心肌球培养基血清浓度优化研究

摘要

Objective To compare the effects of different concentrations of fetal bovine serum (excell) on culturing of rats' cardiac stem cells (CSCs), and to explore the optimal concentration of fetal bovine serum in culture medium for CSCs.Methods Fifteen healthy newborn Wistar rats (1 day after birth) were selected randomly, and their hearts were picked out carefully under sterile condition, which were digested with trypsin and collagenaseⅡ repeatedly and then planted into culture flasks.The tissue-derived cardiac progenitor cells (CPCs) were cultured in complete medium containing 20% fetal bovine serum, and the external plant derived cells (EDCs) were randomly divided into three groups which were cultured with cardiacl sphere growth medium of 10%, 11%, 15% fetal bovine serum respectively.After the comparison of the number of CSCs, growth cycle and growth curve, the optimal serum concentration of cardiac sphere culture medium was screened.The third generation of CSCs were used for test of cellular surface antigen staining of C-kit to identify the percentage of C-kit positive CSCs.Results CSCs were successfully separated from myocardial tissues of neonatal rats, and the positive rate of c-kit was 87%.Cell number was represented by cellular population doubling level(PDL), the PDL of each group showed a uniform increase, and the comparative result of PDL and rate of growth among the three groups (growth curve) was: B>C>A.The CSCs obtained from EDCs cultured with medium containing 11% fetal calf serum had a short growth period (P<0.05) and a greater cell number (P<0.05).Conclusion Rat cardiac stem cells could be isolated and cultured more rapidly and more largely using the culture medium containing 11% fetal bovine serum.%目的 通过比较含有不同胎牛血清浓度的心肌球培养基分离培养大鼠心肌干细胞(CSCs)的效果,探讨最优的心肌干细胞血清培养浓度.方法 选取15只健康新生Wistar大鼠(出生1天龄),无菌条件下取出心脏,经胰酶和Ⅱ型胶原酶反复消化后,种植于培养瓶中,应用含20%胎牛血清的完全培养基培养组织块源性心脏祖细胞(CPCs),将所得的培养外植物源性细胞(EDCs)随机分为A、B、C 3组,各组5瓶细胞(25 mL塑料培养瓶),分别应用含10%、11%、15%不同浓度胎牛血清的心肌球生长培养基培养,培养至第三代CSCs时行细胞表面抗原C-kit染色,鉴定C-kit阳性CSCs占所获得细胞的比率,比较所得的细胞数量,生长周期和生长曲线.结果 从新生大鼠心肌组织中成功分离培养出CSCs,C-kit阳性率87%.培养后所得细胞数量以细胞倍增水平(PDL)表示,各组细胞PDL呈匀速递增,PDL水平和其递增速率(生长曲线所示)比较:B组>C组>A组.应用含11%胎牛血清的心肌球生长培养基培养EDCs获得的CSCs,与其他两组相比,具有生长周期短(P<0.05),生长数量多的特点(P<0.05).结论 含11%胎牛血清浓度是心肌球生长培养基培养原代大鼠心肌干细胞的最优血清培养浓度.