【摘要】：神經(jīng)病理性疼痛，由外周神經(jīng)，背根神經(jīng)節(jié)，脊髓或中樞神經(jīng)系統(tǒng)的損傷引起，其特征是痛覺過敏、異常性疼痛及自發(fā)性疼痛。神經(jīng)病理性疼痛的治療依舊是個難題目前，臨床主要用阿片類止痛藥物治療，但其效果有限且副作用較多，這對患者的生活帶來了很大的困擾。外周神經(jīng)損傷以后，脊髓背角神經(jīng)膠質(zhì)細(xì)胞的激活在神經(jīng)病理性疼痛的產(chǎn)生和維持中發(fā)揮了重要作用。Toll樣受體4（TLR4）是一種模式識別受體，參與了膠質(zhì)細(xì)胞的激活，是使中樞敏化導(dǎo)致疼痛的一個重要蛋白。然而，目前尚未有針對TLR4蛋白的可利用的抑制劑。RNA干擾技術(shù)為基因功能的研究提供了可靠的途徑。由慢病毒介導(dǎo)的RNA干擾技術(shù)已被批準(zhǔn)用于人類研究。長期的基因下調(diào)可以通過利用慢病毒攜帶小發(fā)夾RNA(shRNA)導(dǎo)入受感染細(xì)胞來實現(xiàn), shRNA的發(fā)卡結(jié)構(gòu)可被細(xì)胞機制切割成siRNA，然后siRNA結(jié)合到RNA誘導(dǎo)沉默復(fù)合物上(RNA-inducedsilencing complex，RISC)，該復(fù)合物能夠結(jié)合到目的mRNAs并將其降解。在本研究中我們構(gòu)建攜帶TLR4小發(fā)夾RNA的慢病毒，經(jīng)鞘內(nèi)注射進入蛛網(wǎng)膜下腔，擬達(dá)到治療大鼠神經(jīng)病理性疼痛的目的。為了防止慢病毒對體內(nèi)TLR4蛋白的過度抑制而產(chǎn)生副作用，我們對慢病毒加入了四環(huán)素tet-on調(diào)控系統(tǒng)。用四環(huán)素類似物強力霉素來啟動慢病毒的表達(dá)。 大鼠的神經(jīng)病理性疼痛模型由結(jié)扎右側(cè)坐骨神經(jīng)（CCI）來構(gòu)建。慢病毒是通過鞘內(nèi)置管給予,強力霉素溶于動物飲用水中通過口服的方式給予。 該研究分為兩個部分： 1.四環(huán)素可調(diào)控的干擾TLR4蛋白的慢病毒包裝與構(gòu)建。 2.鞘內(nèi)注射慢病毒LvOn-siTLR4對坐骨神經(jīng)結(jié)扎的大鼠熱痛閾和機械痛閾的影響，此部分實驗將SD雄性大鼠分為6組，每組7只。分別為:sham組（假手術(shù)大鼠鞘內(nèi)注射生理鹽水）；CCI組（CCI大鼠鞘內(nèi)注射生理鹽水）；CCI+LvOn-siTLR4+DOX組（CCI+鞘內(nèi)注射LvOn-siTLR4+口服強力霉素）；CCI+LvOn-siTLR4組（CCI+鞘內(nèi)注射LvOn-siTLR4）CCI+Lv-mismatch siRNA（CCI+攜帶錯配序列的慢病毒）；CCI+DOX組（CCI+鞘內(nèi)注射生理鹽水+口服強力霉素）。坐骨神經(jīng)結(jié)扎手術(shù)和鞘內(nèi)注射慢病毒在一次麻醉中先后實施。通過測量大鼠的機械痛閾和熱痛閾來評估慢病毒對大鼠疼痛的影響。術(shù)后第7天處死大鼠取脊髓腰膨大節(jié)段，分別用RT-PCR技術(shù)及western-blot分析TLR4mRNA的表達(dá)及蛋白表達(dá)的情況。 主要研究結(jié)果如下： 1.四環(huán)素可調(diào)控的攜帶TLR4shRNA的慢病毒成功構(gòu)建并完成滴度測定，體外轉(zhuǎn)染成功。 2.①與假手術(shù)組相比，，CCI組的大鼠機械刺激痛和熱刺激痛閾值明顯降低（P＜0.05），脊髓腰L4-L6節(jié)段TLR4的mRNA及蛋白表達(dá)升高（P＜0.05）。②在坐骨神經(jīng)結(jié)扎后的第3，5，7天，LvOn-siTLR4+DOX組的大鼠機械痛閾與熱痛閾值比CCI組、Lv-mismatch siRNA組及CCI+DOX組相比有明顯的升高（P＜0.05），脊髓腰膨大TLR4mRNA及蛋白的表達(dá)降低。③CCI組與CCI+DOX組相比，大鼠機械痛閾值與熱刺激痛閾值無明顯差異（P0.05）,脊髓腰膨大TLR4mRNA表達(dá)及蛋白表達(dá)無明顯差異（P0.05）. 結(jié)論： 1.四環(huán)素可調(diào)控的攜帶TLR4小干擾RNA的慢病毒成功構(gòu)建。 2.在CCI大鼠鞘內(nèi)注射可調(diào)控的干擾TLR4受體的慢病毒可以緩解大鼠的神經(jīng)病理性疼痛，大鼠服用強力霉素后熱痛閾和機械痛閾升高，脊髓TLR4蛋白的表達(dá)量明顯降低。
[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
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R614

【參考文獻】

相關(guān)期刊論文 前1條

1 ;Tetracycline-inducible Expression Systems: New Strategies and Practices in the Transgenic Mouse Modeling[J];Acta Biochimica et Biophysica Sinica;2007年04期

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