| 刘美丽 刘丹 刘艳霞 田孝祥 闫承慧.成年SD大鼠心肌原代成纤维细胞分离及培养的优化方案[J].现代生物医学进展英文版,2016,16(27):5223-5226. |
| 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 |
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| DOI： |
| 中文关键词: 成年SD大鼠 原代成纤维细胞 原代培养 |
| 英文关键词: Adult SD rat Primary fibroblast Primary culture |
| 基金项目:国家重点基础研究发展计划（973 计划）（2012CB517804)；国家自然科学基金青年基金项目(81500282) |
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| 中文摘要: |
| 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60
min，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。 |
| 英文摘要: |
| 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 digestion
60 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 SDrats is single enzyme digestion 60 min which provides an ideal experimental cell model for the cardiac basic and clinical research. |
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