Liu Feng-Hua, Yang Dong-Zi, Wang Yi-Feng, Liang Xiao-Ping, Peng Wen-Ming, Cao Chang-An, Chen Xi-Gu, Guo Zhong-Min
Center of Reproductive Medicine, the Second Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510120, China.
Zhonghua Nan Ke Xue. 2005 May;11(5):350-5.
To isolate, culture and purify mouse bone marrow mesenchymal stem cells (MSCs) and observe the main biological characteristics of MSCs cultured in conditions for spermatogonia in vitro.
The tibias and femurs were dissected from 5 - 6-week old mice and the marrow in the tibias and femurs was flushed out with medium. MSCs were isolated, cultured, purified in vitro by Percoll density gradient centrifugation combined with adherent method and identified by dynamic observation of stem cell characteristics by transmission electron microscope, HE staining, and immunohistochemical detection of cell markers. The quantities of such cytokines as IL-6, IL-8, G-CSF and SCF in culture liquid with MSCs were measured by ELISA, and compared with those of the control group. MSCs of the third generation were divided into two groups to be induced and cultured. MSCs of the control group were cultured with basal medium, while those of the experimental group with conditional medium. The results were analysed by microscopic observation, HE staining and immunohistochemical methods.
Pure MSCs were obtained. The cultured cells, with stem cell characteristics, shuttle-shaped at HE staining, immature under the transmission electron microscope and CD44 and CD90 positive by immunohistochemical detection, could be identified as MSCs. Compared with the control group, the quantities of IL-6, IL-8, G-CSF and SCF in the experimental group increased significantly (P < 0.05). The shapes of MSCs changed and immunohistochemical staining for CD27, CD119 and Oct-4 was positive in the experimental group, but both were just the opposite in the control group.
Pure MSCs can be obtained by Percoll density gradient centrifugation combined with adherent method and identified by dynamically observing stem cell characteristics, HE staining, observation under the transmission electron microscope and immunohistochemical detection of cell markers. MSCs can secrete cytokines such as IL-6, IL-8, G-CSF, SCF, and so on. MSCs cultured in conditions for spermatogonia may show some biological characteristics of spermatogonia.
分离、培养和纯化小鼠骨髓间充质干细胞（MSCs），并观察在体外精原细胞培养条件下培养的MSCs的主要生物学特性。
从5至6周龄小鼠中取出胫骨和股骨，用培养基冲洗出胫骨和股骨中的骨髓。通过Percoll密度梯度离心结合贴壁法在体外分离、培养和纯化MSCs，并通过透射电子显微镜动态观察干细胞特征、HE染色以及细胞标志物的免疫组织化学检测进行鉴定。用ELISA法检测含MSCs的培养液中IL-6、IL-8、G-CSF和SCF等细胞因子的含量，并与对照组进行比较。将第三代MSCs分为两组进行诱导培养。对照组的MSCs用基础培养基培养，而实验组的用条件培养基培养。通过显微镜观察、HE染色和免疫组织化学方法分析结果。
获得了纯化的MSCs。培养的细胞具有干细胞特征，HE染色呈梭形，透射电子显微镜下不成熟，免疫组织化学检测CD44和CD90呈阳性，可鉴定为MSCs。与对照组相比，实验组中IL-6、IL-8、G-CSF和SCF的含量显著增加（P<0.05）。实验组中MSCs的形态发生变化，CD27、CD119和Oct-4的免疫组织化学染色呈阳性，而对照组则相反。
通过Percoll密度梯度离心结合贴壁法可获得纯化的MSCs，并通过动态观察干细胞特征、HE染色、透射电子显微镜观察和细胞标志物的免疫组织化学检测进行鉴定。MSCs可分泌IL-6、IL-8、G-CSF、SCF等细胞因子。在精原细胞培养条件下培养的MSCs可能表现出一些精原细胞的生物学特性。
