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| 原代人心包间质细胞的分离培养方法及鉴定 |
| 龚德军1△,吴洪玉2△,张锡武1,袁扬1,陶婧1,陆方林1,韩林1,徐志云1,刘晓红1* |
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(1. 第二军医大学长海医院胸心外科，上海 200433;
2. 第二军医大学长海医院消化内科，上海 200433
△共同第一作者
*通信作者) |
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| 摘要: |
| 目的 建立原代人心包间质细胞分离、培养方法。方法 将心包组织标本剪切成1 mm×1 mm×1 mm大小，采用Ⅱ型胶原酶与透明质酸酶联合胰蛋白酶消化分离、培养获得细胞。观察细胞形态，测定细胞倍增时间，应用流式细胞术与免疫组织化学染色对细胞进行鉴定。结果 分离培养的细胞接种24 h后有细胞贴壁生长，10 d左右细胞融合，可进行传代培养。细胞呈梭形或纺锤样外观，细胞核明显、核仁清晰、核质比例大。流式细胞术检测显示细胞表面标记物CD34（-）、CD45（-），免疫组织化学检测显示CK（-）、vimentin（+）、α-SMA（ ）。10例心包组织采用上述方法分离培养细胞，其中8例获得成功。结论 本方法可高效地分离、培养原代人心包间质细胞，为缩窄性心包炎体外研究提供了途径。 |
| 关键词: 心包间质细胞 细胞培养技术 缩窄性心包炎 |
| DOI：10.3724/SP.J.1008.2014.00500 |
| 投稿时间：2013-09-06修订日期：2013-11-06 |
| 基金项目:国家自然科学基金（81170217，81370336）. |
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| Isolation, culture and identification of primary human pericardial interstitial cells |
| GONG De-jun1△,WU Hong-yu2△,ZHANG Xi-wu1,YUAN Yang1,TAO Jing1,LU Fang-lin1,HAN Lin1,XU Zhi-yun1,LIU Xiao-hong1* |
(1. Department of Cardiothoracic Surgery, Changhai Hospital, Second Military Medical University, Shanghai 200433, China;
2. Department of Gastroenterology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
△Co-first authors.
* Corresponding authors.) |
| Abstract: |
| Objective To establish a method for isolation, culture and identification of primary human pericardial interstitial cells (PICs). Methods Human pericardial tissues were cut into 1 mm×1 mm×1 mm sized pieces, digested with type Ⅱ collagenase, hyaluronic acid enzyme and trypsin to isolate the interstitial cells. The cells were cultured and cell morphology was observed. The cell doubling time, flow cytometry and immunohistochemical staining were applied for cell identification. Results Adherent cells were observed 24 h after culturing. The cells reached 80% confluences and could be subcultured after about 10 days. The cells isolated and cultured were fibroblast-like or spindle-like, with obvious nuclei, clear nucleolus, and a greater proportion of nucleus and cytoplasm. Flow cytometry results showed no cell surface markers CD34 and CD45. Immunocytochemistry showed negative CK staining, and positive vimentin and α-SMA staining. Cells isolated from 8 of the 10 pericardial tissues were successfully cultured. Conclusion The present method can effectively isolate and culture primary human PICs, which lays a foundation for further in vitro study of constrictive pericarditis. |
| Key words: pericardial interstitial cells cell culture techniques constrictive pericarditis |
