Shh通路介导损伤后反应性星形胶质细胞获得干细胞潜能

目的 探讨Shh信号通路对大脑皮质反应性星形胶质细胞（RAS）获得神经干细胞潜能的影响。方法 体外原代培养SD大鼠大脑皮质星形胶质细胞，以肿瘤坏死因子（TNF）-α、白细胞介素（IL）-1α和C1q三种因子诱导炎症型RAS，划痕实验制备创伤型RAS，使用免疫荧光染色法观察细胞形态及纯度。实验分为普通星形胶质细胞组（对照组）、炎症组、划痕组（创伤组）。炎症组培养至干预后 24 h，对照组和划痕组培养至第 5天进行免疫荧光染色观察巢蛋白（Nestin）阳性细胞，运用Western blot技术检测不同损伤模型的细胞中Shh蛋白的表达量。之后各组细胞培养 至第7天更换神经干细胞培养基，将前述3组各自再分为激活组（各组细胞更换神经干细胞培养基后添加Shh激活剂SAG）和抑制组（各组细胞更换神经干细胞培养基后添加Shh抑制剂环巴胺）两个亚组，培养14 d后采用qRT-PCR检测各亚组 RAS源性神经干细胞相关基因的表达量。结果 细胞损伤模型中，免疫荧光染色证明各组细胞中划痕组Nestin阳性细胞明显多于炎症组和对照组。Western blot结果显示2组损伤细胞中划痕组Shh蛋白表达量高于炎症组（P＜0.05）。更换神经干细胞培养基后，激活组中 2 组损伤细胞的干细胞相关基因 Nestin、Pax-6、Sox-2、Oct-4 和Map-2的表达水平均有所升高，其中由划痕诱导的RAS神经干细胞相关基因Nestin、Pax-6、Sox-2、Oct-4和Map-2的表达水平高于炎症诱导的 RAS神经干细胞和普通星形胶质细胞（P＜0.01）；添加 Shh抑制剂后，3组上述基因表达量皆明显降低，但划痕组的表达量仍高于其他 2组（P＜0.01）。结论 在体外，Shh信号通路可以介导损伤后大脑皮质RAS获得干细胞潜能。

Shh pathway mediated the potential of reactive astrocytes to acquire stem cell properties after injury

Objective To explore the effect of sonic hedgehog (Shh) on the stem cell potential in cerebral cortical reactive astrocytes (RAS). Methods The astrocytes of cerebral cortex of SD rats were cultured in vitro. The inflammatory RAS were induced by tumor necrosis factor (TNF)-α, interleukin (IL)-1 α and C1q. The traumatic RAS were prepared by scratch test. The morphology and purity of cells were observed by immunofluorescence staining. The cells were divided into the normal astrocytes group (control group), the inflammation group, and the scratch group (trauma group). The inflammation group was cultured for 24 h after the intervention, control group and scratch group were cultured for 5 days. The positive rate of Nestin was observed by immunofluorescence staining. The expressions of Shh in different injured groups were detected by Western blot assay. Then, the cells in each group were replaced with neural stem cell culture medium on the 7th day, and cells were subdivided into the activation group (the group was replaced with neural stem cell culture medium and added with smoothened agonist) and inhibition group (the cells in each group were replaced with neural stem cell culture medium and then added with cyclopamine). After 14 days, the expression levels of RAS -induced neural stem cell related genes were verified by qRT-PCR. Results In cell injury model, the result of immunofluorescence staining showed that Nestin-positive cells were significantly more in the scratched group than those in the inflammatory group and control group. Western blot assay showed that the expression of Shh was significantly higher in the scratched group than that in the inflammatory group(P＜0.05). After replacing the neural stem cell conditioned medium, the expressions of neural stem cell genes (Nestin, Pax-6, Sox-2, Oct-4 and Map-2) were significantly higher in the scratch group than those in the inflammatory group and the control group (P＜0.01). After adding the cyclopamine, the gene expression levels were significantly reduced in the three groups, but the expressions of neural stem cell genes were significantly higher in the scratch group than those of the other two groups (P＜0.01). Conclusion In vitro, the Shh signaling pathway can mediate the potential of stem cells in the cerebral cortex RAS after injury.