再程序化脂肪干细胞体外定向神经分化效率和机制

Efficiency and mechanism of neural differentiation of reprogrammed adipose-derived stem cells in vitro

目的探讨再程序化脂肪干细胞（adipose—derived stem cells，ADSCs）体外定向神经分化的效率和机制。方法体外培养大鼠ADSCs并纯化、鉴定，将第3代ADSCs分为三组：未进行慢病毒介导基因转染ADSCs组（空白组）；不含神经元生成素-2（neurogenin-2，Ngn2）基因慢病毒空载体转染ADSCs组（空病毒组）；慢病毒载体介导Ngn2基因转染ADSCs组（Ngn2组）；各组细胞均用含细胞生长因子的诱导培养基诱导15d。免疫荧光检测各组细胞神经元特异性蛋白（NeuN）阳性表达以观察神经元分化效率；Western blot检测各组细胞Mash1、Hes1、Dll1蛋白表达水平的差异，探索分化机制。结果诱导培养基诱导15d后，Ngn2组、空病毒组、空白组阳性表达NeuN分别为90．12％、45．34％、40．26％，各组比较差异均有统计学意义（P〈0．01）。Western blot结果显示，Ngn2组Dll1蛋白表达水平明显高于空病毒组和空白组（P〈0．01），Mashl、Hesl蛋白表达水平明显低于空病毒组和空白组（P〈0．01）；空病毒组和空白组Dll1、Mash1和Hes1蛋白表达差异均无统计学意义（P〉0．05）。结论再程序化ADSCs诱导后神经元分化的比例较单纯ADSCs提高近99％，再程序化ADSCs定向分化神经元机制可能是通过上调Dll1蛋白水平，同时下调Mash1、Hes1蛋白水平，抑制notch信号通路，从而促进细胞的定向神经分化。

Objective To investigate the efficiency and mechanism of differentiation of reprogrammed adipose-derived stem ceils （ADSCs） to neurons in vitro. Methods ADSCs from rats were cultured in vitro and then purified and identified. ADSCs at the third passage were divided into three groups： ADSCs without lentivirns-mediated gene transfeetion （blank group ）, ADSCs transfected with lentivirus carrying no neurogenin2 （Ngn2） （empty virus group ） and ADSCs with lentivirus-mediated transfection of Ngn2 （ Ngn2 group）. All groups were induced in the medium containing cell growth factor for 15 days. The positive expression of neuron-specific nuclear protein （NeuN） in three groups was detected using immunofluorescence method so as to observe the efficiency of neuron differentiation. Expression variances of Mash1, Hes1 and Dll1 in each group were detected by Western blot analysis and the mechanism of differentiation was also discussed. Results After 15 days of induction, positive expression rate of NeuN in Ngn2 group, empty virus group, blank group was 90.12% , 45.34% and 40.26% respectively, with significant differences among groups （P 〈 0.01 ）. Western blot analysis showed that Ngn2 group had a significantly higher expression of Dll1 （P 〈 0.01 ） and obvious lower expressions of Hesl and Mashl （P 〈0.01 ） , as compared with empty virus group and blank group. However, there were no significant differences of expression levels of Dll1, Mash1 and Hes1 between empty virus group and blank group （ P 〉 0.05）. Conclusions After induction, the ratio of neuron differentiation of reprogrammed ADSCs is increased by almost 99%, as compared with simple ADSCs. The increased dfferentiation of reprogrammed ADSCs to neurons may be associated with the inhibition of notch signaling through up-regulating Dll1 and down-regulating Mashl and Hesl.