神经细胞缺氧实验装置的研制及其评价

摘要

Objective To develop a hypoxic experimental apparatus for nerve cells with good function and easy to use, which can be used to explore the pathogenesis and management of neonatal brain injury by simulating a hypoxic-ischemia process on a cellular level. Methods A set of hypoxic experimental apparatus was designed and developed. Using this hypoxic experimental apparatus, the oxygen-glucose deprivation (OGD) model of nerve cells were respectively established with various OGD time of 30 min, 45 min, 60 min, 2 h and 3 h, respectively. The influence of different OGD time on the survival rates of nerve cells was assessed 24 hours after OGD by using Cell Counting Kit-8 (CCK-8) and Hoechst33342/PI double fluorescence dying method. Results The set of hypoxic experimental apparatus consisted of four parts including a hypoxic chamber, a commercial source of non-oxygen gas, a water-sealed bottle for draining air, and a CO2 incubator. The hypoxic chamber developed was lucid with good closure property and easiness to open and carry. The results for evaluating the influence of different OGD time on the survival rates of nerve cells established by using the hypoxic experimental apparatus showed that the apoptotic and necrotic cells tended to increase significantly with the extension of OGD period gradually. The survival rates of nerve cells were from 85% with 30min OGD reduced to 22% with 3 h OGD. About 45-60 min OGD could cause 40-55% of nerve cells to die. Conlusions The set hypoxic experimental apparatus developed is with good function and easy to use, which can ensure the successful establishment of OGD model of nerve cells.%目的 研制一种性能良好、简便易用的神经细胞缺氧实验装置,以便从细胞水平模拟神经细胞的缺氧缺血过程,探讨新生儿脑损伤的发病机理和防治方法.方法 设计研制一套细胞缺氧实验装置.应用这套装置,分别建立了缺氧30 min、45 min、60 min、2h及3h的氧糖剥夺(OGD)神经细胞模型.应用WST-8方法和Hoechst 33342/PI荧光双染方法,分别评估不同OGD时间对神经细胞成活率的影响.结果 所研制的细胞缺氧实验装置包括细胞缺氧舱、市售缺氧气源、出气水封瓶以及市售二氧化碳培养箱.其中研制的细胞缺氧舱外形精巧透明,便于携带,开启方便,封闭性能好.对用这套装置所建立的不同OGD时间神经细胞成活情况的评估结果,显示随着OGD时间的延长,凋亡和坏死细胞逐渐增多,细胞成活率由OGD 30 min时的85％渐降至OGD3h时的22％,其中OGD 45～60 min可造成40％～55％的神经细胞死亡.结论 所研制的这套细胞缺氧实验装置性能良好、简便易用,缺氧时间精准,可确保OGD神经细胞模型的成功建立.