实验在培养的乳鼠心肌细胞缺氧复氧损伤模型上观察碱性纤维细胞生长因子对Ａ／Ｒ损伤及蛋白激酶活性的影响,以探讨ｂＥＧＦ作为药物预处理心肌保护的可行性及其机理。结果表明,ｂＥＧＦ预处理呈浓度依赖地提高Ａ／Ｒ后心肌细胞存活率,减少细胞内ＡＴＰ消耗及胞浆乳酸脱氢酶漏出;ＰＫＣ抑制剂Ｈ７完全消除ｇＦＧＦ的上述保护作用;实验结果表明,ｂＦＧＦ可以直接激活心肌细胞ＰＫＣ,其激活时相的变化与缺氧预处理者相近,提示     

NOS和PTEN在血管紧张素Ⅱ诱导心肌细胞肥大中的作用

目的: 研究NOS和PTEN在AngⅡ诱导的心肌细胞肥大中的作用及其机制.方法: 采用AngⅡ诱导新生大鼠心肌细胞肥大模型,应用RT-PCR、Western blot及免疫组化等方法,分别检测各组心肌细胞eNOS、iNOS、PTEN mRNA表达和PTEN蛋白表达的变化,以及PTEN蛋白定位.结果: ①应用AngⅡ培养1 d的心肌细胞,心肌细胞蛋白质含量未见明显变化,但eNOS mRNA表达显著减少,iNOS mRNA表达显著增加.②应用AngⅡ培养5 d的心肌细胞,心肌细胞蛋白质含量显著增加;eNOS mRNA和iNOS mRNA表达未见明显变化;心肌细胞PTEN蛋白表达显著减少.③免疫组化结果显示,心肌细胞核内有棕黄色细颗粒状的免疫产物生成.结论: NOS和PTEN参与了血管紧张素Ⅱ诱导心肌细胞肥大的发生发展过程.     

The enhancement of growth and differentiation of rat adrenal nerve cells by the addition of conditioned medium from human fibroblast cultures

The growth of rat adrenal nerve cells was remarkably enhanced by supplementing the cultured medium from the human fibroblast cell line, Hs 68. Maximum specific growth rate and length of the neurites were observed as 0.076 (1/hr) and 0.026 mm, respectively in 20% supplement of five day old medium. In adding more than 20% of the cultured medium both cell and neurite growth was severely decreased. It was interesting that the cultured medium from Hs 68 cells could play a role in the extension of the neurites rather than in the growth of neurite cells. It was also found that molecules lower than 50,000 daltons in the conditioned medium could improve the growth of neurite bearing cells and the extension of the neurites than larger molecules. The efficacy of the proteins (<50,000 MW) was similar to that of human nerve growth factor and much better than that of basic fibroblast growth factor which was mainly secreted from human fibroblast cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Differential expression and localization of annexin V in cardiac myocytes during growth and hypertrophy

Recently it was shown that annexin V is the most prominent member of the annexin family in the adult heart [1]. Amongst others, annexin V has been suggested to play a role in developmental processes. The aim of the present study was to explore whether in the heart annexin V content and localization change during maturational and hypertrophic growth, in order to obtain indications that annexin V is involved in cardiac growth processes. First, in the intact rat heart annexin V content and localization were studied during perinatal development. It was clearly demonstrated that annexin V content in total heart transiently increased in the first week after birth, from 0.79 ± 0.06 µg/mg protein at l day before birth to a peak value of 1.24 ± 0.08 µg/mg protein 6 days after birth, whereafter annexin V protein levels declined to a value of 0.70 ± 0.06 µg/mg protein at 84 days after birth (p < 0.05). Differences in annexin V content were also observed between myocytes isolated from neonatal and adult hearts [0.81 ± 0.09 and 0.17 ± 0.08 µg/mg protein, respectively (p < 0.05)]. Moreover, during cardiac maturational growth the subcellular localization of annexin V might change from a cytoplasmic to a more prominent sarcolemmal localization. Second, in vivo hypertrophy induced by aortic coarctation resulted in a marked degree of hypertrophy (22% increase in ventricular weight), but was not associated with a change in annexin V localization or content. The quantitative results obtained with intact hypertrophic rat hearts are supported by findings in neonatal ventricular myocytes, in which hypertrophy was induced by phenylephrine (10^-5 M). In the latter model no changes in annexin V content could be observed either. In conclusion, the marked alterations in annexin V content during the maturational growth in the heart suggest a possible involvement of this protein in this process. In contrast, the absence of changes in annexin V content and localization in hypertrophied hearts compared to age matched control hearts suggests that annexin V does not play a crucial role in the maintenance of the hypertrophic phenotype of the cardiac muscle cell. This notion is supported by observations in phenylephrine-induced hypertrophied neonatal cardiomyocytes.     

Accumulation of pyrroline 5-carboxylic acid in conditioned medium of cultured fibroblast: Stimulatory effects of serum,insulin, and IGF-1

Summary Pyrroline 5-carboxylate, an intermediate of amino acid metabolism, is released into medium by cultured normal human fibroblasts. With cells made quiescent by serum starvation, the addition of 10% fetal bovine serum augmented the release of pyrroline 5-carboxylate into medium by 2.5-fold. Although platelet-derived growth factor was without effect, both insulin and insulinlike growth factor-1 nearly reproduced the serum effect. The dose-dependence of insulin and insulinlike growth factor 1 effects suggested their mediation by their own respective receptors. Although the mechanism for the stimulatory effect remains unknown, these effects of insulin and insulinlike growth factor 1 on pyrroline 5-carboxylate suggest hormonal regulation of pyrroline 5-carboxylate release.     

A simple method for preparation of cultured cardiac fibroblasts from adult human ventricular tissue

Cardiac fibroblasts constitute greater than 90% of non-myocyte cells in the heart. Because they are responsible for synthesis of components of the extracellular matrix, growth factors and cytokines in the myocardium, they play an important role in normal and pathologic performance of the heart. An understanding of their biology requires in depth studies in a stable and reliable system in which the biological responses of cardiac fibroblasts to various stimuli can be determined. With the exception of few, all studies have been performed on cardiac fibroblasts obtained from rodent hearts. We present a method for isolation and subsequent culture of viable cardiac fibroblasts from ventricular tissue of adult human. This method allows rapid and reliable isolation and subsequent culture of cardiac fibroblasts from adult heart tissue without the need for cumbersome isolation techniques and complex nutrient-enriched and hormone-supplemented culture media for maintenance.     

Growth factor for cardiac hypertrophy

Cardiac size can be regulated by the balance in activity between cardiac growth factors and inhibiting factors, chalones. This study was undertaken to verify the role of the cardiac growth factor and its purification from hypertrophied hearts. For this propose the hypertrophied hearts of renovascular hypertensive rats were used. The purification was made by using an isoelectric focusing chromatography and the HPLC method. We examined the cardiac growth effect of the isolated fractions with cultured chicken embryonic cardiac myocytes. Simultaneously, the influence of these fractions on the cardiac cell cycle was examined by DNA analysis with the flow cytometric method. If the hearts were overloaded due to hypertension, the growth of the cardiac size could be induced by increased the level of five proteins with different molecular weight and with an isoelectric point of 8.3. The significant growth activities were observed at these five proteins compared to the absence of the fractions. For the appearance of these growth effect, it is necessary that the structure of the protein is there fundamentally as a form with a molecular weight of 27 k dalton. After addition of these isolated fractions, BrdU content is S and G2 phases by flow cytometry was increased. This change indicates that the cardiac myocytes are stimulated in form DNA synthesis.     

Stimulation of ribosomal RNA synthesis during hypertrophic growth of cultured heart cells by phorbol ester

Primary cultures of neonatal cardiac myocytes were used to determine the effects of tumor-promoting phorbol esters on ribosomal RNA (rRNA) synthesis during myocyte growth. Treatment of myocytes with phorbol-12,13-dibutyrate (PDBu) increased protein accumulation by 25% and RNA content by 20%. Rates of rRNA synthesis were measured to assess the mechanism by which rRNA accumulated during myocyte growth. Rates of rRNA synthesis were determined from the incorporation of [³H]uridine into UMP of purified rRNA and the specific radioactivity of the cellular UTP pool. After 24h of PDBu treatment, cellular rates of 18S and 28S rRNA synthesis were accelerated by 67% and 64%, respectively. The increased rate of rRNA synthesis accounted for the net increase in myocyte rRNA content after PDBu treatment.     

Impulse conduction and gap junctional remodelling by endothelin-1 in cultured neonatal rat ventricular myocytes

Endothelin-1 (ET-1) is an important contributor to ventricular hypertrophy and failure, which are associated with arrhythmogenesis and sudden death. To elucidate the mechanism(s) underlying the arrhythmogenic effects of ET-1 we tested the hypothesis that long-term (24 hrs) exposure to ET-1 impairs impulse conduction in cultures of neonatal rat ventricular myocytes (NRVM). NRVM were seeded on micro-electrode-arrays (MEAs, Multi Channel Systems, Reutlingen, Germany) and exposed to 50 nM ET-1 for 24 hrs. Hypertrophy was assessed by morphological and molecular methods. Consecutive recordings of paced activation times from the same cultures were conducted at baseline and after 3, 6 and 24 hrs, and activation maps for each time period constructed. Gap junctional Cx43 expression was assessed using Western blot and confocal microscopy of immunofluorescence staining using anti-Cx43 antibodies. ET-1 caused hypertrophy as indicated by a 70% increase in mRNA for atrial natriuretic peptide ( P < 0.05), and increased cell areas ( P < 0.05) compared to control. ET-1 also caused a time-dependent decrease in conduction velocity that was evident after 3 hrs of exposure to ET-1, and was augmented at 24 hrs, compared to controls ( P < 0.01). ET-1 increased total Cx43 protein by ∼40% ( P < 0.05) without affecting non- phosphorylated Cx43 (NP-Cx43) protein expression. Quantitative confocal microscopy showed a ∼30% decrease in the Cx43 immunofluorescence per field in the ET-1 group ( P < 0.05) and a reduced field stain intensity ( P < 0.05), compared to controls. ET-1-induced hypertrophy was accompanied by reduction in conduction velocity and gap junctional remodelling. The reduction in conduction velocity may play a role in ET-1 induced susceptibility to arrhythmogenesis.