慢病毒干扰TNF-α对脊髓损伤大鼠Caspase-3和Bcl-2表达的影响

摘要

Objective To explore the change of apoptosis factor Caspase-3 and Bcl-2 in the injured segment of rat with spinal cord injury after inhibiting lentivirus expression of inflammation factor TNF-α. To study the relationship between Caspase-3, Bcl-2, Bax and TNF-α in spinal cord injury. Mthods Spinal cord contusion model was prepared by Allen method. The relation between tumor necrosis factor alpha and Bcl-2, was predicted by the method of GeneMANIA bioinformatics. The RNA which was packaged by lentivirus constructed the RNA interference model of tumour necrosis factor alpha. After interference of tumor necrosis factor alpha, we used the method of QRT-PCR to assays the mRNA expression of Caspase-3 and Bcl-2 in spinal cord and detect of the localization of Caspase-3 and Bcl-2 by immunohistochemistry. Statistical analysis with SPSS17.0. Results SD rats had paraplegia and urinate retentaion because of spinal cord injury. The result of QRT-PCR showed that in the seventh day after SCC, the expression of Caspase-3 reduced significantly (P < 0.05) and Bcl-2 did not change significantly (P > 0.05). Immunohistochemistry experiment results showed that Caspase-3 Bcl-2 and Bax immunoreactive cells were observed in the neurons and glial cells of both white matter and gray matter in the spinal cord. The results were the same with QRT-PCR.. Conclusion TNF-α in rats after SCC can effectively regulate the ratio of Bcl-2 and Bax , and then regulate the expression of Caspase-3 , which may affect the function of apoptosis and function recovery after spinal cord injury.%目的：采用慢病毒抑制炎症因子TNF-α的表达，观察凋亡相关因子Caspase-3和Bcl-2、Bax 在脊髓损伤大鼠损伤段的表达变化，探究在脊髓损伤中TNF-α与Caspase-3、Bcl-2、Bax 的相互关系。方法：采用Allen 法制备脊髓钝挫伤模型；GeneMANIA 生物信息学方法预测TNF-α与Bcl-2的关系。用慢病毒包装的RNA ，构建 TNF-α的 RNA 干扰模型；TNF-α的表达被抑制后，用 QRT-PCR 检测 Caspase-3和 Bcl-2 mRNA的表达，用免疫组织化学技术检测Caspase-3和Bcl-2、Bax 蛋白质的定位分布，用SPSS 17．0进行统计分析。结果：脊髓钝挫伤后SD 大鼠双后肢截瘫，无法自行排便排尿。慢病毒干扰 TNF-α后 QRT-PCR 结果显示， Caspase-3表达显著减少（P ＜0．05），Bcl-2表达下降，但是没有显著变化（P ＞0．05）。免疫组织化学实验结果显示在脊髓白质和灰质的神经元和神经胶质细胞中观察到 Caspase-3、Bcl-2、Bax 的免疫阳性反应细胞，并与QRT-PCR变化结果相同。结论：在脊髓损伤后的大鼠体内TNF-α能有效调控Bcl-2和Bax 的比值，进而调节Caspase-3的表达，最终可能影响凋亡而作用于脊髓损伤后的功能恢复。