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目的研究基因bcl-2对神经元的生物活性作用,为神经系统疾病的保护治疗提供有效的途径。方法构建真核表达载体pcDNA3-bcl-2,采用脂质体介导将重组质粒导入PC12细胞,WesternBlot检测外源基因表达;10,50和100μmol/L顺铂处理转染重组质粒的实验A组细胞,同样条件处理转染空质粒的对照B组细胞,72h后比较两者存活的细胞数;流式细胞仪检测分析两者的细胞周期指数。结果成功构建了真核表达载体pcDNA3-bcl-2,并用脂质体介导的方法获得了稳定表达Bcl-2的细胞克隆;10,50和100μmol/L顺铂处理后,实验A组存活的细胞数分别为276±13,185±11和108±10,而对照B组中存活的细胞数分别为100±9,12±3和2±2,两者相比差异有显著性意义;流式细胞仪检测显示,实验A组S期细胞所占百分比为8.81%比对照B组25.79%明显减少(χ2=22.53,P<0.01)。结论bcl-2能够拮抗顺铂对神经元的损害作用,促进神经细胞存活,其神经保护作用机制可能通过调控细胞周期来完成。
Objective To study the biological activity of gene bcl-2 on neurons and provide an effective approach for the protection and treatment of nervous system diseases. Methods The eukaryotic expression vector pcDNA3-bcl-2 was constructed and transfected into PC12 cells by lipofectamine. Western Blot was used to detect the expression of exogenous gene. 10,50 and 100μmol / L cisplatin treatment group A Cells were treated under the same conditions as control group B cells transfected with empty plasmid. After 72 hours, the number of surviving cells in both groups was compared. The cell cycle index was analyzed by flow cytometry. Results The eukaryotic expression vector pcDNA3-bcl-2 was successfully constructed and the cell clone stably expressing Bcl-2 was obtained by liposome-mediated method. After treated with 10, 50 and 100 μmol / L cisplatin, The number of cells were 276 ± 13,185 ± 11 and 108 ± 10, respectively, while the number of surviving cells in control group B was 100 ± 9,12 ± 3 and 2 ± 2, respectively, the difference was significant; flow cytometry The results showed that the percentage of S phase cells in experimental group A was significantly lower than that in control group B (8.81% vs 25.79%, χ2 = 22.53, P <0.01). Conclusion bcl-2 can antagonize the damaging effect of cisplatin on neurons and promote the survival of nerve cells. The mechanism of neuroprotection may be through the regulation of cell cycle.