摘要
目的建立制备骨髓间充质干细胞胞质体的方法，以用于体细胞核移植研究。方法（1）培养骨髓间充质十细胞，用流式细胞仪检测骨髓间充质干细胞表面标志物CD44、CD90、CD71、CD11b；（2）用梯度离心法制备骨髓间充质干细胞胞质体，并用HE染色、Giemsa染色、荧光双标染色、透射电镜检查等方法进行鉴定，用台盼蓝染色检测其活性。结果（1）骨髓间充质干细胞呈长梭形、漩涡状生长，骨髓间充质干细胞表面标志物CD44、CD90、CD71表达阳性，CD11b表达阴性；（2）HE染色、Giemsa染色、荧光双标染色和透射电镜检查显示骨髓间充质干细胞经梯度离心后在15％－18％Ficoll400梯度层可收集到大量胞质体，胞质体可快速贴壁，表达CD71，与完整骨髓间充质干细胞相似，体积与完整细胞相比差别不大。有活性的胞质体比例达99．5％。结论用梯度离心法可有效获得骨髓间充质干细胞胞质体。
Objective To establish a method for separating the cytoplasts from rat bone marrow-derived mesenchymal stem cells （BMSCs） for nuclear transfer. Methods Rat BMSCs were cultured and identified using flow cytometry for the surface markers CD44, CD90, CD71 and CD11b, and cytoplasts were separated from these BMSCs by centrifugation through a Ficoll density gradient. The cytoplasts were identified by HE staining, Giemsa staining, fluorescence double labeling and transmission electron microscopy, and their viability was examined with trypan blue exclusion test. Results By Ficoll gradient centrifugation of the verified BMSCs, a large number ofcytoplasts were collected from the 15%-18% Ficoll layer, and were identified by HE staining, Giemsa staining, immunofluorescence double labeling and transmission electron microscopy. With a viable rate of 99.5%, the isolated cytoplasts showed similar adhesive property, cell volume and CD71 expression with the intact BMSCs. Conclusion The cytoplasts of BMSCs can be obtained by gradient centrifugation.
出处
《中华神经医学杂志》
CAS
CSCD
2008年第11期1110-1113,1123,共5页
Chinese Journal of Neuromedicine
基金
国家自然科学基金（30270490）
重庆市科技计划项目（7452）
关键词
骨髓间充质干细胞
梯度离心法
胞质体
Bone marrow-derived mesenchymal stem cells
Gradient centrifugation
Cytoplast
