褪黑素对脂多糖所致新生乳鼠心肌细胞能量代谢障碍的影响？？

目的：探讨褪黑素（melatonin,MT）对脂多糖（I。PS）诱导新生乳鼠心肌细胞能量代谢障碍的影响。方法：培养12d龄乳鼠心肌细胞,建立体外培养模型,随机分为4组：对照组、I。PS组、低剂量MT预处理组和高剂量MT预处理组,检9n．4各组ATP、ADP及AMP水平,乳酸脱氢酶（I。DH）和线粒体琥珀酸脱氢酶（MSDH）活性;利用荧光分光光度计测定线粒体膜电位（mitochondrialmembranepo—tentiaI,MMP）;电子显微镜下观察线粒体形态改变。结果：与对照组相比,I。PS组ATP、ADP及AMP水平明显下调,乳酸脱氢酶活性明显上调,线粒体琥珀酸脱氢酶活性及线粒体膜电位明显下调,差异均有统计学意义（P〈0．05）。以上指标在低剂量MT预处理组和高剂量MT预处理组均被抑制（P〈0．050．01）。同时,MT能改善LPS诱导的心肌超微结构的异常改变。结论：褪黑素可抑制LPS诱导的新生乳鼠心肌细胞能量代谢障碍。     

S100B immunoreactivity: a new marker of hypoxia-related cardiac damage in newborn piglets

Abstract

Objective: The evaluation of the expression of S100B protein, in the swine heart in an experimental model of hypoxia - reoxygenation.

Methods: Normocapnic hypoxia was induced in 40 male Landrace/Large White neonatal piglets by decreasing the inspired concentration of oxygen to 6–8%. When animals developed bradycardia or severe hypotension, reoxygenation was initiated. Piglets were allocated in four groups of 10, according to the oxygen concentration they were reoxygenated with: Group 1, 2, 3 and 4 resuscitated with 18%, 21%, 40% and 100% oxygen, respectively. The animals were further classified into 4 groups according with the time required for reoxygenation: group A (<15?min); group B (16–60?min); group C (>60?min); group D (deceased animals).

Results: Immunostaining for S100B protein was detected in 14 out of the 40 heart samples (35%), both inside the cytoplasm of cardiomyocytes and as globular deposits in the interstitial spaces. Significant differences were observed among groups 1–4 regarding S100B expression. Reactivity for S100B in cardiac cells was detected in 50%, 50%, 10% and 33% of animals in groups 1 and 2, 3 and 4, respectively. Marked differences were also observed among groups A–D: 75%, 33%, 12% and 22% of the animals in group 1, 2, 3 and 4, respectively, showed reactivity for S100B in the heart.

Conclusions: Expression of S100B protein occurred in the heart of some of newborn piglets following severe hypoxia. S100B storage in cardiomyocytes correlates with the different oxygen concentration used during reoxygenation, being higher in piglets reoxygenated with 18% and 21%, and lower in animals reoxygenated with 40% oxygen. Intermediate levels of S100B expression were found in 100% O₂-treated animals. The finding of a higher percentage of S100B-immunoreactive hearts in piglets with a fast recovery and the detection of a decreased reactivity in animals with a slow and a very slow recovery clearly indicates S100B protein as an early protective factor with a positive prognostic value in asphyxiated newborn piglets.     

Metallothionein-I/II null cardiomyocytes are sensitive to Fusarium mycotoxin butenolide-induced cytotoxicity and oxidative DNA damage

Previous studies revealed butenolide (BUT), a Fusarium mycotoxin distributes extensively, induced myocardial oxidative damage, which could be abated by antioxidants such as glutathione. Metallothionein (MT) has proved to attenuate several oxidative cardiomyopathies via its potent antioxidant property. The present study is therefore undertaken to investigate the protective potential of the endogenous expression of MT against BUT-induced myocardial toxicity. Primary cultures of neonatal cardiomyocytes from MT-I/II null mice along with the corresponding wild-type mice will be utilized to determine the possible mechanistic properties of MT. BUT treatment to the cardiomyocytes evoked significant cytotoxicity as evidenced by morphological changes and concentration- and time-dependent reductions in cell viability. Additionally, BUT treatment remarkably increased reactive oxygen species (ROS) production in the cardiomyocytes of both MT-I/II null and wild-type mice. As a result, noticeable DNA damage in both cardiomyocytes was detected by alkaline comet assay. Furthermore, the comparison between the MT-I/II null and wild-type cardiomyocytes indicated that ROS production in the cardiomyocytes from the MT-I/II null mice was higher than from wild-type mice. DNA damage as evaluated by percentage of comet tail DNA, tail length and tail moment was more severe in the MT-l/II null cardiomyocytes than in wild-type myocytes. And in agreement with those results mentioned above, the MT-l/II null cardiomyocytes were more sensitive to BUT-induced cytotoxicity than wild-type cardiomyocytes. Taken together, these findings clearly show that basal MT can efficiently attenuate BUT-induced cytotoxic injuries in cardiomyocytes via the inhibition of intracellular ROS production, and associated DNA damage.     

Expression of cTnI-R145G affects shortening properties of adult rat cardiomyocytes

The R145G amino acid exchange in the inhibitory subunit (cTnI) of cardiac troponin, which regulates muscle contraction, is related to familial hypertrophic cardiomyopathy. Information on its impact on contractility of adult cardiomyocytes is scarce. We studied shortening of adult rat cardiomyocytes before and during ss-adrenergic stimulation using adenovirus-driven expression of human cTnI-wild type (wt) and cTnI-R145G. Baseline sarcomere shortening was significantly decreased by cTnI-R145G expression. Upon ss-adrenergic stimulation using isoproterenol (ISO), nearly identical amplitudes of shortening were obtained with cells expressing cTnI-R145G and control cardiomyocytes (native and cTnI-wt). However, rates of shortening and relengthening were depressed in cTnI-R145G-expressing cells but were comparable to those of control cells upon addition of forskolin or ISO and ICI118,551. This indicates that cTnI-R145G expression influences the response to ss-adrenergic stimulation dependent on the receptor subtype.     

Isolation and preparation of single mouse cardiomyocytes for confocal microscopy

Myofibril degeneration is a common hallmark of cardiomyopathies leading to cardiac failure. However, information regarding myofibrillar organization in histologic analysis of sectioned tissue is limited by both large size and variable orientation of the cardiomyocytes. In order to observe individual cardiomyocytes from a single mouse heart, we have developed a new procedure for trapping dissociated cardiomyocytes on a polyester mesh. Cells can then be visualized by fluorescence confocal microscopy and three-dimensional reconstruction of cells can be performed without distortion of intracellular organization resulting from cytofugation. Since the cells are trapped between the mesh and coverslip, the mesh is not seen in reconstructed images. Sarcomeric protein organization in cardiomyocytes can now be conveniently studied using this procedure. Furthermore, individual atrial or ventricular cardiomyocytes are easily identified in the preparation and decreased expression of -myosin heavy chain protein expression is apparent in propythiouracil (PTU) treated mice. This procedure will be a valuable asset in the analysis of intracellular changes occurring within the hearts of cardiomyopathic mice.     

circRNA_100395通过结合miR-31-5p抑制心肌细胞肥大相关基因的表达

目的:探讨环状RNA(circRNA)_100395对心肌细胞肥大表型的影响及其作用机制.方法:通过RT-qPCR检测健康器官捐献者(n=8)与心衰患者(n=14)心肌组织中circRNA_100395及其宿主基因Kelch蛋白样家族成员20(KLHL20)的表达水平.原代分离、培养乳小鼠心室肌细胞(NMVCs),分别...     

黄芪甲苷对过氧化氢致损心肌细胞的保护作用及机制研究

目的 探讨黄芪甲苷对过氧化氢致损心肌细胞的保护作用及机制.方法 选取新生Wistar 大鼠3只,体外分离大鼠原代心肌细胞,按0.6×106/ml密度接种至75 cm2培养瓶培养,待80%融合后予0.5 mmol/L过氧化氢培养基培养4 h,分别加入100、10、1、0.1 μmol/L黄芪甲苷培养基6 ml培养24 h;设立对照组,仅加入等量普通培养基.观察心肌细胞存活率,乳酸脱氢醇(LDH)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)酶活性情况,bax和bcl-2基因的表达,bax和bcl-2蛋白的表达情况.结果 与不同浓度黄芪甲苷共培养24 h的过氧化氢致损心肌细胞的成活率(0.1、1、10、100 μmol/L黄芪甲苷组分别为48%、55%、57%、52%)均明显高于对照组(30%),(均P＜0.01);黄芪甲苷可降低H2O2致损心肌细胞内的bax mRNA和蛋白的表达,提高bcl-2 mRNA和蛋白的表达.结论 黄芪甲苷对过氧化氢致损心肌细胞有保护作用,其机制可能是通过调节bax/cbl-2基因和蛋白的表达实现的.

Abstract：

Objective To study the effects of Astragaloside Ⅳ on injured rat cardiomyocytes caused by hydrogen peroxide (H2O2). Methods Three clean Wistar rats were supplied by Experimental Animal Center,Xingjiang medical university. Primary rat cardiomyocytes were isolated in vitro and cultured in 75 cm2 flasks. When cells were at 80% confluence, they were cultured with 0.5 mmol/L H2O2 medium for 4 hours and then treated with 6 ml 100, 10, 1,0. 1 μmol/L Astragaloside Ⅳ medium for24 hours. Cells in the control group were treated with an equal volume of normal medium. Cell survival rate were examined by trypan blue exclusion assay. Enzyme activities of lactate dehydrogenase(LDH), superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px) were assayed by Elisa. The mRNA and protein expression were detected with realtime polymerase clain reaction(PCR) and Westernhlot method separately. Results Astragaloside Ⅳ promoted cardiomyocytes survival rate in a dose-dependent manner, while high concentration showed an inhibition tendency. Astragaloside Ⅳ decreased LDH activity (P ＜0.01 ) but increased SOD and GSH-Px activities (P ＜0.05 ). The results of Realtime PCR and Westernblot showed that Astragaloside Ⅳ could suppress expression of bax mRNA and protein, promote expression of cbl-2 mRNA and protein. Conclusions Astragaloside Ⅳ has protective effects on injured rat cardiomyocytes caused by H2O2, which is probably associated with regulating expression of bax/bcl-2 gene and protein.     

体外转染Cyclin A2基因对原代大鼠心肌细胞增殖的影响

目的探讨体外转染细胞周期素A2(Cyclin A2)基因对原代大鼠心肌细胞增殖的影响,为心脏再生提供细胞学依据。方法 SD乳鼠12只,分离、培养、鉴定原代乳鼠心肌细胞并分为3组,实验组:转染带有强化绿色荧光蛋白(GFP)和Cyclin A2基因的重组腺病毒(Ad-Cyclin A2-GFP);空病毒组:转染不含目的基因带有GFP的重组腺病毒(Ad-Null-GFP);阴性对照组:未做转染处理,仅加入等量的培养基。利用GFP示踪技术,评估原代心肌细胞转染效率;转染后的细胞继续体外培养3~5d,利用免疫荧光技术分别检测Cyclin A2、磷酸化组蛋白H3(H3P)、心肌肌钙蛋白-T(c Tn T)。结果 1.荧光GFP示踪表明原代心肌细胞的转染效率高达(97±0.74)%;2.免疫荧光标记显示,空病毒组和对照组结果相似;心肌细胞特异性标记蛋白c Tn T分布于细胞质,原代心肌细胞纯度高达(95±0.62)%;心肌细胞Cyclin A2主要在胞核内聚集,少数分布于细胞质。H3P为核蛋白在细胞核内分布。3.转染Cyclin A2后,实验组H3P阳性率明显高于空病毒组和对照组,差异具有统计学意义(P0.05);实验组可见大量多核细胞,以双核为主,伴少量3核细胞。结论腺病毒作为转染载体对原代心肌细胞有很好的侵染效率;Cyclin A2超表达促进原代心肌细胞形成双核。     

体外培养状态下心肌细胞有丝分裂的证据

目的探讨体外培养状态下心肌细胞有丝分裂、增殖的证据。方法分离新生大鼠心肌细胞,分别原代培养0h至12d;免疫荧光分别双标心肌肌钙蛋白-T(c Tn T)、磷酸化组蛋白H3(H3P)和微管蛋白(tubulin),利用活细胞工作站和荧光显微镜观察处于分裂期的心肌细胞。结果原代培养的心肌细胞生长状态好、纯度高,可观察到分裂各期的心肌细胞及其微管蛋白的变化。处于分裂各期的心肌细胞不隆起变圆仍维持扁平状。分裂前期细胞核形状规则,染色质凝集,H3P开始表达;前中期核膜破裂,纺锤体形成;中期染色体排列在赤道面,纺锤体呈典型纺锤样;后期染色单体分开并移向两极;末期子核形成,平行微管聚结在两子核之间;胞质分裂期形成两个子细胞,两个细胞分开后,可见有少量微管呈丝状连接。原代培养1~12d的心肌细胞在整个细胞分裂图像中很少见到双核心肌细胞。培养第3天开始发现分裂象的心肌细胞,占总心肌细胞数的2/127(1.57%)和1/92(1.09%),培养第7天,分裂细胞比率达3/69(4.35%)和5/163(3.07%),培养第12天有2/100(2%)和0/60(0)的心肌细胞处于分裂期。结论体外培养的心肌细胞存在一定数量的完全有丝分裂,是心肌增殖和心肌再生的细胞学证据。