Conclusion
After sciatic nerve injury in rats, the up-regulation of the miR-221 expression targets the down-regulation of PTEN expression, which results in the difference of expression levels of miR-221 and PTEN in proximal and distal stumps. This phenomenon may play a role in promoting nerve repair after peripheral nerve injury. 目的: 研究大鼠坐骨神经损伤后近、远侧断端组织中微小 RNA-221（microRNA-221，miR-221）以及磷酸酶与张力蛋白同源物（phosphatase and tension protein homologue，PTEN）的表达情况，及其与周围神经损伤修复的相关性，以期为临床诊治周围神经损伤提供新靶点。. 方法: 取 SPF 级雄性 SD 大鼠 96 只建立坐骨神经损伤模型，分别于术后 0（术后即刻）、1、4、7 d 处死 24 只大鼠，无菌条件下取坐骨神经近、远侧断端组织。实时荧光定量 PCR 检测 miR-221 表达，Western blot、免疫荧光染色检测 PTEN 蛋白表达，采用双荧光素酶报告基因验证 miR-221 与 PTEN 的调控关系；同时结合透射电镜观察神经组织超微结构。. 结果: 实时荧光定量 PCR、Western blot 及免疫荧光染色检测示，术后大鼠坐骨神经近、远侧断端组织中 miR-221 相对表达量均逐渐增加，PTEN 蛋白相对表达量均逐渐减少，术后各时间点间比较差异均有统计学意义（ P<0.05）。术后 1、4、7 d 近侧断端 miR-221 相对表达量均显著高于远侧断端，PTEN 蛋白相对表达量均显著低于远侧断端，差异均有统计学意义（ P<0.05）。双荧光素酶报告基因示 PTEN 为 miR-221 作用靶点。透射电镜观察示随术后时间延长，雪旺细胞增殖及轴突、髓鞘溃变逐渐增加；术后 1 d 近、远侧断端无明显差异，4、7 d 时近侧断端雪旺细胞增殖较远侧断端增多，轴突、髓鞘溃变程度降低。. 结论: 大鼠坐骨神经损伤后，miR-221 表达上调，靶向调控 PTEN 表达降低，进而产生近、远侧断端 miR-221 及 PTEN 表达差异，这一现象可能对促进周围神经损伤后修复发挥一定作用。.
Methods
Ninety-six male Sprague-Dawley rats of SPF grade were selected to establish sciatic nerve injury models. Twenty-four rats were sacrificed at 0 (immediately after operation), 1, 4, and 7 days after operation. The proximal and distal sciatic nerve fragments were taken under aseptic conditions. The expression of miR-221 was detected by real-time fluorescent quantitative PCR, and the expression of PTEN protein was detected by Western blot and immunofluorescent staining. The relationship between miR-221 and PTEN was verified by dual-luciferase reporter gene. At the same time, the ultrastructure of nerve stump was observed by transmission electron microscopy.
Objective
To study the expressions of microRNA-221 (miR-221) and the protein of phosphatase and tension protein homologue (PTEN) in the proximal and distal stumps after sciatic nerve injury in rats and their correlation with the repair of peripheral nerve injury, so as to provide a new target for clinical diagnosis of peripheral nerve injury.
Results
The results of real-time fluorescent quantitative PCR, Western blot, and immunofluorescence staining showed that the relative expression of miR-221 in the proximal and distal stumps increased gradually with time, and the relative expression of PTEN protein decreased gradually, and the differences between different time points after operation were significant ( P<0.05). At 1, 4, and 7 days after operation, the relative expression of miR-221 in proximal stump was significantly higher than that in distal stump, and the relative expression of PTEN protein in proximal stump was significantly lower than that in distal stump ( P<0.05). Dual-luciferase reporter gene suggested that PTEN was the target for miR-221. Transmission electron microscopy observation showed that the normal morphological structure was observed at 0 day after operation, and the proliferation of Schwann cells and degeneration of axons and myelin sheaths gradually increased with time. There was no significant difference between proximal and distal stumps at 1 day after operation. At 4 and 7 days, Schwann cells proliferated more in proximal stump than in distal stump, and the degeneration of axons and myelin sheaths was less.