不同消化时间对原代心肌细胞的影响

Influence of digestion time on the primary culture of cardimyocytes

目的原代心肌细胞培养是一项重要的体外实验技术,为建造心血管疾病相关模型提供了重要的基础保障。文中旨在通过探讨不同消化时间对原代心肌细胞培养的影响,得到一套简便易行并且能获得较高成活率、纯度及活性的原代心肌细胞的培养方法。方法采用单消化酶多次消化心肌组织,根据消化时间分为8 min组和10 min组,差速贴壁法和5-溴-2-脱氧尿苷抑制法获得纯度较高的心肌细胞。光学显微镜下观察心肌细胞形态,记录镜下不同区域心肌细胞搏动频率测定细胞活力。用台盼蓝染色法检测培养细胞的存活率,横纹肌肌动蛋白鉴定心肌细胞。最后建立心肌细胞肥大模型,比较实验组(血管紧张素Ⅱ)和对照组[10%FBS DMEM(H+)培养基]的细胞截面平均面积。结果与8 min组比较,10 min组原代心肌细胞大部分伸出伪足相互接触交织成网,逐渐形成细胞簇,立体感明显,细胞搏动及收缩更为明显而有力。分离纯化后原代心肌细胞的平均存活率均>90%,10 min组平均存活率显著高于8 min组[(95.4±0.8)%vs(93.0±0.8)%,P<0.05]。α-actin免疫细胞化学染色鉴定,8 min组和10 min组原代心肌细胞的阳性率均>95%。血管紧张素Ⅱ作用48 h后,10 min组原代心肌细胞实验组心房钠尿肽(atrial natriuretic peptide,ANP)、细胞截面平均面积较对照组表达均显著升高[(20.8±0.7)vs(15.8±0.5),(34.77±8.43)μm2vs(14.11±4.29)μm2,P<0.05],而消化时间为8 min的原心肌细胞截面平均面积和ANP值未见显著变化。结论采用消化时间为8 min的原代心肌细胞培养方法具有简便、省时、易操作等特点,最重要的是能够获得较高质量的心肌细胞。

Objective Primary myocardial cell culture,as an important technique for in vitro experiment,provides an essential basis for the establishment of models of cardiovascular diseases. This study aimed to investigate the influence of digestion time on primary myocardial cell culture and to find some simple primary culture methods for obtaining myocardial cells with high survival rate,purity,and activity. Methods We used only one collagenase for repeated digestion of myocardial tissue for 8 and 10 minutes,respectively,and obtained high-purity cardiomyocytes by differential adhesion and Brdu inhibition. We observed the morphological changes of the cardiomyocytes under the optical microscope,measured their vitality according to the beating frequency in different regions,and counted the living cells after typan blue staining. Finally,we established a model of myocyte hypertrophy for verification of cell activity and compared the cell surface area between the experimental（ angiotensinⅡ） and control（ 10% FBS DMEM[H＋] medium）groups. Results After cultured for 48 hours, the primary cardimyocytes in the 10 min group mostly extended pseudopodia,interwoven into a network and gradually forming cell clusters,with obviously powerful beating and contraction. After isolation and purification,the average survival rate of the cells was 90%,significantly higher in the 10 min group than in the 8 min（ [95. 4 ± 0. 8]%vs [93. 0 ± 0. 8]%,P〈0. 05）. The positive expression rate of cardiac α-actin was 95% in both of the groups. After cultured with angiotensinⅡ for 48 hours,the 10 min group showed a significantly increased level of atrial natriuretic peptide（ ANP）（ 20. 8 ± 0. 67 vs15. 8 ± 0. 48,P〈0. 05） and cell surface area（ [34. 77 ± 8. 43] μm2vs [14. 11 ± 4. 29] μm2,P〈0. 05） as compared with the 8 min group. Conclusion Primary culture of cardimyocytes with 8 min digestion is simple and time-saving,and what is most important,can obtain high-quality cardiomyocytes.