Bcl-2基因转染对体外培养大鼠大脑皮层神经细胞氧糖剥夺损伤的保护作用

Protective Effect of bcl-2 Gene Transfection on Oxygen Glucose Deprivation-induced Injury of Rat Cerebral Cortical Neurons Cultured in vitro

目的研究bcl-2基因转染对体外培养大鼠大脑皮层神经细胞氧糖剥夺损伤的保护作用,为基因转染治疗脑血管病寻找实验依据。方法体外培养新生大鼠大脑皮层神经细胞,应用脂质体转染法将bcl-2基因转染入神经细胞,建立氧糖剥夺模型,实验分为正常对照组、氧糖剥夺组和bcl-2转染组(转染bcl-2基因24h后,进行氧糖剥夺),采用Western blot法、MTT法、流式细胞仪分别检测各组细胞的bcl-2蛋白表达、细胞活力及凋亡率。结果 SD大鼠大脑皮层神经细胞可纯化体外培养,纯度达到90%以上。成功建立了大鼠神经细胞体外氧糖剥夺模型,成功进行了质粒pc-DNA3.1-hbcl-2的转化、扩增及基因转染。对照组神经细胞bcl-2少量表达,氧糖剥夺组bcl-2蛋白微弱表达,转染组细胞bcl-2蛋白高表达,各组间差异均有统计学意义(均P<0.01)。MTT法检测结果显示,对照组细胞的吸光度值(A)为(0.851±0.034);氧糖剥夺组的A值为(0.648±0.030),与对照组相比明显降低(P<0.01);转染组的A值为(0.787±0.004),低于对照组(P<0.05),但明显高于氧糖剥夺组(P<0.05)。对照组神经细胞凋亡率为4.6%;氧糖剥夺损伤后细胞凋亡率明显增加,为19.6%;转染组细胞凋亡率与氧糖剥夺组相比有明显下降,为9.6%,差异有统计学意义(均P<0.01)。结论脂质体介导的bcl-2基因转染对氧糖剥夺损伤的大鼠大脑皮层神经细胞有明显保护作用。

Objective To investigate the neuroprotective effects of bcl-2 gene transfection on rat cortical neurons injury by oxygen glucose deprivation in vitro.Methods Neonatal rat cerebral cortical neurons were cultured in vitro and the oxygen glucose deprivation（OGD）models were established.Bcl-2 gene was transfected into rat cortical neurons with the application of liposomes.The experimental cells were randomly divided into three groups：control group,OGD group and bcl-2 gene transfection group（the oxygen-glucose was deprived 24 h after bcl-2 gene was transfected）.Western blot,MTT assay and flow cytometry were used to assess the protein expression of bcl-2 gene,cell viability and apoptosis of each group.Results SD rat cerebral cortical neurons could be purified（more than 90%）and cultured in vitro.OGD models of rat cerebral cortical neurons were successfully established in vitro,and the conversion,amplification and transfection of plasmid pcDNA3.1-hbcl-2 were successfully carried out.The results showed that the protein expression of bcl-2 gene in control group,OGD group and bcl-2 gene transfection group was different,respectively,with the difference being statistically significant among three groups（P〈0.01）.Absorbance（A）value in control group was（0.851±0.034）,significantly higher than in OGD group（0.648±0.030）（P〈0.01）.The A value in bcl-2 gene transfection group was（0.787±0.004）,lower than in control group（P〈0.05）,but significantly higher than in OGD group（P〈0.05）.Apoptosis rate in control group,OGD group and bcl-2 gene transfection group was 4.6%,19.6% and 9.6% respectively.There was significant difference between OGD group and bcl-2 gene transfection group（P〈0.01）.Conclusion Bcl-2 gene transfection mediated by liposomes vector can protect rat cortical neurons from injury by OGD in vitro.