心肌样细胞和聚乳酸-聚乙醇酸共聚物在体内构建工程化心肌组织
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摘要
目的探索以骨髓间充质干细胞(BMMSCs)诱导分化的心肌样细胞为种子细胞、以聚乳酸-聚乙醇酸共聚物(PLGA)作为支架材料,在大鼠体内构建工程化心肌组织的可实施性。方法分离培养大鼠BMMSCs,取第3代细胞用5-氮胞苷和血管紧张素Ⅱ联合诱导24 h继续培养3周,将诱导分化的心肌样细胞种植到PLGA支架上形成移植物,在孵箱里孵育3 d,然后将其移植到预先制备好的大鼠腹膜腔囊袋之中。4周以后,取出移植物并采用HE染色观察心肌样细胞的形态特征、用免疫组织化学染色法检测工程化心肌细胞肌钙蛋白(c Tn) I的表达、透射电镜下观察心肌样细胞的形态和结构。结果 HE染色结果显示,在PLGA支架上可见到梭形的细胞核,且心肌样细胞分布均一;免疫组织化学染色结果显示PLGA-心肌样细胞组绝大多数移植细胞表达心肌特异性蛋白c Tn I;透射电镜观察显示,在体内构建的工程化心肌组织中,可以看到肌丝沿细胞的长轴平行排列,胞浆中富含大量的线粒体和内质网,以及桥粒结构、缝隙连接和Z线样物质。结论成功的建立了在大鼠体内构建工程化心肌组织的方法。这种体内微环境有助于移植组织或细胞的存活,在大鼠体内构建的工程化心肌组织具有与天然心肌组织相似的结构。
AIM To explore the practicability of constructing engineered myocardial tissues( EMTs) in vivo by cardiomyocyte-like cells derived from bone marrow mesenchymal stem cells( BMMSCs) for seeded cells and polylactic acid-co-glycolic acid( PLGA) for scaffolds. METHODS By density-gradient centrifugation,BMMSCs were isolated from the tibia and femur of Sprague-Dawley( SD) rats. BMMSCs were cultured for the third passage and cells were induced by the combination of 5-azacytidine and angiotensin II for 24 h. Cells were cultured by replacement of the medium for 3 weeks. The differentiated cardiomyocytelike cells were seeded on PLGA scaffolds to form grafts which were incubated in the incubator for three days,and then transplanted into the prefabricated peritoneal cavity of SD rats. After four weeks,the graft was removed and its morphology and structure were detected by HE staining,immunohistochemical staining( troponin I,cTnI) and transmission electron microscopy. RESULTS HE staining showed that the cardiomyocyte-like cells were evenly distributed in the PLGA scaffold and the nuclei were spindle shaped. The results of immunohistochemical staining demonstrated that most of the transplanted cells in PLGA-cardiomyocyte-like cells group were positive for specific protein cTnI. Transmission electron microscopy showed that in the engineered myocardial tissue constructed in vivo,the myofilaments were arranged parallel to the long axis of the cells and there were abundant mitochondria and endoplasmic reticulum in the cytoplasm. Desmosomes,gap junctions and Z line-like substances were also observed.CONCLUSION The present investigation explored a method of constructing engineered myocardial tissue in vivo. The microenvironment in vivo can help engrafted tissues or cells to survive. In vivo,engineered myocardial tissues which share similarities with the native heart tissues can be constructed in rats in vivo.
AIM To explore the practicability of constructing engineered myocardial tissues( EMTs) in vivo by cardiomyocyte-like cells derived from bone marrow mesenchymal stem cells( BMMSCs) for seeded cells and polylactic acid-co-glycolic acid( PLGA) for scaffolds. METHODS By density-gradient centrifugation,BMMSCs were isolated from the tibia and femur of Sprague-Dawley( SD) rats. BMMSCs were cultured for the third passage and cells were induced by the combination of 5-azacytidine and angiotensin II for 24 h. Cells were cultured by replacement of the medium for 3 weeks. The differentiated cardiomyocytelike cells were seeded on PLGA scaffolds to form grafts which were incubated in the incubator for three days,and then transplanted into the prefabricated peritoneal cavity of SD rats. After four weeks,the graft was removed and its morphology and structure were detected by HE staining,immunohistochemical staining( troponin I,cTnI) and transmission electron microscopy. RESULTS HE staining showed that the cardiomyocyte-like cells were evenly distributed in the PLGA scaffold and the nuclei were spindle shaped. The results of immunohistochemical staining demonstrated that most of the transplanted cells in PLGA-cardiomyocyte-like cells group were positive for specific protein cTnI. Transmission electron microscopy showed that in the engineered myocardial tissue constructed in vivo,the myofilaments were arranged parallel to the long axis of the cells and there were abundant mitochondria and endoplasmic reticulum in the cytoplasm. Desmosomes,gap junctions and Z line-like substances were also observed.CONCLUSION The present investigation explored a method of constructing engineered myocardial tissue in vivo. The microenvironment in vivo can help engrafted tissues or cells to survive. In vivo,engineered myocardial tissues which share similarities with the native heart tissues can be constructed in rats in vivo.
引文
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[16]Tiburcy M,Meyer T,Soong PL,et al. Collagen-based engineered heart muscle[J]. Methods Mol Biol,2014,1181:167-176.
[17]Fujita B,Zimmermann WH. Myocardial Tissue Engineering for Regenerative Applications[J]. Curr Cardiol Rep,2017,19(9):78.
[18]Lee SJ,Yang CS,Kim DD,et al. Microenvironmental interactions and expression of molecular markers associated with epithelial-tomesenchymal transition in colorectal carcinoma[J]. Int J Clin Exp Pathol,2015,8(11):14270-14282.
[2] Eschenhagen T,Didie M,Munzel F,et al. 3D engineered heart tissue for replacement therapy[J]. Basic Res Cardio,2002,97(4):146-152.
[3]Leor J,Amsalem Y,Cohen S. Cells,scaffolds,and molecules for myocardial tissue engineering[J]. Pharm acol Ther,2005,105(2):151-163.
[4]Zimmermann WH,DidieM,D9ker S,et al. Heart muscle engineering:an update on cardiac muscle replacement therapy[J]. Cardiovasc Res,2006,71(3):419-429.
[5] Leor J,Landa N,Cohen S. Renovation of the injured heart with myocardial tissue engineering[J]. Expert Rev Cardiovasc Ther,2006,4(2):239-252.
[6] Zimmermann WH,Schneiderbanger K,Schubert P,et al. Tissue engineering of a differentiated cardiac muscle construct[J]. Circ Res,2002,90(2):223-230.
[7]Vilquin JT,Rosset P. Mesenchymal stem cells in bone and cartilage repair:current status[J]. Regen Med,2006,1(4):589-604.
[8]Fukuda K. Development of regenerative cardiomyocytes from mesenchymal stem cells for cardiovascular tissue engineering[J]. Artif Organs,2001,25(3):187-193.
[9] Makino S,Fukuda K,Miyoshi S,et al. Cardiomyocytes can be generated from marrow stromal cells in vitro[J]. J Clin Invest,1999,103(5):697-705.
[10]Lattanzi L,Salvatori G,Coletta M,et al. High efficiency myogenic conversion of human fibroblast by adenoviral vector-mediated Myo D gene transfer[J]. Clin Invest,1998,101(10):2119-2128.
[11]Jeong SI,Kim SH,Kim YH,et al. Manufacture of elastic biodegradable PLCL scaffolds for mechano-active vascular tissue engineering[J]. J Biomater Sci Polym Ed,2004,15(5):645-660.
[12]Allison DD,Grande-Allen KJ. Hyaluronan:a powerful tissue engineering tool[J]. Tissue Eng,2006,12(8):2131-2140.
[13]Kadner A,Hoerstrup SP,Zund G,et al. A new source for cardiovascular tissue engineering:human bone marrow stromal cells[J].Eur J Cardiothorac Surg,2002,21(6):1055-1060.
[14]Li XK,Cai SX,Liu B,et al. Characteristics of PLGA-gelatin complex as potential artificial nerve scaffold[J]. Colloids Surf B Biointerfaces,2007,57(2):198-203.
[15]Hu X,Shen H,Yang F,et al. Preparation and cell affinity of microtubular orientation-structured PLGA(70/30)boold vessel scaffold[J]. Biomaterials,2008,29(21):3128-3136.
[16]Tiburcy M,Meyer T,Soong PL,et al. Collagen-based engineered heart muscle[J]. Methods Mol Biol,2014,1181:167-176.
[17]Fujita B,Zimmermann WH. Myocardial Tissue Engineering for Regenerative Applications[J]. Curr Cardiol Rep,2017,19(9):78.
[18]Lee SJ,Yang CS,Kim DD,et al. Microenvironmental interactions and expression of molecular markers associated with epithelial-tomesenchymal transition in colorectal carcinoma[J]. Int J Clin Exp Pathol,2015,8(11):14270-14282.