摘要
目的利用RNA干扰技术分别沉默神经元中缺氧诱导因子1α(HIF-1α)和磷酸酶并和张力蛋白同源物(PTEN)基因,论证它们在神经元体外氧糖剥夺(OGD)损伤后的功能调控作用。方法构建并筛选靶向HIF-1α及PTEN基因的shRNA慢病毒载体;提取并将原代神经元分为4组:(1)NC组,仅加空载病毒;(2)NC+OGD组,加空载病毒后缺氧;(3)Si-hif-1α+OGD组,加Si-hif-1α病毒后缺氧;(4)Si-pten+OGD组,加Si-pten病毒后缺氧,均在培养第3天感染病毒,第7天OGD损伤模拟细胞缺氧。缺氧后24 h行荧光实时定量PCR(qRT-PCR)法检测干扰效率,行乳酸脱氢酶(LDH)检测及AnnexinV-FITC/PI检测神经元细胞损伤、凋亡变化,Western blot检测相应蛋白表达变化。结果慢病毒介导的shRNA能有效沉默目的基因mRNA的表达;HIF-1α沉默后,缺氧细胞的损伤及凋亡加重,PTEN蛋白的表达升高,p-PTEN、p-AKT、NR2A及VEGFa表达降低(P<0.05);PTEN沉默后,缺氧细胞的损伤及凋亡有所减轻,p-PTEN、p-AKT表达升高(P<0.05),HIF-1α、NR2A及VEGFa的表达无显著变化(P>0.05)。结论慢病毒介导的shRNA能有效沉默神经元HIF-1α及PTEN的表达,逆转神经元缺氧损伤中HIF-1α升高对PTEN活性的抑制,参与神经元损伤调控。
Objective To observe the effects of lentivirus-mediated RNA interference(RNAi)of hypoxia-inducible factor 1α(HIF-1α)and phosphatase and tensin homolog on chromosome ten(PTEN)on oxygen-glucose deprivation(OGD)injury in primary cultured rat neurons.Methods Primary cultures of neonatal SD rat neurons were infected by lentiviral vectors carrying short hairpin RNA(shRNA)targeting HIF-1α or PTEN followed 4 days later by hypoxic exposure,and the control neurons were infected with the empty virus only with or without subsequent hypoxic exposure.Twenty-four hours after hypoxia,the interference efficiency was assessed with qRT-PCR,and lactate dehydrogenase(LDH)assay and AnnexinV-FITC/PI assay were performed to detect neuronal damage and apoptosis.The expressions of the related proteins were determined with Western blotting.Results Lentivirus-mediated RNAi effectively silenced the mRNA expression of the target genes.HIF-1α silencing obviously aggravated the hypoxia-induced damage and apoptosis of the neurons,enhanced the expression of PTEN protein and significantly lowered the expressions of p-PTEN,p-AKT,NR2A and VEGFa(P<0.05).PTEN silencing significantly alleviated hypoxia-induced damage and apoptosis of the neurons and increased the cellular expressions of p-PTEN and p-AKT(P<0.05)without obviously affecting the expressions of HIF-1α,NR2A or VEGFa(P>0.05).Conclusion An up-regulated expression of HIF-1α causes down-regulation of PTEN expression to protect primary cultured rat neurons against OGD injury.
作者
唐静华
杨颖
陈洁
李廷玉
代英
TANG Jinghua;YANG Ying;CHEN Jie;LI Tingyu;DAI Ying(Department of Child Health Care,Children's Hospital of Chongqing Medical University//National Clinical Research Center for Child Health and Disorders//Ministry of Education Key Laboratory of Child Development and Disorders//Chongqing Key Laboratory of Child Health and Nutrition,Chongqing 40014,China)
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2021年第12期1795-1800,共6页
Journal of Southern Medical University
基金
国家自然科学基金(81672248)。
