成年兔心耳肌細胞的分離標記與自體移植

成年兔心耳肌細胞的分離標記與自體移植

作者：張浩，蔡振杰，俞世強，趙璧君，張近寶
【關鍵詞】 心肌/細胞學
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細胞的急性分離和熒光標記的方法，觀察心耳肌細胞移植到自體左室前壁的存活狀況. 方法： 成年新西蘭白兔20只，隨機分為移植組和對照組（n=10）. 結扎并剪取自體左心耳組織，急性消化分離為單細胞，經DAPI標記后分別將細胞懸液和培養基注射到移植組和對照組自體正常左室前壁內. 4 wk后處死兔子，取移植區組織進行組織學觀察. 結果： 急性分離的細胞中絕大部分為桿狀，形態結構符合心耳肌細胞的特征，且細胞活性好. 移植組梗死區內可以觀察到“細胞島”狀結構，行熒光檢測可見藍色熒光的細胞核，而對照組梗死區內無細胞結構，證明移植的心耳肌細胞在移植區存活以及DAPI標記的可靠性. 與對照組相比,移植區血管密度增高（P=0.027）. 結論： 酶消化法和DAPI熒光標記是一套可靠的心耳肌細胞急性分離和標記方法；急性分離的心耳肌細胞移植到自體左室前壁后可以存活，并能夠促進周圍血管生長.
　　【關鍵詞】 心肌/細胞學；分離；標記；移植，自體
　　0引言
　　目前正在研究的心肌細胞移植技術是通過向已梗死的心肌組織內移入新的細胞，以改善心臟功能［1］. 本文旨在建立一套可靠的成年兔心耳肌細胞的分離標記方法，并對其移植到自體心肌組織進行研究, 為進一步應用心耳肌細胞移植治療缺血性心臟病提供實驗基礎.
　　1材料和方法
　　1.1材料
　　成年健康新西蘭白兔20只, 雌雄不限,體質量2.0~2.5 kg, 購自第四軍醫大學實驗動物中心. 將實驗動物隨機分為移植組和對照組（n=10）. 小牛血清(Gibco) ,DMEM高糖培養基(Gibco公司), 胰蛋白酶(Gibco公司) , 膠原酶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自體左心耳心肌組織的取材取成年兔經耳緣靜脈注射戊巴比妥鈉（30 mg/kg），左側胸骨旁切口，切斷第4肋骨進胸，向上推開胸腺，剪開心包，顯露并牽引左心耳，用4號絲線結扎左心耳基底部后，剪取左心耳并立即置于預冷（4℃）的普通臺式液中. 用鹽水紗布覆蓋傷口.
　　1.2.2左心耳心肌細胞的消化分離參照文獻［2］，在無鈣臺式液(mol/L: NaCl 140, KCl 5.4, MgCl2 1, 酶糖10, HEPES 5, pH 7.4)中洗去血凝塊，用眼科剪將心耳組織剪碎，移入含有1.25 g/L胰酶、0.25 g/L膠原酶的無鈣臺式液中，37℃消化10 min. 輕柔吹打，自然沉淀后棄上清. 再加入酶消化10 min，輕柔吹打后，吸取上清，移入等體積含100 mL/L新生牛血清的DM

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