小鼠乳鼠原代心肌细胞的改良培养  

Modified primary culture of neonatal mouse myocardial cells

作　　者：孟琳琳[1] 黄莺[1] 马依彤[1] 刘芬[2] 陈邦党[2] 陈小翠[2] 盖敏涛 

机构地区：[1]新疆医科大学第一附属医院心脏中心冠心病二科,新疆维吾尔自治区乌鲁木齐市830054 [2]新疆医科大学第一附属医院心脏中心科技楼冠心病实验室,新疆维吾尔自治区乌鲁木齐市830054

出　　处：《中国组织工程研究》2015年第37期5993-5997,共5页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金资助项目(81260022);新疆维吾尔自治区自然科学基金资助项目(2014211C040)~~

摘　　要：背景:大鼠心肌细胞原代培养技术日渐成熟,但小鼠心肌细胞在同样实验条件下不易获得,而小鼠基因组与人类基因组有更多相似之处,具有更高的研究价值。目的:改进小鼠乳鼠原代心肌细胞的培养方法,得到纯度高、活力强、保持心肌细胞原有结构和功能的心肌细胞。方法:将小鼠心肌组织利用酶消方法分离为单个的心肌细胞,实验步骤中将经典的胰酶、胶原蛋白酶混合酶消法分解开,先用胰酶消化心肌组织,使心肌组织变得松散,再用胶原蛋白酶,作用于细胞间质的胶原纤维,使心肌组织分离为单个的心肌细胞。调整胰酶和Ⅱ型胶原蛋白酶的浓度和作用时间,严格控制试剂p H值和各步骤的温度。结果与结论:心肌细胞在接种24 h后开始贴壁,48 h后可观察到部分心肌细胞出现自主搏动,72 h后彼此形成交联,96 h后可观察到心肌细胞形成细胞簇,并出现一致性搏动。心肌细胞的存活率和纯度均达95%以上。结果证实,实验采用改良方法成功培养出小鼠乳鼠心肌细胞,并且保留心肌细胞的结构和功能,纯度和成活率高,是可行的培养方法。BACKGROUND;A lot of work has been carried out on the development of the primary cultured rat myocardial cells at home and abroad.The primary culture technology of rat myocardial cells becomes more mature,but myocardial cells from neonatal mice are not easy to be obtained under the same experimental conditions.The mouse genome has more similarities with the human genome,which has a higher research value.OBJECTIVE;To improve the primary culture method of neonatal mouse myocardial cells,and to obtain myocardial cells with high purity,vitality and original structure and function.METHODS;The mouse cardiac tissues were treated using an enzyme digestion method to isolate isolated single myocardial cells;first,the cardiac tissues were digested using trypsin,and then collagenous fibers were treated with collagenase to isolate single myocardial cells.The concentration and action time of trypsin and type II collagenase were adjusted,and the p H values of reagents and temperature of each step were strictly controlled.RESULTS AND CONCLUSION;At 24 hours after inoculation,the myocardial cells began to be adherent; at 48 hours,independent pulsation of myocardial cells could be observed; at 72 hours,myocardial cells were cross-linked; and at 96 hours,myocardial cells formed cell clusters and presented with consistent beating.Thesurvival rate and purity of myocardial cells were both over 95%.This modified method could successfully culture myocardial cells with high purity and viablility from neonatal mice,and the structure and function of myocardial cells could be retained.Therefore,it is a feasible culture method.

关 键 词：组织工程 肌细胞 心脏 小鼠 组织构建 心肌细胞 小鼠乳鼠 原代细胞培养 酶消法 Ⅱ型胶原蛋白酶 作用时间 心肌搏动 细胞簇 温度 p H值 国家自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]