G蛋白偶联受体激酶5对大鼠胚胎神经干细胞抗凋亡作用及其机制  

A Study on relationship between GRK5 and PKA on neural stem cell apoptosis

作　　者：张运[1] 沈光莉[1] 上官丽娟[1] 于洋[1] 鲍金[1] 贺茂林[1] 

机构地区：[1]首都医科大学附属北京世纪坛医院神经内科,北京100038

出　　处：《中风与神经疾病杂志》2014年第11期964-968,共5页Journal of Apoplexy and Nervous Diseases

基　　金：国家自然科学基金资助项目(81200849);北京市自然科学基金资助项目(7112068)

摘　　要：目的探讨培养大鼠胚胎神经干细胞G蛋白偶联受体激酶5(GRK5)基因沉默后蛋白激酶A(PKA)活性变化及其与神经干细胞凋亡的关系。方法采用分别或联合RNA干扰沉默GRK5基因表达与PKA激动剂forskolin干预大鼠胚胎神经干细胞,用荧光素标记的膜联蛋白V(FITC-Annexin V)联合PI染色检测早期凋亡率,并检测神经干细胞中PKA与caspase-3的活性,Western blot检测Bcl-2/Bax蛋白与磷酸化LKB1蛋白的表达。结果 GRK5基因沉默后神经干细胞内PKA活性及磷酸化LKB1蛋白表达显著减低(P<0.01),Bcl-2/Bax蛋白表达比值降低(P<0.05),caspase-3活性升高(P<0.01),Annexin V荧光染色阳性细胞比率显著升高(P<0.01)。联合应用PKA激动剂forskolin后细胞凋亡显著减少(P<0.01)。结论 GRK5基因沉默增加了大鼠胚胎神经干细胞早期凋亡,其机制可能与抑制PKA活性相关。Objective To explore the effects of GRK5 on the apoptosis of the neural stem cells （NSCs） in vitro from brain cortex of embryonic Sprague-Dawley rats. Methods Based on the model of rat embryonic neural stem cells （NSCs） in vitro, we Silenced GRK5 Gene or control gene each of two groups for 24 hours. After that we added forskolin, which specifically activate PKA,into the culture media of two different group according to Gene Silencing for 3 hours. Then we tested enzyme activity of PKA and caspase-3. Moreover, we observed relationship between changes of the expression of Bcl-2/Bax and NSCs apoptosis. Results With GRK5 gene scilence,the PKA activity obviously decreased and simultaneously Bcl-2/Bax ratio decreased. And Annexin V-positive NSCs in GRK5 siRNA groups were increased compared with the control siRNA group. Afterwards, treatment of forskolin could obviously increase PKA activity and Bcl-2 expression, which reduced Annexin V-positive NSCs significantly. Conclusion GRK5 and PKA are involved in the process of NPCs apoptosis. Express of GRK5 can be beneficial to NPCs.

关 键 词：神经干细胞 凋亡 G蛋白偶联受体激酶5 蛋白激酶A 

分 类 号：Q813[生物学—生物工程]