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目的体外分离、培养、纯化小鼠骨髓间充质干细胞(MSCs),并观察MSCs在精原细胞培养条件下的生物学特征,为体内诱导MSCs分化为精原细胞奠定基础。方法①分离5～6周龄的BALB/c小鼠胫骨、股骨,冲出骨髓,以Percoll密度梯度离心法和贴壁法相结合的方法分离、培养、纯化MSCs;②通过动态观察、苏木精伊红(HE)染色、透射电镜观察、免疫组化检测细胞表面标记等鉴定MSCs;③用ELISA法检测MSCs培养上清中白细胞介素6(IL6)、IL8、粒细胞集落刺激因子(G CSF)、干细胞因子(SCF)等细胞因子的相对含量,与相应对照组比较;④取第3代MSCs,分组进行诱导培养观察,对照组用基本培养液培养MSCs,实验组用条件培养液诱导培养MSCs。通过显微镜下动态观察、HE染色、免疫组化等方法观察诱导结果。结果①获得纯化的MSCs;②动态观察培养细胞具有不断增殖的干细胞特性,HE染色细胞呈梭形,透射电镜观察细胞较幼稚,免疫组化检测细胞表面标记CD44、CD90呈阳性,从而鉴定了MSCs;③MSCs培养上清中IL6、IL8、G CSF、SCF等的含量显著高于对照组(P<0.05);④实验组细胞形态发生变化,CD29、CD117和Oct4免疫组化染色阳性;对照组细胞形态不变,CD29、CD117和Oct4免疫组化染色阴性。结论①用Percoll密度梯度离心法和贴壁法相结合的方法可获得纯化的MSCs;②
OBJECTIVE: To isolate, culture and purify mouse bone marrow mesenchymal stem cells (MSCs) in vitro and to observe the biological characteristics of MSCs under the condition of spermatogonia culturing, which lays a foundation for inducing MSCs to differentiate into spermatogonia in vivo. Methods ①The tibia and femur of 5 to 6-week-old BALB / c mice were isolated and washed out of bone marrow. MSCs were isolated, cultured and purified by Percoll density gradient centrifugation and adherent method. ②Through dynamic observation, MSCs were identified by HE staining, transmission electron microscopy and immunohistochemical staining of cell surface markers. ③The levels of interleukin 6 (IL6), IL8, granulocyte colony-stimulating factor (G CSF ), The relative content of cytokines such as stem cell factor (SCF), compared with the corresponding control group; (4) The third generation of MSCs was taken and induced by sub-culture. MSCs were cultured in basal medium in the control group. MSCs. Dynamic observation under microscope, HE staining, immunohistochemistry and other methods to observe the induction results. Results ① The purified MSCs were obtained. ② The characteristics of stem cells proliferated continuously were observed. The cells stained with spindle were observed by transmission electron microscopy. The surface markers CD44 and CD90 were positive by immunohistochemistry. MSCs were identified ; ③ The content of IL6, IL8, G CSF and SCF in the supernatant of MSCs was significantly higher than that of the control group (P <0.05); ④ The morphological changes of the cells in the experimental group were positive in immunohistochemical staining of CD29, CD117 and Oct4; Morphological changes, CD29, CD117 and Oct4 immunohistochemical staining negative. Conclusion ① purified MSCs can be obtained by Percoll density gradient centrifugation combined with adherent method; ②