胶原海绵复合新生大鼠原代心肌细胞构建工程化心肌组织  

Construction of engineered myocardial tissue using collagen sponges and primary cardiocytes in neonatal rats

作　　者：李军杰[1] 吕安林[1] 刁繁荣[1] 苑媛[1] 张荣庆[1] 胡小菁[1] 

机构地区：[1]解放军第四军医大学西京医院心内科,陕西省西安市710032

出　　处：《中国组织工程研究与临床康复》2007年第9期1609-1612,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的:探索以胶原海绵为支架、新生大鼠原代心肌细胞为种子细胞,于体外构建工程化心肌组织的方法。方法:实验于2005-12/2006-11在解放军第四军医大学西京医院心内科实验室完成。Ⅰ型胶原海绵剪切成方形片状(2.0cm×1.4cm×0.2cm),经60Co照射消毒,于DMEM培养液中水化1h左右。另取1d龄SD大鼠心脏,剪成小碎块,然后用2.5g/L胰蛋白酶于37℃中消化,吸取上清至含胎牛血清的DMEM中,重复消化四五次,用差速贴壁法除去大部分成纤维细胞,将细胞沉淀用DMEM培养液以2×109L-1的密度悬浮备用。将上述的心肌细胞悬液1mL缓慢滴注于玻璃模型中的胶原海绵上,然后置于细胞培养中培养。肉眼及显微镜主要观察工程化心肌组织在培养期间的自发收缩情况,包括收缩的部位、强度、频率、一致性以及收缩随时间变化的情况。苏木精-伊红染色观察工程化心肌组织内胶原纤维的变化,细胞形态,胞核的形状及细胞之间的连接。免疫组织化学染色和透射电镜观察工程化心肌组织片的形态和功能。结果:①细胞接种于胶原海绵上1d后,细胞/胶原复合物的凝胶化过程基本完毕,体积保持恒定,维持至培养结束,第3天细胞/胶原复合物局部出现点片状自发收缩,第5天整个细胞/胶原复合物出现同步化自发收缩,收缩频率61～199次/min。2周后37.5%的工程化心肌组织的自发收缩活动减弱,但75%的工程化心肌组织的自发收缩活动持续至培养结束。②苏木精-伊红染色、免疫组织化学染色和透射电子显微镜显示,工程化心肌组织内细胞间连接广泛存在,细胞多呈纵向分布,胞核呈长圆形,胞浆内α-肌节肌动蛋白阳性,胞内肌原纤维排列整齐,可见到心肌特异性的肌小节结构和Z线,多数细胞具有分化的心肌细胞表型。结论:用新生大鼠原代心肌细胞为种子细胞、以Ⅰ型胶原海绵为支架材料,构建出的工程化心肌组织,于体�AIM： To construct engineered myocardial tissue （EMT） in vitro using collagen sponge and primary cardiocytes in neonatal rat. METHODS： The exporiment was performed in the Cardiovascular Laboratory of Xijing Hospital of Fourth Military Medical University of Chinese PLA from December 2005 to November 2006. Collagen sponge type I was sheared to square piece （2.0 cm×1.4 cm×0.2 cm）, sterilized by ^60 Co irradiation, and hydrated in DMEM for 1 hour. SD rats of one day old were used for broken bits, which were digested by 2.5 g/L trypsin at 37 ℃ and then by DMEM containing supematant for 4-5 times. Neonatal rat cardiocytes were isolated with differential adhesion technique, precipitated and suspended at the density of 2×10^9 L^-1, then added to the collagen sponge segment gradually and cultured in the incubator. Microscopy and naked eye observation were used to determine the spontaneous contraction of EMT, including location, strength, frequency and consistency of contraction as well as its change with time. Routine hematoxylin-eosin （HE） staining was adopted for the change of collagen fibers, morphous, shape of nuclei and cellular connection. Immunohistochemical staining and transmission electron microscopy were applied to assess the morphous and the functions of the EMT. RESULTS： （1）At first, gelatiflcation of cell/collagen compound finished and pontaneous beating areas in the EMTs were seen not long after seeding. Synchronous contraction of the EMT was observed on the 3^rd day of culture at the frequency of 61-199 beats per minute. Two weeks later, synchronous beating attenuated in 37.5% of EMT but 75% of EMT continued the beating until culture was over. （2）HE staining, immunohistochemical staining and transmission electron microscopy analyses revealed that intercellular contacts were very widespread, the majority of the cardiocytes stretched in the EMTs, nuclei were elliptic, α-actins in most cardiocytes were positive and cardiocytes in the EMTs contained arranged myofibrils as well

关 键 词：工程化心肌组织 新生大鼠原代心肌细胞 胶原海绵 

分 类 号：R318.08[医药卫生—生物医学工程]