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目的:探讨三七总皂苷在骨髓间质干细胞(MSCs)分化为神经元样细胞中的调解作用。方法:①实验材料:成年健康Wister大鼠(雌雄不限),SRF级,体质量(150±30)g;②实验方法:无菌的条件下从大鼠的股骨中分离并继代培养MSCs细胞,当传至第5代时,先用完全培养液[含10μg/L碱性成纤维细胞生长因子(bFGF)]进行预诱导,再更换无血清培养液(含三七总皂苷0.25g/L)进行诱导,采用免疫组织化学SABC法检测神经丝蛋白(NF-M)、神经元特异性烯醇化酶(NSE)、巢蛋白(nestin)、微管联合蛋白-2(MAP-2)、生长相关蛋白43(GAP-43)以及胶质纤维酸性蛋白(GFAP)的表达。结果:①采用三七总皂苷诱导后,大多数MSCs呈神经元样细胞分化。经免疫组织化学鉴定,上述细胞表现为NSE、MAP-2和NF染色阳性,呈棕黄色,而GFAP染色阴性。②采用三七总皂苷进行诱导时,MSCs的胞体逐渐增大,并伸出细长的突起,在突起的末端出现分支,有些相邻细胞的突起还成网状连接。③三七总皂苷诱导MSCs转变为类似于神经元样的形态,经免疫组织化学鉴定,大多数细胞表现为NF-M、NSE、nestin、MAP-2和GAP-43染色阳性,并在胞质和突起中有棕黄色的物质,而GFAP染色均为阴性。SABC法显示NSE和MAP-2阳性细胞数分别为(72.64±2.04)%和(73.5±2.17)%。结论:三七总皂苷在体外能使MSCs向神经元样细胞转化,且具有神经元样细胞的特性,这为MSCs治疗相关疾病提供了理论基础。
Objective: To investigate the role of Panax Notoginseng saponins in the differentiation of bone marrow mesenchymal stem cells (MSCs) into neuron-like cells. Methods: ① Materials: Adult healthy Wister rats (male and female were not limited), SRF grade, body weight (150 ± 30) g; ②Experimental methods: MSCs were isolated and subcultured from rat femur under aseptic conditions The cells, when passing on passage 5, were pre-induced with complete medium containing 10 μg / L basic fibroblast growth factor (bFGF), and then replaced with serum-free medium (containing Panax notoginseng saponins 0.25 g / L), the expression of NF-M, NSE, nestin, MAP-2, Expression of growth-associated protein 43 (GAP-43) and glial fibrillary acidic protein (GFAP). Results: ① After induced by Panax notoginseng saponin, most MSCs showed neuron-like cell differentiation. Immunohistochemistry identified the above cells as NSE, MAP-2 and NF-positive staining, showing a yellowish brown, but negative for GFAP staining. ② When using Panax notoginseng saponin for induction, the cell bodies of MSCs gradually increased, and elongated slender protrusions appeared on the distal ends of the protrusions, and the protrusions of some adjacent cells also became reticular connections. (3) Panax notoginseng sapogenin-induced changes of MSCs to neuron-like morphology. Immunohistochemistry showed that most of the cells showed positive staining of NF-M, NSE, nestin, MAP-2 and GAP-43, And protuberances have a brownish substance, whereas GFAP staining is negative. SABC method showed that the number of NSE and MAP-2 positive cells were (72.64 ± 2.04)% and (73.5 ± 2.17)%, respectively. Conclusion: Panax notoginseng can transform MSCs to neuron-like cells in vitro and has the characteristics of neuron-like cells, which provides a theoretical basis for MSCs treatment-related diseases.