TRPC1沉默对脑缺血大鼠神经干细胞迁移的影响

The effects of TRPC1 silencing on neurogenesis after cerebral ischemia of rats

目的探讨瞬时受体电位通道1(TRPC1)沉默对脑缺血大鼠内源性神经干细胞增殖、迁移和分化的影响。方法 SD大鼠分为假手术组、脑缺血组、TRPC1沉默组(脑缺血+TRPC1沉默)和阴性对照组,以脑室内注射siRNA沉默TRPC1,以线栓法制作大鼠大脑中动脉脑缺血(MCAO)模型,脑缺血模型制作成功后,腹腔内注射Brdu标记内源性神经干细胞,分别于48 h、4 w后处死大鼠,行Brdu免疫组化染色、免疫荧光双标染色(Brdu/GFAP、Brdu/Neun)观察NSC的增殖、迁移和分化情况。结果脑缺血后48 h,脑缺血组和TRPC1沉默组SVZ区Brdu阳性表达均较假手术组明显增多(P<0.01),但TRPC1沉默组Brdu阳性细胞数较缺血组低(P<0.01)。脑缺血4 w后,缺血组与假手术组相比,皮质区具有更多的Brdu阳性细胞(P<0.01),同样TRPC1沉默组Brdu阳性细胞数多于假手术组,但明显低于脑缺血组(P<0.01);免疫荧光双标染色发现,脑缺血各组Brdu/GFAP、Brdu/Neun双阳性细胞的数量均比假手术组明显增高,但TRPC沉默组双阳性细胞显著少于脑缺血组和阴性对照组(P<0.01)。结论 TRPC1沉默显著影响脑缺血大鼠SVZ区NSC的增殖、向脑缺血区迁移及向成熟细胞的分化。

Objective To investigate the effects of transient receptor potential canonical 1（ TRPC1） silencing on proliferation,migration and differentiation of endogenous neural stem cells（ NSCs） after cerebral ischemia. Methods SD rats were divided into sham group,cerebral ischemia group,TRPC1 silent group（ cerebral ischemia ＋ TRPC1 silence） and negative control group. siRNA was injected to intraventricular to silence TRPC1. Middle cerebral artery occlusion models were produced using the method of intraluminal monofilament. After cerebral ischemia models were made,endogenous neural stem cells were marked by intraperitoneal injection of Brdu. The rats were sacrificed after 48 hours and 4 weeks,then Brdu immunohistochemistry and immunofluorescence double staining（ Brdu/GFAP,Brdu/Neun） were performed to observe the proliferation,migration and differentiation of NSCs. Results At 48 hours after cerebral ischemia,the Brdu expression in SVZ region of cerebral ischemia group and TRPC1 silent group increased significantly compared with the sham group（ P〈0. 01）,but the number of Brdu positive cells in TRPC1 silencing group were lower than the ischemic group（ P〈0. 01）.However,the number of Brdu positive cells were lower in TRPC1 blocking group than ischemia group（ P〈0. 01）. Four weeks after cerebral ischemia,there were more Brdu positive cells in the cortex in ischemia group and TRPC1 blocking group than sham group（ P〈0. 01）,but the number was significantly lower in TRPC1 blocking group than cerebral ischemia group（ P〈0. 01）. Double immunofluorescence staining found that,the numbers of Brdu/GFAP,Brdu/Neun double positive cells in all cerebral ischemia groups were significantly higher than the sham group. The numbers in TRPC1 silent group were significantly less than the cerebral ischemia group and negative control group（ P〈0. 01）. Conclusion TRPC1 silencing significantly affected the proliferation in SVZ,migration to cortex and differentiation into mature cells of NSCs after cerebral ischemia.