南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (12): 1795-1800.doi: 10.12122/j.issn.1673-4254.2021.12.06

• • 上一篇    下一篇

沉默神经元HIF-1α和PTEN的表达对体外培养的大鼠神经元氧糖剥夺损伤的影响

唐静华，杨 颖，陈 洁，李廷玉，代 英   

  1. 重庆医科大学附属儿童医院儿童保健科//国家儿童健康与疾病临床医学研究中心//儿童发育疾病研究教育部重点实验室//儿童营养与健康重庆市重点实验室，重庆 400014
  • 出版日期:2021-12-20 发布日期:2022-01-05

Effects of lentivirus-mediated RNA interference of HIF-1α and PTEN on oxygen-glucose deprivation injury in primary cultured rat neurons

TANG Jinghua, YANG Ying, CHEN Jie, LI Tingyu, DAI Ying   

  1. 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
  • Online:2021-12-20 Published:2022-01-05

RichHTML

23

PDF

504

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。

关键词: 缺氧；HIF-1α；PTEN-PI3K/AKT信号通路；RNA干扰

Abstract: 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.

Key words: hypoxia; hypoxia-inducible factor-1α; PTEN-PI3K/AKT signal path; RNA Interference