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成年兔心耳肌细胞的分离标记与自体移植
作者：张浩，蔡振杰，俞世强，赵璧君，张近宝【关键词】 心肌/细胞学
Isolation, labelling and autotransplantation of left auricle myocytes in adult rabbit
【Abstract】 AIM: To develop a reliable method for isolation and fluorescent labeling of adult rabbit left auricle myocytes and to investigate the survival of grafted autologous left auricle myocytes. METHODS: Twenty adult New Zealand rabbits were randomly assigned to transplant group and control group (n=10). The left auricle of rabbit was ligated and harvested and the auricular cells were isolated and labeled with DAPI ex vivo. Either a cell suspension (transplant group) or culture medium (control group) was injected into the normal left ventricular anterior wall. The rabbits were sacrificed after 4 weeks and specimens were harvested and observed by histologic methods. RESULTS: Most of the isolated cells were observed rodshaped. The morphologic feature of these cells corresponded to that of auricle cardiomyocytes and the cell activity was good. The “cell island” could be found in myocardial infarction (MI) area of transplant group, and the nuclei with blue fluorescence could be found in transplant group, but not in control group, which confirmed the survival of implanted cells and the reliability of DAPI labeling. Vascular density in transplant group was better than that in control group (P=0.02). CONCLUSION: Enzyme digestion and DAPI labeling are reliable methods for the isolation and labeling of left auricle myocytes in adult rabbits. Isolated and DAPI labeled auricle myocytes can survive after autografted into normal ventricular anterior wall and may improve peripheral vascular proliferation.
【Keywords】 myocardium/cytology;isolation; label; transplantation, autologous
【摘要】 目的： 建立可靠的成年兔心耳心肌细胞的急性分离和荧光标记的方法，观察心耳肌细胞移植到自体左室前壁的存活状况. 方法： 成年新西兰白兔20只，随机分为移植组和对照组（n=10）. 结扎并剪取自体左心耳组织，急性消化分离为单细胞，经DAPI标记后分别将细胞悬液和培养基注射到移植组和对照组自体正常左室前壁内. 4 wk后处死兔子，取移植区组织进行组织学观察. 结果： 急性分离的细胞中绝大部分为杆状，形态结构符合心耳肌细胞的特征，且细胞活性好. 移植组梗死区内可以观察到“细胞岛”状结构，行荧光检测可见蓝色荧光的细胞核，而对照组梗死区内无细胞结构，证明移植的心耳肌细胞在移植区存活以及DAPI标记的可靠性. 与对照组相比,移植区血管密度增高（P=0.027）. 结论： 酶消化法和DAPI荧光标记是一套可靠的心耳肌细胞急性分离和标记方法；急性分离的心耳肌细胞移植到自体左室前壁后可以存活，并能够促进周围血管生长.
【关键词】 心肌/细胞学；分离；标记；移植，自体
0引言
目前正在研究的心肌细胞移植技术是通过向已梗死的心肌组织内移入新的细胞，以改善心脏功能［1］. 本文旨在建立一套可靠的成年兔心耳肌细胞的分离标记方法，并对其移植到自体心肌组织进行研究, 为进一步应用心耳肌细胞移植治疗缺血性心脏病提供实验基础.
1材料和方法
1.1材料
成年健康新西兰白兔20只, 雌雄不限,体质量2.0~2.5 kg, 购自第四军医大学实验动物中心. 将实验动物随机分为移植组和对照组（n=10）. 小牛血清(Gibco) ,DMEM高糖培养基(Gibco公司), 胰蛋白酶(Gibco公司) , 胶原酶Ⅱ型（Sigma公司）, 4,6二脒基2苯茚二酮(Sigma公司).
1.2方法
1.2.1自体左心耳心肌组织的取材取成年兔经耳缘静脉注射戊巴比妥钠（30 mg/kg），左侧胸骨旁切口，切断第4肋骨进胸，向上推开胸腺，剪开心包，显露并牵引左心耳，用4号丝线结扎左心耳基底部后，剪取左心耳并立即置于预冷（4℃）的普通台式液中. 用盐水纱布覆盖伤口.
1.2.2左心耳心肌细胞的消化分离参照文献［2］，在无钙台式液(mol/L: NaCl 140, KCl 5.4, MgCl2 1, 酶糖10, HEPES 5, pH 7.4)中洗去血凝块，用眼科剪将心耳组织剪碎，移入含有1.25 g/L胰酶、0.25 g/L胶原酶的无钙台式液中，37℃消化10 min. 轻柔吹打，自然沉淀后弃上清. 再加入酶消化10 min，轻柔吹打后，吸取上清，移入等体积含100 mL/L新生牛血清的DM
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