黄芪诱导大鼠骨髓间充质干细胞的分化特点

背景:黄芪提取物有较好的抗氧化、清除氧自由基的作用,加之骨髓间充质干细胞的多向分化潜能及其自体移植的优越性,可能为神经退行性疾病的治疗提供新的方式.目的:探讨黄芪诱导后大鼠骨髓间充质干细胞的分化特点.设计:细胞学体外观察.材料:清洁级6周龄雄性大鼠1只,购自中国医科院实验动物中心.黄芪注射液批号060105,为大理药业有限公司产品.方法:全骨髓法体外分离培养、纯化大鼠骨髓间充质干细胞,传至第4代时,按4×108L-1密度接种于置有盖玻片的12孔板内,制备细胞爬片.盖玻片上细胞达80%～90%融合后全量换液,加入含200 g/L黄芪注射液、体积分数为15%胎牛血清的DMEM培养基连续诱导6 d;对照组DMEM培养基中不加入黄芪注射液.主要观察指标:倒置显微镜观察诱导前后骨髓间充质干细胞的形态变化,诱导后免疫细胞化学染色鉴定特异性标志物的表达.结果:原代培养的骨髓间充质干细胞多呈圆形,扩增至第4代后细胞形态多呈梭形或成纤维细胞状,诱导后细胞形态发生改变,自胞体长出突起,随诱导时间延长,长出突起的细胞数量逐渐增多,部分突起末端呈分叉状,可见网络状连接.免疫细胞化学染色结果显示,诱导第3天巢蛋白阳性细胞、神经元特异性烯醇化酶阳性细胞和神经胶质纤维酸性蛋白阳性细胞较多,诱导第6天微管相关蛋白2阳性细胞较多.结论:黄芪首先诱导骨髓间充质干细胞向神经干细胞分化,然后促进其向神经元或神经胶质细胞的非特异性分化,并能够使已分化细胞的进一步成熟、老化.

Astragalus mongholicus-induced differentiation of rat bone marrow mesenchymal stem cells

BACKGROUND: Extract of astragalus mongholicus has good effects on antioxidization and oxygen free radical scavenge. Bone marrow mesenchymal stem cells (BMSCs) have multiple directional differentiation potential and superiority of autologous transplantation. This can provide a new way for treating neurodegenerative diseases.OBJECTIVE: To explore the differentiation of BMSCs of rats following astragalus mongholicus induction.DESIGN: A cytological in vitro study.MATERIALS: One clean male rats aged 6 weeks were purchased from Experimental Animal Center of Chinese Academy of Medical Sciences. Astragalus mongholicus injection (lot number 060105) were obtained from Dali Pharmaceutical Co., Ltd.METHODS: Rat BMSCs were in vitro isolated, cultured and purified by the whole bone marrow method. At the fourth passage, BMSCs at a density of 4×10<'8>/L were incubated in 12-well plate with a coverslip. After 80% 90% cells were confluent, the medium was changed. BMSCs were then incubated in DMEM, supplemented with 200 g/L astragalus mongholicus injection and 15% fetal bovine serum, for 6 days. BMSCs in the control group were not treated with astragalus mongholicus injection.MAIN OUTCOME MEASURES: Morphological changes in BMSCs were observed under the inverted microscope before and after induct.ion. Expression of specific markers was determined by immunocytochemical staining following induction.RESULTS: Primary cultured BMSCs were round. At the fourth passage, BMSCs were spindle. BMSCs after induction showed processes, which became more with prolonged time. Some processes were branch-shaped, forming a network structure. Results of immunocytochemical staining demonstrated that many cells were positive for nestin, neuron specific enolase and glial fibrillary acidic protein on day 3, and abundant cells were positive for microtubule-associated protein-2 on day 6.CONCLUSION: Astragalus mongholicus induces differentiation of BMSCs into neural stem cells, and then promotes the differentiation into neurons or glial cells, and makes differentiated cells mature and aging.