解放军医学杂志2017，Vol.42Issue(8)：686-691,6.DOI:10.11855/j.issn.0577-7402.2017.08.05
Tat-LK15介导siRNA干扰大鼠脊髓背角nNOS表达对神经病理性疼痛的治疗作用
Therapeutic effect of Tat-LK15-mediated the interference of siRNA with nNOS expression in the spinal dorsal horn on neuropathic pain in rats
摘要
Abstract
Objective To investigate the therapeutic effect of cell penetrating peptide Tat-LK15 mediating small interfering RNA (siRNA) interference with the expression of neuronal nitric oxide synthase (nNOS) in rat spinal dorsal horn on neuropathic pain. Methods The transfection reagent, Tat-LK15, was used to mediate the transfection of rat spinal dorsal horn (SDH) neuronal cells with carboxyfluorescein (FAM), and then the transfection effect was observed under inverted fluorescence microscope. Fifty healthy male SD rats were randomly divided into 5 groups (n=10): control group, sham operation group (sham group), neuropathic pain group (SNL group), Tat-LK15-nNOS siRNA group (TS group) and Tat-LK15-NC siRNA group (TN group). Neuropathic pain was induced by spinal nerve ligation (SNL), rats in control group did not receive operation and only the spinal nerve was exposed in sham group. Groups SNL, TS and TN were made into the models by SNL and implanted intrathecal catheter, intrathecal administration was performed from the 7th day after model establishment, and 10μl normal saline, 10μl TS complex (including 5μg siRNA) and 10μl TN (including 5μg siRNA) were injected intrathecally each day for 7 days. Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured at 1 day before (baseline) and 3, 7, 10 and 14 days after model establishment. Then animals were sacrificed on the 14th day after the operation and the lumbar segment (L4-6) of the spinal cord was removed to detect the expressions of nNOS mRNA and protein using q-PCR and Western blotting analysis. Results Tat-LK15 effectively mediated FAM-siRNA into SDH neuronal cells. Compared with sham group, SNL significantly decreased PWMT and PWTL and increased expressions of nNOS mRNA and protein from the 3rd day (P<0.01), but there was no significant difference between the sham and control group. Tat-LK15-nNOS siRNA complex significantly increased PWMT and PWTL and down-regulated nNOS mRNA and protein expressions in TS group compared with SNL group on the days 10 and 14. There was no significant difference between TN and SNL group. Conclusion Tat-LK15 not only can mediate successful nNOS siRNA transfection and inhibit the expression of nNOS, but also effectively relieve SNL-induced neuropathic pain in rats.关键词
Tat-LK15/RNA,小分子干扰/神经元型一氧化氮合酶/神经病理性疼痛/基因治疗Key words
Tat-LK15/RNA/small interfering/neuronal nitric oxide synthase/neuropathic pain/gene therapy分类
医药卫生引用本文复制引用
饶云,彭捷,陆建华,吴昊澎,杨秀环,屠伟峰..Tat-LK15介导siRNA干扰大鼠脊髓背角nNOS表达对神经病理性疼痛的治疗作用[J].解放军医学杂志,2017,42(8):686-691,6.基金项目
国家自然科学基金(81100817,81371233) （81100817,81371233）
广东省自然科学基金面上项目(S2012010009271)This work was supported by the National Natural Science Foundation of China (81100817, 81371233) and the Natural Science Foundation of Guangdong Province (S2012010009271) （S2012010009271）
