脂质体介导下pcDNA3．1／BDNF基因转染对缺糖缺氧-再给氧损伤神经细胞保护作用的实验研究

The protection of gene pcDNA3.1 / BDNF transfection mediated liposome on neural cells under glucose deprivation, hypoxia/reoxygenation

目的 研究脂质体介导下pcDNA3.1/BDNF基因转染对缺糖、缺氧-再给氧损伤神经细胞的保护作用.方法 建立缺糖、缺氧-再给氧损伤SH-SY5Y神经细胞模型,应用显微镜观察细胞形态、MTT比色法测定细胞存活率、苔盼蓝染色排斥试验检测细胞活力、测定SH-SY5Y细胞 LDH 释放率、Annexin V-FITC和Propidium Iodide双染色法荧光显微镜下观察细胞凋亡以及流式细胞仪检测细胞死亡和凋亡等方法,观察应用基因转染方法导入表达BDNF的基因片段（pcDNA3.1/BDNF）对缺糖、缺氧-再给氧损伤神经细胞的保护作用.结果 显微镜下观察到,在转染了表达BDNF基因的SH-SY5Y细胞中,经ATRA诱导后,即使实施了缺糖、缺氧-再给氧处理,其死亡、凋亡的细胞数和对照组相比明显减少,且细胞的形态改善显著;在细胞存活率、死亡率、LDH 释放率的测定结果中,基因转染组和对照组相比,显著提高了细胞的存活率,降低细胞死亡率和LDH 释放率（P＜0.05）;在Annexin V-FITC和Propidium Iodide双染色法荧光显微镜观察及流式细胞仪的检测中,转染了表达BDNF基因的SH-SY5Y细胞,经ATRA诱导后,其保护作用最好,和正常对照组比较,死亡、凋亡的细胞数显著减少.结论 应用基因转染方法导入表达BDNF的基因片段对缺糖、缺氧-再给氧损伤神经细胞具有保护作用.

Objective To investigate the protection of gene pcDNA3. 1/BDNF transfection mediated liposome on neural cells under glucose deprivation, hypoxia/ reoxygenation. Methods The glucose deprivation, hypoxia/reoxygenation injured SH- SY5Y neural cell model was established. The morphology of cells was observed under microscope. The cell survival rate was determined using MTr colorimetry. The cell vitality was detected using trypan blue exclusion test. The LDH release rate of SH- SY5Y cells was determined. The cell apoptosis was observed using Annexin V -FITC and Propidium Iodide double staining methods under a fluorescent microscope. The cell death and apoptosis were detected using flow cytometry. The protective effects of pcDNA3. 1/BDNF （ the gene fragment expressed by gene transfection method） on glucose deprivation, hypoxia/reoxygenation injured SH - SY5Y neural cells were invesitageted. Results Under microscope showed huge amount of died cells in the injury model group, while cells survived more in the protective group with BDNF. After ATRA induction, compared with those of the control group, the numbers of died and apoptotic SH - SY5Y ceils with transfection of expressed BDNF gene were obviously reduced when they received the glucose deprivation, hypoxia/reoxygenation. In the tests of the survival rate, death rate, and LDH release rate of SH-SY5Y ceils, the cell survival rate of the injury model group was obviously lower than that of the normal control group, while its cell death rate and LDH release rate were obviously raised（ P 〈 0.05 ）. After ATRA induction, compared with those of the control group, the numbers of died and apoptotic SH -SY5Y ceils with transfection of expressed BDNF gene were obviously reduced in the experiment of detecting SH -SY5Y cell death and apoptosis by the flow cytometry and Annexin V -FITC and Propidium Iodide double staining methods under a fluorescent microscope. Conclusion The BDNF gene fragment introduced by gene transfection method show the protective effects on glucose deprivation, hypoxia/reoxygenation injured cells.