大鼠心肌干细胞体外分离培养的方法研究

Study on method for isolation and culture of rat cardiac stem cell in vitro

目的对比不同温度下采用纤维黏连蛋白（FN）包被分离培养大鼠心肌干细胞（CSC）的效果，以筛选体外大鼠心肌干细胞的最佳分离培养方法。方法选取健康新生Wistar大鼠，在无菌条件下取出心脏，分别经胰酶和Ⅱ型胶原酶反复消化后于FN包被的培养瓶中培养，将第3代CSC进行免疫组化细胞表面抗原鉴定。将在37℃及室温（22℃-25℃）下用FN包被培养的原代细胞分别设为A组及B组，在37℃及室温（22℃～25℃）下用FN包被培养的CSC分别设为C组及D组，比较A组与B组、C组与D组的细胞贴壁时间、首次传代时间及细胞倍增水平。结果从新生大鼠心肌组织内成功分离培养出CSC，细胞表面抗原c-kit阳性率为83％。A组及C组的贴壁时间、首次传代时间分别均短于B组、D组（P〈0．05）。细胞倍增水平呈C组〉A组〉D组〉B组。结论应用37℃FN包被培养瓶的方法分离培养原代大鼠心肌干细胞效果更优。

Objective To compare the efficacies of fibronectin （FN） under different temperatures on isolation and culture of rat cardiac stem cell（CSC） , thereby to screen the optimum method for isolation and culture of rat CSC in vitro. Methods The hearts of healthy newborn Wistar rats were removed under aseptic condition. And the heart tissues were digested by trypsin and collagenase Ⅱ separately and repeatedly, and then were cultured in cell cuhure flasks coated by FN. Cell surface antigen identification was performed in the CSCs of the third generation by immunohistochemistry. The primary cells cultured by FN coating under 37℃ and room temperature（22℃ -25℃ ） were taken as Group A and Group B respectively. The CSCs cultured by FN coating under 37℃ and room temperature（22℃ -25℃ ） were taken as Group C and Group D respectively. The time for cellular adherence, the first generation time and cellular multiplication level were compared between Group A and Group B,as well as Group C and Group D. Results CSCs were separated from heart tissues of newborn rats successfully ,and the positive rate of cell surface antigen c-kit was 83%. The time for cellular adherence and the first generation time in Group A were shorted than those in Group B,and the times in Group C were also shorter than those in Group D （P 〈 0.05）. The cellular multiplication levels decreased in the order of Group C,Group A,Group D and Group B. Conclusion The efficacy of culture flasks coated by FN under 37℃ is superior in the isolation and culture of rat CSCs.