新生小鼠Sca-1^+心脏干细胞的分离与鉴定

Isolation and identification of Sca-1 positive cardiac stem cells in newborn mice

目的:分离、纯化新生小鼠干细胞抗原-1(Sca-1)阳性心脏干细胞,观察其特异性标记物。方法 :将新生小鼠的心脏剪碎后用Ⅱ型胶原酶及胰酶反复消化。组织法培养10~14d后结合免疫磁珠进行阳性分选出Sca-1^+心脏干细胞。分选纯化后的细胞通过流式细胞术和免疫荧光的方法进行Sca-1的鉴定,并通过免疫荧光的方法鉴定心源性转录因子GATA-4和Nkx-2.5的相关蛋白。结果:组织培养和磁珠分选法结合成功分离并纯化了小鼠Sca-1^+心脏干细胞。分选后流式结果显示Sca-1^+心脏干细胞可达(62.8±1.3)%。免疫荧光显示Sca-1、GATA-4及Nkx-2.5均为阳性表达。结论:组织培养结合磁珠分选能成功分离纯化新生小鼠Sca-1^+心脏干细胞,且能稳定培养。细胞表达心源性转录因子相关蛋白GATA-4及Nkx-2.5。

Objective：To isolate and purify the stem cell antigen 1（Sca-1）positive cardiac stem cells from mouse,and to observe its specific markers.Methods：Mice cardiac stem cells were generated with tissue block culture method.After 10-14 days,Sca-1 positive cardiac stem cells were isolated by magnetic cell sorting（MACS）,and identified by flow cytometry and immunofluorescence,respectively.Cardiac-specific transcription factors GATA-4 and Nkx-2.5 were detected by immunofluorescence.Results：Sca-1 positive cardiac stem cells were successfully separated and purified.Flow cytometry demonstrated that the proportion of Sca-1 positive cardiac stem cells was（62.8±1.3）%.Immunofluorescence showed positive expressions of Sca-1,GATA-4 and Nkx-2.5.Conclusion：Sca-1 positive cardiac stem cells in newborn mice could be isolated by MACS and stably cultured.The cardiac-specific transcription factors GATA-4 and Nkx-2.5 could be detected.