成年SD大鼠心肌原代成纤维细胞分离及培养的优化方案

Optimized Isolation and Culture Method of Primary Fibroblast in Adult SD Rat

目的:摸索及优选成年SD大鼠心肌原代成纤维细胞的体外分离、培养及鉴定的实验方法。方法:将成年SD大鼠心脏剪成小组织块,采用以下四种方案(A:0.08%胰酶+0.1%胶原酶II消化15 min,B:0.2%胶原酶II消化15 min,C:0.2%胶原酶II消化60min,D:0.2%胶原酶II消化90 min)提取成年大鼠心脏原代成纤维细胞,再通过差速贴壁分离方法培养原代成纤维细胞。采用倒置显微镜观察成纤维细胞的基本形态特征,并进行Vimentiin免疫荧光染色对培养的原代细胞进行荧光鉴定;采用台盼兰染色对培养的原代成纤维细胞存活率进行鉴定;采用细胞计数对培养的成纤维细胞生长趋势进行鉴定。结果:四种方法均能培养成纤维细胞,但单酶消化60 min可一次性提取较多细胞,并且细胞状态佳,3 d即可传代。72 h成纤维细胞Vimentin免疫荧光染色阳性率高达97%。台盼兰染色可见其细胞死亡率明显降低,并且细胞计数可见细胞生长状态极佳。结论:单酶消化60 min是提取成年SD大鼠心肌原代成纤维细胞的高效、快速、稳定的实验方法,为心脏疾病的基础及临床研究提供了较为理想的细胞学实验模型。

Objective： To explore and optimize the isolation, culture and identification of cardiac primary fibroblasts of adult SD rats in vitro. Methods： The heart of Adult SD rat is cuted into small tissue blocks then using the following four methods（A： 0.08% trypsin＋0.1% collagenase II, digestion 15 min; B： 0.2% collagenase II, digestion 15 min; C： 0.2% collagenase II, digestion 60 min; D： 0.2% collagenase II, digestion 90 min） to isolate primary cardiac fibroblast. At last primary cultured fibroblasts are cultured through differential adherent method. The basic morphological characteristics of fibroblasts are observed by inverted microscope, and the primary cells are identified by Vimentin immunofluorescence staining. The survival rate of the cultured fibroblasts is identified by using the method of cell counting. Results： The primary cardiac fibroblast can be isolated through above four methods, but the method of single enzyme digestion60 min can culture more cells in good condition, and can go down to next generation at 3 d. The positive rate of Vimentin immunofluorescence staining is nearly up to 97% at 72 h. The cell death rate is significantly decreased by Blue staining, and cell count reveals that the cell growth status is excellent. Conclusion： The most efficient, fast and stable method for the extraction of the primary fibroblasts of adult SD rats is single enzyme digestion 60 min which provides an ideal experimental cell model for the cardiac basic and clinical research.