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The objectives of this study were to establish a protocol for the isolation of metabolically viable ventricular cardiomyocytes from the rainbow trout and to determine which measures may best reflect viability 24 h after isolation. Cardiomyocytes were isolated by enzymatic digestion and maintained in cell suspension. Viability was assessed using Trypan blue dye exclusion, ATP content, oxygen consumption and lactate dehydrogenase (LDH) leakage into the medium. Viability, assessed by these measures did not significantly change over the time period of this study. ATP content did correlate significantly with oxygen consumption but not with Trypan blue exclusion. We conclude that primary cultured cardiomyocytes remain metabolically viable for at least 24 h after isolation. Also, it appears that ATP content and oxygen consumption most adequately reflect metabolic cell viability. To be confident with a culture, however, a combination of viability measures is necessary when isolating cardiomyocytes from fish. 相似文献
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目的 观察加味丹参饮含药血清对缺氧/复氧（hypoxia/reoxygenation,H/R）乳鼠心肌细胞p38 MAPK蛋白表达的影响,以探讨其保护心肌的作用机制。方法 将20只SD乳鼠的心肌细胞进行原代培养,建立H/R模型并随机分为H/R组、SB203580（p38MAPK阻断剂组）、加味丹参饮含药血清组,正常心肌细胞组作为对照组。采用T淋巴细胞化学染色法鉴定心肌细胞,罗丹明B（Rhodamine B）染色法观察细胞形态,MTT比色法检测含药血清对乳鼠心肌细胞的毒性,Western-blot法检测MKK3（促分裂原活化蛋白激酶-激酶3）、MKK6（促分裂原活化蛋白激酶-激酶6）、p38MAPK、phospho-p38MAPK蛋白表达。结果 与正常血清对照组比较,H/R组MKK3、MKK6的蛋白表达增多,p38MAPK通路阻断剂SB203580和加味丹参饮均不能减少其表达;p38MAPK、phospho-p38MAPK在H/R时表达均增加（P<0.01）,而SB203580和加味丹参饮含药血清均能减少其表达（P<0.01）。结论 加味丹参饮含药血清预处理可通过抑制缺氧,复氧心肌细胞p38MAPK信号通路发挥保护作用,抑制p38MAPK表达及其磷酸化,减轻心肌细胞损伤,起到保护心肌细胞的作用。 相似文献
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本试验旨在研究银杏叶提取物（Ginko biloba extract,GBE）对热应激致鸡心肌细胞氧化损伤的保护作用。取12日龄AA肉鸡心脏制备鸡原代心肌细胞,向原代心肌细胞培养板中分别加入0、5、10、50、100、150 mg/mL GBE,CCK-8试剂盒检测心肌细胞存活率;将原代心肌细胞以1×104个/孔接种于96孔板,培养48 h,分别加入0、5、10、50、100、200、500、1 000 μg/mL GBE,Hoechst 33258检测心肌细胞凋亡率,确定最适GBE浓度。42℃热应激处理（0、0.5、1、2和4 h）建立心肌细胞损伤模型,ELISA试剂盒检测乳酸脱氢酶（LDH）、细胞丙二醛（MDA）和谷胱甘肽过氧化物酶（GSH-Px）含量,以及超氧化物歧化酶（SOD）活性;Western blotting检测Hsp72蛋白表达。结果显示,高浓度GBE具有细胞毒性,50、100、150 mg/mL GBE能极显著抑制细胞生长（P<0.01）,50 μg/mL GBE能够极显著提高细胞增殖率（P<0.01）。热应激后细胞发生凋亡,细胞内MDA、LDH含量增加,SOD活性、GSH-Px含量减少,产生氧化损伤;GBE可以降低热应激时细胞LDH和MDA的含量,增加SOD活性和GSH-Px的含量,热应激2、4 h,GBE处理效果明显。此外,GBE可增加心肌细胞中Hsp72蛋白的表达。综上所述,GBE能降低热应激状态下心肌细胞的氧化损伤,其机制可能与提高抗氧化酶活性及提高Hsp72的表达有关。 相似文献
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采用大鼠心肌细胞条件培养基对兔胚胎干细胞(Embryonic stem cells,ESC)进行分离、传代培养,研究大鼠心肌细胞条件培养基对兔ESC分离培养效果的影响。结果显示,用SD大鼠心肌细胞条件培养基培养的兔ESC集落呈岛屿状生长,碱性磷酸酶染色呈强阳性,体外分化可形成类胚体状结构,贴壁的类胚体周边会出现许多分化的上皮样细胞或单个散在的细胞;常规冻存后再传代的兔ESC集落具有较为一致的生长特征,并呈现胚胎干细胞集落所特有的岛屿状生长形态;传至第5代的兔ESC集落核型正常率>75％。表明SD大鼠心肌细胞条件培养基可用于兔胚胎干细胞分离培养。 相似文献
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为解析热应激对大菱鲆心脏损伤及其机制,实验从组织形态、生理生化反应及凋亡基因表达等多个水平,分别使用H.E染色法、电镜观察法、酶活性检测法、qPCR检测基因表达法开展了本研究。结果显示,随着温度升高,心肌纤维肿胀,断裂,间质宽度增加,炎性细胞浸润,线粒体结构破坏等组织损伤现象加重,但在24°C-24 h时组织损伤明显减轻;CK活性随着热应激加剧显著升高;LDH、SOD活性,MDA含量在24°C时达到峰值,表明大菱鲆遭受到热应激,心肌防御酶发挥抵抗作用,维持机体稳态。qPCR显示,大菱鲆心肌细胞Bax基因和Caspase-3基因变化趋势一致,随着热应激的加剧,表达量降低,而Bcl-2基因逐渐升高。表明在热应激程度较轻时,大菱鲆心肌通过降低Bax、Caspase-3基因表达,促进抗凋亡基因Bcl-2的表达,减少心肌细胞丢失来减少热应激损伤。当热应激加剧至28°C时,热应激超过自身生理调节阈值,损伤加重,机体防御系统自身也受损,造成大菱鲆心脏结构严重损伤甚至机体死亡。研究表明,随着温度升高,大菱鲆心肌损伤加重,机体通过调节心肌防御酶活性以及使细胞凋亡,最大限度维持稳态,减少组织损伤。超过24... 相似文献
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Effects of aflatoxin B1 on mitochondrial respiration,ROS generation and apoptosis in broiler cardiomyocytes 
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下载免费PDF全文 Aflatoxin B1 (AFB1) develops various toxic effects in the liver by impairing mitochondrial function, inducing cell apoptosis. However, little is focused on its toxicity to broiler cardiomyocytes (BCMs). Here, the mitochondrial membrane potential (MMP), reactive oxygen species (ROS) generation, cardiac troponin T (cTnT) location, apoptosis induced by AFB1, and antioxidative genes were investigated in BCMs. It was found that AFB1 evoked intracellular ROS generation, and induced apoptosis in BCMs. AFB1 treatment resulted in increased percentage of apoptotic cells, increased location range of cTnT in cytoplasm, upregulated messenger RNA (mRNA) expression of nuclear factor erythroid 2‐related factor 2 (Nrf2) and downregulated mRNA expressions of Mn‐superoxide dismutase in BCMs. These findings suggested AFB1 treatment caused significant cardiomyocyte damage and cardiotoxicity, impairment of mitochondrial functions, activated ROS generation, and induced apoptosis, and probably was involved in the Nrf2 signal pathway in BCMs. 相似文献
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对采用组织块法分离培养的大鼠心肌细胞进行了鉴定及相关的生物学特性研究,以进一步用于心脏细胞工程、组织工程的种子细胞及其他方面的研究。结果显示,分离培养出的心肌细胞呈多种形态,整体形如铺路石样,生长中的心肌细胞常伸出伪足,形状为星形,培养至第4天的心肌细胞常可相互交织形成细胞簇,出现节律性的搏动;心肌细胞的PAS糖原染色呈阳性,胞浆内大量的糖原颗粒被染成品红色;细胞凋亡-Hoechst检测试剂盒荧光染色显示,心肌细胞核内荧光物质分布均匀一致,没有出现细胞凋亡时所特有的荧光致密浓染或碎块状致密浓染现象;对第1代和第6代心肌细胞生长曲线测定显示,心肌细胞生长曲线呈"S"型,2代心肌细胞生长曲线趋于一致;心肌细胞α-sarcomaricactin(α-SA)免疫组化鉴定显示,心肌细胞呈强阳性且具有较高纯度。上述结果表明,利用该方法培养的心肌细胞具有较高的增殖能力和纯度,可作为种子细胞用于心脏的细胞工程和组织工程及其他方面的研究。 相似文献
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LIANG Qing LI Zi-cheng KUANG Su-hua HUANG Wei-qing HUANG Min-jian LIN Jun-min LIANG Zi-jing 《园艺学报》2013,29(11):2024-2029
AIM: To study the effects of metoprolol (Meto) on the apoptosis of neonatal rat cardiomyocytes and the phosphorylation of connexin 43 (Cx43) induced by norepinephrine (NE). METHODS: Neonatal SD rat cardiomyocytes were divided into the following five groups (n=6 in each group): (1) control (Con) group: no treatment; (2) NE group: treatment with NE at 0.1 μmol/L for 24 h; (3) NE+Meto group: simultaneous treatment with NE and Meto both at 01 μmol/L for 24 h; (4) NE+Meto+PD98059 group: pretreatment with extracellular signal-regulated kinase (ERK) phosphorylation inhibitor PD98059 at 10 μmol/L for 30 min and then treatment with NE and Meto both at 01 μmol/L for 24 h; (5) NE+PD98059 group: pretreatment with PD98059 at 10 μmol/L for 30 min and then treatment with NE at 01 μmol/L for 24 h. The beating rates of cardiomyocytes in various groups were calculated, and the viability of cardiomyocytes was assayed by MTT method. The Cx43 mRNA expression was detected by RT-PCR, and the protein expression of phosphorylated Cx43 (p-Cx43), phosphorylated ERK1/2 (p-ERK1/2) and cleaved caspase-3 was detected by Western blotting. RESULTS: (1) Separate NE treatment could significantly increased the beating rate of cardiomyocytes and reduced cell viability, while Meto showed the opposite effects. PD98059 treatment had no significant effect on cardiomyocyte beating rate, but suppressed Meto to improve cell viability to some extent. (2) Compared with Con group, separate NE treatment significantly increased the Cx43 mRNA expression (P<001). Compared with NE group, Meto or PD98059 intervention could significantly inhibited Cx43 mRNA expression (both P<001), and simultaneous treatment with Meto and PD98059 could further suppress Cx43 mRNA expression up-regulated by NE (P<001). (3) Compared with NE group, Meto significantly inhibited the increased p-Cx43, p-ERK1/2 and cleaved caspase-3 expression induced by NE (P<001), and simultaneous treatment with Meto and PD98059 could further enhance the inhibition of p-Cx43, p-ERK1/2 and cleaved caspase-3 expression by Meto (P<001). PD98059 treatment had no significant effect on the increased p-Cx43 and cleaved caspase-3 expression induced by NE (P>005). CONCLUSION: The inhibitory effect of Meto on NE-induced cardiomyocyte apoptosis is related to the inhibition of Cx43 phosphorylation, which may be partly mediated via ERK1/2 pathway. 相似文献
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【目的】研究牛诱导性多能干细胞(Bovine induced pluripotent stem cells,biPSCs)向心肌细胞的分化能力。【方法】将biPSCs悬浮培养制备类胚体,采用类胚体介导体外自由分化与定向诱导分化(添加RA、DMSO和RA+DMSO3种诱导物)的方法,使biPSCs在体外分化,比较了biPSCs在3种不同心肌诱导条件下定向分化为心肌细胞的诱导效率;分别提取biPSCs、类胚体及不同诱导体系分化细胞的总RNA,用RT-PCR检测心肌细胞发育标志基因的表达。【结果】biPSCs在悬浮条件下培养7d,能够形成典型球形和囊腔样类胚体。类胚体再贴壁培养7d,经过免疫细胞化学检测,贴壁细胞大量分化为α-actin阳性的心肌样细胞。在体外诱导分化体系中,RA+DMSO共同诱导的效率较RA和DMSO单独诱导显著增高(P0.05)。RT-PCR结果显示,各组分化后的细胞中心肌细胞发育特异性基因ACTA2与GATA4均有高水平表达。【结论】biPSCs在体外悬浮培养能够形成类胚体,类胚体贴壁培养后可以分化为α-actin阳性的心肌样细胞;分化形成的心肌样细胞均表达心肌特异性基因。 相似文献
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为研究牦牛血低聚肽对缺氧所致H9c2心肌细胞损伤以及对运动后小鼠抗疲劳能力的影响,本研究通过建立H9c2 细胞缺氧损伤模型以及小鼠负重游泳试验,研究其抗缺氧能力以及抗疲劳能力。结果表明,与正常对照组相比,缺氧组的H9c2细胞存活率极显著降低(P<0.01),表明缺氧对H9c2心肌细胞有一定损伤;与缺氧组相比,低聚肽干预组的细胞存活率增加且存在剂量依赖性;与常用抗缺氧药物红景天苷相比,牦牛血低聚肽高剂量组细胞存活率为75.1%,而红景天苷组为88.0%。牦牛血低聚肽及其复合肽均能延长小鼠负重游泳时间,提高运动后小鼠肝脏肝糖元(HG)含量,降低运动后小鼠血清乳酸(BLA)和血清尿素氮(BUN)含量。综上,牦牛血低聚肽对缺氧介导的心肌细胞损伤具有一定的保护作用,能提高小鼠的抗疲劳能力,且牦牛血低聚肽与大豆低聚肽按照 5∶5 比例复配后的抗疲劳能力优于单一使用牦牛血低聚肽。本研究结果为耐缺氧活性因子的研究提供了一条新途径。 相似文献