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作 者:董悦[1] 张雷[1] 邵素霞[1] 尹青[1] 陈炜[1] 赵春芳[1]
机构地区:[1]河北医科大学组织胚胎教研室,石家庄050017
出 处:《中国细胞生物学学报》2010年第3期361-369,共9页Chinese Journal of Cell Biology
基 金:河北省自然科学基金(No.C2008001030)资助项目~~
摘 要:采用5μmol/L 5-氮胞苷(5-azacytidine,5-aza)诱导Wistar大鼠缺血心脏成纤维细胞(fibroblasts of ischemia myocardium,FIMs)分化为心肌样细胞,利用抗心肌特异性肌钙蛋白(cardiacTroponin T,C-TnT)和抗α-横纹肌肌动蛋白(α-sarcomeric actin,α-SA)间接免疫荧光标记,透射电镜以及Real-time PCR检测血管紧张素原(angiotensinogen,AGT)和脑钠肽(brain natriuretic peptide,BNP)mRNA含量,鉴定诱导分化的心肌样细胞。采用扫描电镜鉴定两种不同支架组织(脱细胞大肠肌膜和脱细胞胶原膜),将心肌样细胞分别接种于两种支架组织上生长5~7天,利用抗C-TnT,α-SA间接免疫荧光和扫描电镜,观察工程化心肌样组织生长情况。结果发现,诱导分化的心肌样细胞胞质内含有丰富的肌微丝,对抗C-TnT和α-SA间接免疫荧光标记反应阳性,Real-time PCR检测显示心肌样细胞内AGT和BNP mRNA表达明显高于对照组FIMs,差别有显著性(P<0.05)。在两种不同支架组织上生长,心肌样细胞呈多边形有突起或柱状分支状片状平铺排列,对抗C-TnT和α-SA反应阳性,细胞胞浆内可见肌微丝。研究表明,FIMs能诱导分化为心肌样细胞,在不同支架组织上生长,构建工程化心肌样组织。Fibroblasts of ischemia myocardium (FIMs) were obtained from the ischemia myocardium of adult Wistar rats and induced by 5-azacytidine (5-aza) to be differentiated to cardiomyocyte-like cells (CLCs), which were identified by indirect immunofluorescence against cardiac Troponin T (C-TnT) and α-sarcomeric actin (α- SA) antibodies and transmission electron microscope (TEM). Real-time PCR was performed to assay expressions of both angiotensinogen (AGT) and brain natriuretic peptide (BNP)mRNA in CLCs and FIMs. Scanning electron microscope (SEM) was used to evaluate two different scaffolds of acellular colon muscle film (ACMF) scaffold and Bio-Gide/Bio-Gide Perio (Bio-Gide) scaffold. CLCs were seeded on both ACMF scaffold and Bio-Gide scaffold and cultured for 5-7d. Indirect immunofluorescence against C-TnT and α-SA antibodies, SEM observation was done respectively to identify the myocardial-like tissue engineering. The results found that the cytoplasms of CLCs contained rich myofilaments, and were positive for C-TnT and α-SA. Real-time PCR revealed that the expressions of both AGT and BNP mRNA were much higher in CLCs than those in FIMs (P〈0.05). CLCs showed elongated and/or polyhedral type, arranged in multilayers and stretched on ACMF scaffold and Bio-Gide scaffold to form myocardial-like stratums displayed rich myofilaments in the cytoplasms and were positive for C-TnT and tx-SA. It was suggested that FINIs could differentiate into CLCs and be constructed myocardial-like tissue engineering on the different scaffolds.
分 类 号:R318[医药卫生—生物医学工程]
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