乳鼠心肌细胞的体外培养及生物学特性研究  

Primary culturing and biological identification of neonate rat myocardial cells

作　　者：何蕾[1] 张莉[2] 黄靖香[2] 赵斌[2] 崔雪梅[2] 宋光[3] 江朝光[1] 

机构地区：[1]解放军总医院外科重症监护科,北京100853 [2]解放军总医院骨科研究所,北京100853 [3]解放军总医院普外科,北京100853

出　　处：《解放军医学杂志》2007年第5期506-507,共2页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金资助项目(30572427)

摘　　要：目的探讨乳鼠心肌细胞的体外培养方法并对其生物学特性进行观察。方法1～3日龄新生SD大鼠10只,用无酶消化法行含血清培养基原代心肌细胞培养,连续观察细胞生长及传代情况40天。α-横纹肌肌动蛋白抗体标记培养细胞,以流式细胞仪行心肌细胞纯度鉴定,Giemsa染色观察培养心肌细胞形态,吖啶橙-碘化丙啶(AO-PI)染色法检测培养细胞活性。记录培养心肌细胞总数,计算单个乳鼠心脏细胞产量。结果原代组织块和单个心肌细胞在接种后24～48h贴壁,其中部分出现搏动并持续至观察期末。培养细胞群鉴定心肌细胞纯度为94.1%,活细胞比例95.3%,95%可信区间(CI)为91.6%～99.0%。平均每个乳鼠心脏单细胞产量为3.3×106个,95%CI为2.1×106～4.5×106个。结论无酶消化法培养的乳鼠心肌细胞纯度、活力及产量能够满足心肌细胞研究的需要。Objective To find out a way for culturing the myocardial cells of neonatal rats in vitro, and to study their biological characters. Methods Cardiomyocytes from the heart of 1--3 days old Sprague-Dawley rats were prepared by a modified protocol. Hearts were harvested and cut into pieces of about 1mm^3 in size, and placed into cell culture bottles with nylon without pre-treatment of digestive enzyme. DMEM supplemented with 10% （v/v） FCS was used for culture, with thymidine （6mg/ml） added to inhibit flbroblast growth. Cells in monolayer were formed on the gauze and the bottom of the culture flask, and then cells were isolated by 0. 25% trypsin digestion for 30 seconds. Cell suspension was transferred to 100cm^2 cell culture flasks at a density of 10^4 cells/cm^2 for identification, viability test and morphologic observation. The cells obtained were stained with monoclone anti-a-sarcomeric actinin and FITC to evaluate the purity of the myocardial cell preparation by flow cytometry, AOPI fluorescein stain was used to evaluate the viability and Giernsa staining for examining the morphology of the myocardial cells. Results 24-48 hours after culture, the myocardial cells became adherent to bottle wall to form a sheet, and began to pulsate. The purity of myocardial cells in culture cell population was 94. 13%, the ratio of viable myocardial cells was 95. 3%, and 95%CI was 91. 6%--99%. The output of myocytes from each neonatal heart was 3. 3X106 and 95%CI is 2. 1×10^6 --4. 5×10^6. Conclusion Neonatal myocytes culture in this way can generate large amount of viable single myocardial cells suitable for myocardial research in a short period.

关 键 词：细胞培养 流式细胞术 生物学鉴定法 心肌细胞 无酶消化法 

分 类 号：R54[医药卫生—心血管疾病]