解剖学报

解剖学报 ›› 2013, Vol. 44 ›› Issue (6 ): 784-788.doi: 10.3969/j.issn.0529-1356.2013.06.010

• 细胞和分子生物学 • 上一篇    下一篇

Ⅰ、Ⅱ型胶原酶分离心肌细胞的效果比较及分析

王慧玲1 郭康2 郭志坤1   

  1. 1.新乡医学院，河南省医用组织再生重点实验室; 2.第三附属医院肿瘤科， 河南 新乡 453003
  • 收稿日期:2013-02-04 修回日期:2013-07-02 出版日期:2013-12-06 发布日期:2013-12-06
  • 通讯作者: 郭志坤 E-mail:gzk@xxmu.edu.cn
  • 基金资助:

    河南省21世纪优秀人才支持计划（2005HANCET215）;省自然科学基金资助项目

Comparative ound analysis effects of separated cardiomyocytes by collagenase Ⅰ and Ⅱ

WANG Hui-ling1 GUO Kang2 GUO Zhi-kun 1*   

  1. 1.Key Laboratory for Tissue Regeneration of He’nan Province; 2.Tumor Department, the Third Affiliated Hospital,Xinxiang Medical University, He’nan Xinxiang 453003, China
  • Received:2013-02-04 Revised:2013-07-02 Online:2013-12-06 Published:2013-12-06
  • Contact: GUO Zhi-kun E-mail:gzk@xxmu.edu.cn

摘要：

目的 比较Ⅰ型和Ⅱ胶原酶对大鼠心肌细胞分离的分离效果，并对其成因进行分析。 方法 利用Ⅰ、Ⅱ型胶原酶分别消化心肌组织，比较两种酶对心肌细胞的分离效果；采用免疫组织化学、免疫荧光和Western blotting技术检测成年大鼠心肌组织中是否存在Ⅱ型胶原；用大鼠剑突软骨部组织的Ⅱ型胶原和心肌组织的Ⅲ型胶原作为阳性对照；用SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)检测Ⅱ型胶原酶试剂的成分。 结果 Ⅰ、Ⅱ型胶原酶分别消化心肌组织，所获心肌细胞数量分别为 (5.66±0.83)×106和(5.61±1.01)×106，心肌细胞存活率分别为（74.17±6.27）％及（75.50±9.07）％，两者差异无显著性。分离的心肌细胞大部分不搏动，极少数细胞保持自律性收缩；Ⅱ型胶原在成年大鼠心肌组织中呈阴性表达，在剑突的软骨组织中呈阳性表达，Ⅲ型胶原在心肌组织中呈阳性表达；Western blotting检测显示，心肌组织中并无Ⅱ型胶原表达；SDS聚丙烯酰胺凝胶电泳结果显示，Ⅱ型胶原酶试剂是一种包括5种蛋白的混合物。 结论 成年大鼠心肌组织中无Ⅱ型胶原，用Ⅱ型胶原酶分离大鼠心肌细胞不具有针对性。

关键词: Ⅱ型胶原酶, Ⅱ型胶原, 心肌, 免疫组织化学, 免疫荧光, 大鼠

Abstract:

 Objective To explore the role of collagenase Ⅱ in the separation of adult rat cardiomyocytes. Methods Collagenase Ⅰ and collagenase Ⅱ were used to digest cardiomyocytes and their effect of separating cardiomyocytes was compared. Immunohistochemistry, immunofluorecence and Western blotting were used to detect existence of collagen Ⅱ in adult rat myocardium. Collagen Ⅱ of xiphoid and collagen Ⅲ of myocardium were taken as positive control. SDS-PAGE(polyacrylamide gel electrophoresis) method was used to detect the composition of collagenase Ⅱ. Results After digesting cardiomyocyte with collagenase Ⅰ or collagenase Ⅱ, the number of the obtained myocardial cells were (5.66±0.83)×106 and (5.61±1.01) ×106, the survival rates of cardiomyocyte were（74.17±6.27）％and（75.50±9.07）%, respectively, and no significant difference was found. Most separated cardiocytes did not beat while only very few of them had automaticity systole; Collagen Ⅱ showed negative expression in myocardium of adult rats but positive expression in cartilaginous tissue of xiphoid; Collagen Ⅲ also showed positive expression in myocardium. Western blotting did not show the expression of collagen Ⅱ in myocardium. SDS-PAGE results showed collagenase Ⅱ was a mixture of five kinds of protein. Conclusion The collagen Ⅱ did not exist in myocardium, thus collagenase Ⅱ should not be used to separate adult rat cardiomyocyte.

Key words: Collagenase Ⅱ, Collagen Ⅱ, Myocardium, Immunohistochemistry, Immunofluorecence, Rat