四环素调控的干扰TLR4受体的慢病毒抑制坐骨神经结扎大鼠的伤害性刺激的研究
发布时间：2019-06-21 17:45
【摘要】：神经病理性疼痛，由外周神经，背根神经节，脊髓或中枢神经系统的损伤引起，其特征是痛觉过敏、异常性疼痛及自发性疼痛。神经病理性疼痛的治疗依旧是个难题目前，临床主要用阿片类止痛药物治疗，但其效果有限且副作用较多，这对患者的生活带来了很大的困扰。外周神经损伤以后，脊髓背角神经胶质细胞的激活在神经病理性疼痛的产生和维持中发挥了重要作用。Toll样受体4（TLR4）是一种模式识别受体，参与了胶质细胞的激活，是使中枢敏化导致疼痛的一个重要蛋白。然而，目前尚未有针对TLR4蛋白的可利用的抑制剂。RNA干扰技术为基因功能的研究提供了可靠的途径。由慢病毒介导的RNA干扰技术已被批准用于人类研究。长期的基因下调可以通过利用慢病毒携带小发夹RNA(shRNA)导入受感染细胞来实现, shRNA的发卡结构可被细胞机制切割成siRNA，然后siRNA结合到RNA诱导沉默复合物上(RNA-inducedsilencing complex，RISC)，该复合物能够结合到目的mRNAs并将其降解。在本研究中我们构建携带TLR4小发夹RNA的慢病毒，经鞘内注射进入蛛网膜下腔，拟达到治疗大鼠神经病理性疼痛的目的。为了防止慢病毒对体内TLR4蛋白的过度抑制而产生副作用，我们对慢病毒加入了四环素tet-on调控系统。用四环素类似物强力霉素来启动慢病毒的表达。 大鼠的神经病理性疼痛模型由结扎右侧坐骨神经（CCI）来构建。慢病毒是通过鞘内置管给予,强力霉素溶于动物饮用水中通过口服的方式给予。 该研究分为两个部分： 1.四环素可调控的干扰TLR4蛋白的慢病毒包装与构建。 2.鞘内注射慢病毒LvOn-siTLR4对坐骨神经结扎的大鼠热痛阈和机械痛阈的影响，此部分实验将SD雄性大鼠分为6组，每组7只。分别为:sham组（假手术大鼠鞘内注射生理盐水）；CCI组（CCI大鼠鞘内注射生理盐水）；CCI+LvOn-siTLR4+DOX组（CCI+鞘内注射LvOn-siTLR4+口服强力霉素）；CCI+LvOn-siTLR4组（CCI+鞘内注射LvOn-siTLR4）CCI+Lv-mismatch siRNA（CCI+携带错配序列的慢病毒）；CCI+DOX组（CCI+鞘内注射生理盐水+口服强力霉素）。坐骨神经结扎手术和鞘内注射慢病毒在一次麻醉中先后实施。通过测量大鼠的机械痛阈和热痛阈来评估慢病毒对大鼠疼痛的影响。术后第7天处死大鼠取脊髓腰膨大节段，分别用RT-PCR技术及western-blot分析TLR4mRNA的表达及蛋白表达的情况。 主要研究结果如下： 1.四环素可调控的携带TLR4shRNA的慢病毒成功构建并完成滴度测定，体外转染成功。 2.①与假手术组相比，，CCI组的大鼠机械刺激痛和热刺激痛阈值明显降低（P＜0.05），脊髓腰L4-L6节段TLR4的mRNA及蛋白表达升高（P＜0.05）。②在坐骨神经结扎后的第3，5，7天，LvOn-siTLR4+DOX组的大鼠机械痛阈与热痛阈值比CCI组、Lv-mismatch siRNA组及CCI+DOX组相比有明显的升高（P＜0.05），脊髓腰膨大TLR4mRNA及蛋白的表达降低。③CCI组与CCI+DOX组相比，大鼠机械痛阈值与热刺激痛阈值无明显差异（P0.05）,脊髓腰膨大TLR4mRNA表达及蛋白表达无明显差异（P0.05）. 结论： 1.四环素可调控的携带TLR4小干扰RNA的慢病毒成功构建。 2.在CCI大鼠鞘内注射可调控的干扰TLR4受体的慢病毒可以缓解大鼠的神经病理性疼痛，大鼠服用强力霉素后热痛阈和机械痛阈升高，脊髓TLR4蛋白的表达量明显降低。
[Abstract]:Neuropathic pain is caused by injury to peripheral nerves, dorsal root ganglia, spinal cord or central nervous system, characterized by hyperalgesia, abnormal pain and spontaneous pain. The treatment of neuropathic pain is still a difficult problem. At present, it is mainly used for the treatment of opioid analgesic drugs, but the effect is limited and the side effect is more, which has brought great trouble to the patient's life. After peripheral nerve injury, the activation of the dorsal horn of the spinal cord plays an important role in the generation and maintenance of neuropathic pain. Toll-like receptor 4 (TLR4) is a pattern recognition receptor, which is involved in the activation of glial cells and is an important protein that causes central sensitization to cause pain. However, there are currently no available inhibitors for TLR4 proteins. RNA interference technology provides a reliable way for the study of gene function. RNA interference techniques mediated by lentiviruses have been approved for human studies. Long-term gene downregulation can be achieved by introducing small hairpin RNA (shRNA) into infected cells with lentiviruses, the hairpin structure of shRNA can be cut into siRNA by the cellular mechanism, and then the siRNA binds to the RNA-induced silencing complex (RISC), which can bind to the target mRNAs and degrade it. In this study we constructed lentivirus carrying the small hairpin RNA of TLR4, and injected into the subarachnoid space via intraoral injection, and it is proposed to achieve the purpose of treating the neuropathic pain of the rat. In order to prevent the slow virus from having side effects on the over-inhibition of the TLR4 protein in the body, we have added the tetracycline tet-on control system to the lentivirus. The expression of lentivirus was initiated with a tetracycline analogue of strong mycin. The model of neuropathic pain in rats was constructed by ligation of the right sciatic nerve (CCI). The lentivirus is administered by a built-in tube, and the doxycycline is dissolved in the drinking water of the animal through oral administration. The study is divided into two part: 1.Tetracycline-regulated lentivirus that interferes with TLR4 protein Packaging and construction.2. The effect of slow virus LvOn-siTLR4 on the threshold of thermal pain and mechanical pain in rats with sciatic nerve ligation. The CCI + LvOn-siTLR4 + DOX group (CCI + intraoral injection of LvOn-siTLR4 + oral doxycycline); CCI + LvOn-siTLR4 group (CCI + intra-injection LvOn-siTLR4) CCI + Lv-mismatch siRNA (CCI + carrying mismatch) lentivirus of the sequence); CCI + DOX group (CCI + intraventricular injection of physiological saline + Oral doxycycline). Sciatic nerve ligation and intraoral injection of lentivirus in one time The effect of slow virus was assessed by measuring the mechanical pain threshold and the thermal pain threshold of the rat. The expression of TLR4 mRNA was analyzed by RT-PCR and western-blot respectively. The expression of the protein. The main results are as follows: 1.Tetracycline-regulated lentivirus carrying TLR4shRNA was successfully constructed and completed. 2. Compared with the sham operation group, the mechanical stimulation pain and the thermal stimulation pain threshold in the CCI group decreased significantly (P <0.05), and the mRNA and egg of the L4-L6 segment of the spinal cord L4-L6 were significantly lower (P <0.05). The mechanical pain threshold and the thermal pain threshold of LvOn-siTLR4 + DOX group were significantly higher than that of the CCI group, the Lv-mismatch siRNA group and the CCI + DOX group (P <0.05). The expression of TLR4 mRNA and protein in the spinal cord were not significantly different from that of the CCI + DOX group (P0.05). to be out of sight Significant difference (P0.05). Conclusion:1. Tetracycline can regulate and carry T. LR4 lentivirus with small interfering RNA was successfully constructed.2. The slow virus which can regulate and control the TLR4 receptor in the rat of CCI rats can relieve the neuropathic pain of the rat, and the threshold of heat pain and the threshold of mechanical pain after the administration of the doxycycline in the rat
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R614
本文编号：2504254
[Abstract]:Neuropathic pain is caused by injury to peripheral nerves, dorsal root ganglia, spinal cord or central nervous system, characterized by hyperalgesia, abnormal pain and spontaneous pain. The treatment of neuropathic pain is still a difficult problem. At present, it is mainly used for the treatment of opioid analgesic drugs, but the effect is limited and the side effect is more, which has brought great trouble to the patient's life. After peripheral nerve injury, the activation of the dorsal horn of the spinal cord plays an important role in the generation and maintenance of neuropathic pain. Toll-like receptor 4 (TLR4) is a pattern recognition receptor, which is involved in the activation of glial cells and is an important protein that causes central sensitization to cause pain. However, there are currently no available inhibitors for TLR4 proteins. RNA interference technology provides a reliable way for the study of gene function. RNA interference techniques mediated by lentiviruses have been approved for human studies. Long-term gene downregulation can be achieved by introducing small hairpin RNA (shRNA) into infected cells with lentiviruses, the hairpin structure of shRNA can be cut into siRNA by the cellular mechanism, and then the siRNA binds to the RNA-induced silencing complex (RISC), which can bind to the target mRNAs and degrade it. In this study we constructed lentivirus carrying the small hairpin RNA of TLR4, and injected into the subarachnoid space via intraoral injection, and it is proposed to achieve the purpose of treating the neuropathic pain of the rat. In order to prevent the slow virus from having side effects on the over-inhibition of the TLR4 protein in the body, we have added the tetracycline tet-on control system to the lentivirus. The expression of lentivirus was initiated with a tetracycline analogue of strong mycin. The model of neuropathic pain in rats was constructed by ligation of the right sciatic nerve (CCI). The lentivirus is administered by a built-in tube, and the doxycycline is dissolved in the drinking water of the animal through oral administration. The study is divided into two part: 1.Tetracycline-regulated lentivirus that interferes with TLR4 protein Packaging and construction.2. The effect of slow virus LvOn-siTLR4 on the threshold of thermal pain and mechanical pain in rats with sciatic nerve ligation. The CCI + LvOn-siTLR4 + DOX group (CCI + intraoral injection of LvOn-siTLR4 + oral doxycycline); CCI + LvOn-siTLR4 group (CCI + intra-injection LvOn-siTLR4) CCI + Lv-mismatch siRNA (CCI + carrying mismatch) lentivirus of the sequence); CCI + DOX group (CCI + intraventricular injection of physiological saline + Oral doxycycline). Sciatic nerve ligation and intraoral injection of lentivirus in one time The effect of slow virus was assessed by measuring the mechanical pain threshold and the thermal pain threshold of the rat. The expression of TLR4 mRNA was analyzed by RT-PCR and western-blot respectively. The expression of the protein. The main results are as follows: 1.Tetracycline-regulated lentivirus carrying TLR4shRNA was successfully constructed and completed. 2. Compared with the sham operation group, the mechanical stimulation pain and the thermal stimulation pain threshold in the CCI group decreased significantly (P <0.05), and the mRNA and egg of the L4-L6 segment of the spinal cord L4-L6 were significantly lower (P <0.05). The mechanical pain threshold and the thermal pain threshold of LvOn-siTLR4 + DOX group were significantly higher than that of the CCI group, the Lv-mismatch siRNA group and the CCI + DOX group (P <0.05). The expression of TLR4 mRNA and protein in the spinal cord were not significantly different from that of the CCI + DOX group (P0.05). to be out of sight Significant difference (P0.05). Conclusion:1. Tetracycline can regulate and carry T. LR4 lentivirus with small interfering RNA was successfully constructed.2. The slow virus which can regulate and control the TLR4 receptor in the rat of CCI rats can relieve the neuropathic pain of the rat, and the threshold of heat pain and the threshold of mechanical pain after the administration of the doxycycline in the rat
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R614
【参考文献】
相关期刊论文 前1条
1 ;Tetracycline-inducible Expression Systems: New Strategies and Practices in the Transgenic Mouse Modeling[J];Acta Biochimica et Biophysica Sinica;2007年04期
本文编号：2504254
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