香丹注射液定向诱导大鼠骨髓间质干细胞分化为神经元样细胞

The Study of Committed Differentiation from Adult rMSCs into Neuron-like Cells Induced by Xiangdan Injection in Vitro

目的 :研究香丹注射液对成年SD大鼠骨髓间质干细胞 (rMSCs)的诱导分化作用。方法 :rMSCs取自大鼠的下肢骨 ,贴壁筛选法和营养缺陷培养基 (α MEM完全培养液 )分离纯化。加碱性成纤维生长因子 (bFGF)预诱导第 5代的rMSCs 2 4h ,换无血清培养液 (D/F12 +N2 +bFGF)和香丹注射液 (1%～ 5 % )诱导 6～ 8h ,观察细胞形态变化 ;细胞免疫化学和反转录 PCR法鉴定神经元标记物 (神经元特异性烯醇化酶NSE、神经丝蛋白NF 2 0 0、微管相关蛋白MAP 2及胶质纤维酸性蛋白GFAP)的表达及相对表达量 ,并得到相应的诱导率。结果 :诱导 3h ,rM SCs出现神经元样的形态学变化 ,诱导 6～ 8h ,神经元的诱导率达 83 5 %± 3 8% ;诱导组中 ,神经元样细胞NSE、NF和GFAP的阳性率分别为 82 9%± 2 98%、84 1%± 4 4 5 %、3 4 9%± 1 6 7% ;对照组细胞和诱导组未变化细胞上述染色均呈阴性 ;RT PCR法示 :只有诱导组有NSE、MAP 2 (HWM )、GFAP的表达且前两者的表达量均分别高于后者 (P <0 0 1)。结论 :1%～ 5

Objective:To study the induction of Xiangdan Injection (XDI) on the committed differentiation into neuron from rMSCs.Methods: The 5th passage of rMSCs were pretreated with α-MEM and 10μg/L basic fibroblast growth factor (bFGF)for 24h,and treated with the serum-free induction media (D/F12+N2+bFGF(10μg/L) and 1%～5% XDI).The differentiated cells were observed with phase-contrast microscopy and detected the expression of several specific proteins,such as neurofilament-200(NF-200),neuron-specific enolase(NSE),microtubule-associated protein-2(MAP-2) and glial fibrillary acidic protein (GFAP) with immuno-cytochemistry and semi-quantity reverse transcriptase-polymerase chain reaction (RT-PCR).Results: At 3h of induction,rMSCs gradually turned into the neuron-like cells in the induction groups.The induction rate can reach 83.5%±3.8% at 6～8h of induction.The percentages of responsive cells positive for NSE,NF-200 and GFAP were 82.9%±2.98%,84.1%±4.45% and 3.49%±1.67% respectively.RT-PCR showed that the relative level of expression for NSE and MAP-2 was much higher than that of GFAP(over 33 fold) ((P<0.01)). The unresponsive cells in the induction group and those cultured cells in the control were shown negative for all of the former detected markers.Conclusions: The rMSCs could be differentiated into neuron-like cells in vitro with the induction of XDI in the free-serum media.