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41.
Na+/H+交换体阻滞剂在培养乳鼠心肌细胞/复氧损伤中的保护作用及其机制 总被引:1,自引:0,他引:1
目的:探讨在缺氧/复氧损伤中Na+/H+交换体阻滞剂在不同时相用药的心肌细胞保护作用及其机制。方法:应用乳鼠心肌细胞建立心肌缺血-再灌注(I/R)损伤模型,实验分为对照组、缺氧/复氧全程给药组(EIPA-I组)、复氧前给药1组(EIPA-R1组)和复氧前给药2组(EIPA-R2组)进行缺氧/复氧处理,动态检测单个心肌细胞在缺氧/复氧时细胞胞质Ca2+浓度(即[Ca2+]i)的变化及细胞挛缩程度,并观察细胞的存活率。结果:EIPA-I组和EIPA-R2组显著抑制了[Ca2+]i上升及细胞的挛缩,并提高了细胞存活率。结论:在复氧前给予增大剂量的EIPA与缺氧时常规剂量用药同样有效地抑制复氧期Na+/H+的交换,减轻细胞内钙超载,抑制细胞的挛缩,提高心肌细胞的存活率。 相似文献
42.
目的构建人肌纤生成调节因子1全长的真核表达质粒,并观察其在HEK293T细胞系及Sprague-Daw-ley乳鼠心肌细胞中的表达。方法从NCBI GenBank数据库中克隆得到人肌纤生成调节因子1基因(AF417001)全长序列,与真核表达载体质粒pcDNA3.1/Myc-His(-)B连接并转化大肠杆菌XL1-Blue,筛选阳性克隆,T7引物测序,转染细胞后以逆转录聚合酶链反应、Western Blotting方法检测人肌纤生成调节因子1的表达。结果pcDNA3.1/Myc-His(-)B-hMR-1质粒经测序证实目的基因序列正确,无碱基突变。该质粒转染到HEK293T细胞系和乳鼠心肌细胞后人肌纤生成调节因子1的转录水平及表达水平明显增高。结论成功构建了人肌纤生成调节因子1全长的真核表达载体并确定了简便有效的乳鼠心肌细胞瞬时转染方法。 相似文献
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44.
目的建立新生小鼠心肌细胞(CM)的分离纯化、培养及鉴定的方法。方法采用酶消化法分离消化心室组织,差速贴壁法和化学方法纯化CM;培养7d后,利用免疫荧光技术和RT-PCR技术检测部分CM特异基因在蛋白和mRNA水平的表达;改进透射电镜的常规操作方法,观察CM超微结构。结果采用0.08%胰蛋白酶和0.04%胶原酶Ⅱ消化心室组织后,细胞成活率为98%,贴壁培养第3d出现同簇细胞的同步跳动;抗心肌肌钙蛋白(cTnT)免疫荧光染色阳性率为99.07%;以骨髓间充干细胞为阴性对照,RT-PCR结果显示CMα和β两型心肌肌球蛋白重链、cTnT基因表达呈阳性;经3%~4%琼脂预包埋法处理可以对少量细胞进行超薄切片的制作,CM具有正常的超微结构。结论建立了新生小鼠CM分离纯化培养和鉴定方法。 相似文献
45.
目的:对新生大鼠心肌细胞分离培养方法进行改进,以期获得较高纯度和活力的心肌细胞;方法:采用胶原酶消化法分离大鼠心肌细胞,控制消化时间、消化温度、搅拌速度、离心次数和速度等,结合贴壁分离和Brd U干预纯化心肌细胞,流式细胞仪分析肌钙蛋白Ⅰ(troponin Ⅰ,Tn Ⅰ)表达,荧光探针进行胞浆游离钙离子染色;结果:多数心肌细胞自动收缩,逐渐聚集形成肌管,troponin Ⅰ阳性率达92%,所有心肌细胞钙离子染色阳性,并随细胞收缩呈周期性变化;结论:该方法分离培养的心肌细胞具有较高的纯度和活力。 相似文献
46.
Survival and maturation of human embryonic stem cell-derived cardiomyocytes in rat hearts 总被引:6,自引:0,他引:6
Dai W Field LJ Rubart M Reuter S Hale SL Zweigerdt R Graichen RE Kay GL Jyrala AJ Colman A Davidson BP Pera M Kloner RA 《Journal of molecular and cellular cardiology》2007,43(4):504-516
Human embryonic stem cell (hESC)-derived cardiomyocytes are a promising cell source for cardiac repair. Whether these cells can be transported long distance, survive, and mature in hearts subjected to ischemia/reperfusion with minimal infarction is unknown. Taking advantage of a constitutively GFP-expressing hESC line we investigated whether hESC-derived cardiomyocytes could be shipped and subsequently form grafts when transplanted into the left ventricular wall of athymic nude rats subjected to ischemia/reperfusion with minimal infarction. Co-localization of GFP-epifluorescence and cardiomyocyte-specific marker staining was utilized to analyze hESC-derived cardiomyocyte fate in a rat ischemia/reperfused myocardium. Differentiated, constitutively green fluorescent protein (GFP)-expressing hESCs (hES3-GFP; Envy) containing about 13% cardiomyocytes were differentiated in Singapore, and shipped in culture medium at 4 degrees C to Los Angeles (shipping time approximately 3 days). The cells were dissociated and a cell suspension (2 x 10(6) cells for each rat, n=10) or medium (n=10) was injected directly into the myocardium within the ischemic risk area 5 min after left coronary artery occlusion in athymic nude rats. After 15 min of ischemia, the coronary artery was reperfused. The hearts were harvested at various time points later and processed for histology, immunohistochemical staining, and fluorescence microscopy. In order to assess whether the hESC-derived cardiomyocytes might evade immune surveillance, 2 x 10(6) cells were injected into immune competent Sprague-Dawley rat hearts (n=2), and the hearts were harvested at 4 weeks after cell injection and examined as in the previous procedures. Even following 3 days of shipping, the hESC-derived cardiomyocytes within embryoid bodies (EBs) showed active and rhythmic contraction after incubation in the presence of 5% CO(2) at 37 degrees C. In the nude rats, following cell implantation, H&E, immunohistochemical staining and GFP epifluorescence demonstrated grafts in 9 out of 10 hearts. Cells that demonstrated GFP epifluorescence also stained positive (co-localized) for the muscle marker alpha-actinin and exhibited cross striations (sarcomeres). Furthermore, cells that stained positive for the antibody to GFP (immunohistochemistry) also stained positive for the muscle marker sarcomeric actin and demonstrated cross striations. At 4 weeks engrafted hESCs expressed connexin 43, suggesting the presence of nascent gap junctions between donor and host cells. No evidence of rejection was observed in nude rats as determined by inspection for lymphocytic infiltrate and/or giant cells. In contrast, hESC-derived cardiomyocytes injected into immune competent Sprague-Dawley rats resulted in an overt lymphocytic infiltrate. hESCs-derived cardiomyocytes can survive several days of shipping. Grafted cells survived up to 4 weeks after transplantation in hearts of nude rats subjected to ischemia/reperfusion with minimal infarction. They continued to express cardiac muscle markers and exhibit sarcomeric structure and they were well interspersed with the endogenous myocardium. However, hESC-derived cells did not escape immune surveillance in the xenograft setting in that they elicited a rejection phenomenon in immune competent rats. 相似文献
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48.
The purpose of this essay is to overview our findings that membrane-associated calcium-independent phospholipase A2 is markedly inhibited by low, clinically relevant concentrations of anthracyclines. Our studies suggest that due to the essential
role of this enzyme in membrane homeostasis, its inhibition can be one of the early culprits leading to anthracycline-induced
cardiac dysfunction. The clinical importance and potential pharmaceutical use of this new phenomenon await further studies. 相似文献
49.
50.
目的: 探讨心肌细胞对结缔组织生长因子(CTGF)的基础表达及转化生长因子β1(TGF-β1)对其分泌的诱导作用,并进一步研究以RNA干扰(RNAi)技术靶向抑制CTGF对下游纤维化因子的影响。方法: 实验分为以下5组: 组Ⅰ:单纯心肌细胞组;组Ⅱ:TGF-β1诱导组;组Ⅲ:阴性质粒组;组Ⅳ:RNAi组;组Ⅴ:脂质体组。分离培养Sprague Dawley(SD)大鼠乳鼠心肌细胞,以5 μg/L终浓度的TGF-β1对培养的心肌细胞进行诱导,再通过脂质体法将靶向大鼠CTGF的短发夹RNA(shRNA)干扰质粒转染入心肌细胞,流式细胞技术分选出成功转染的细胞。通过反转录聚合酶链式反应(RT-PCR)和免疫印迹(Western blotting)技术进一步研究各实验组心肌细胞CTGF、纤维连接蛋白(FN)mRNA及蛋白的表达水平。结果: 单纯心肌细胞组对CTGF、FN均有低水平的基础分泌,在予以TGF-β1诱导后表达水平显著升高(P<0.01);而在靶向特异性RNAi后,上述因子的表达均被显著抑制(P<0.01);并且纤维化因子FN的表达水平与促纤维化因子CTGF的表达显著相关。结论: 心肌细胞可自主低水平分泌或诱导后高表达CTGF及下游纤维化因子FN,提示心肌细胞主动参与了心肌纤维化及心脏重塑过程;而通过RNAi靶向抑制CTGF后可阻抑心肌细胞分泌下游纤维化因子,进一步提示CTGF是促心肌纤维化的关键性细胞因子,而RNAi是一种特异高效的很有前途的干预手段。 相似文献