本文選題：神經(jīng)病理性疼痛 + 雙側(cè)坐骨神經(jīng)慢性壓迫　； 參考：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：研究目的miR-146a是炎性免疫反應(yīng)的重要調(diào)節(jié)因子之一,其兩個(gè)主要靶基因IL-1相關(guān)蛋白激酶1(interleukin-1 receptor-associated kinase 1, IRAK1)和腫瘤壞死因子受體活化因子6(TNF receptor-associated factor 6, TRAF6)是Toll樣受體4(toll like receptor 4, TLR4)信號(hào)轉(zhuǎn)導(dǎo)通路中重要銜接因子,而TLR4信號(hào)轉(zhuǎn)導(dǎo)通路的活化與神經(jīng)病理性疼痛密切相關(guān)。本課題擬檢測(cè)雙側(cè)坐骨神經(jīng)慢性壓迫損傷(bilateral chronic constriction injury, bCCI)大鼠L4-6背根神經(jīng)節(jié)(dorsal root ganglion, DRG)中 microRNA-146a的表達(dá)變化及TLR4信號(hào)轉(zhuǎn)導(dǎo)通路活化情況,研究其對(duì)IRAK1和TRAF6參與疼痛相關(guān)TLR4信號(hào)轉(zhuǎn)導(dǎo)通路的調(diào)節(jié)作用,并探討干預(yù)miR-146a表達(dá)后對(duì)bCCl神經(jīng)病理性疼痛大鼠疼痛行為的影響,從而明確miR-146a對(duì)神經(jīng)病理性疼痛的調(diào)控機(jī)制。研究方法1.檢測(cè)bCCI神經(jīng)病理性疼痛大鼠DRG中miR-146a表達(dá)及TLR4信號(hào)轉(zhuǎn)導(dǎo)通路活化情況雌性SD大鼠,體重200-250g,采用隨機(jī)數(shù)字表法隨機(jī)分為①正常對(duì)照組(Naive) (N=12);②假手術(shù)組(Sham) (N=12):進(jìn)行大鼠雙側(cè)假手術(shù),僅暴露雙側(cè)坐骨神經(jīng)不結(jié)扎；③雙側(cè)坐骨神經(jīng)壓迫損傷組(bCCI組)(N=12)：建立大鼠bCCI模型。在建模前1天、后1、3、7、14及21天監(jiān)測(cè)3組大鼠機(jī)械刺激誘發(fā)痛、熱刺激誘發(fā)痛行為學(xué)指標(biāo)。實(shí)時(shí)定量PCR法檢測(cè)大鼠L4-6 DRG中TLR4和miR-146a及其靶基因IRAK1、TRAF6 的mRNA表達(dá)水平；Western-blot法檢測(cè)TLR4信號(hào)轉(zhuǎn)導(dǎo)通路中TLR4、MyD88、TRIF、IRAK1、TRAF6、NF-κB及p-NF-κB蛋白表達(dá)水平變化；免疫組化對(duì)TLR4、IRAK1 和 TRAF6定位檢測(cè)。ELISA檢測(cè)不同時(shí)間點(diǎn)三組大鼠DRG中IL-6和TNF-a的表達(dá)水平。2.干預(yù)miR-146a表達(dá)對(duì)bCCI神經(jīng)病理性疼痛大鼠疼痛行為學(xué)影響及其分子機(jī)制雌性SD大鼠,體重200-250g,采用隨機(jī)數(shù)字表法隨機(jī)分為①miR-146a過(guò)表達(dá)處理組(agomir-146a) (n=6); ②miR-146a低表達(dá)處理組(antagomir-146a)(n=6)；⑧陰性對(duì)照組(negative control, NC) (n=12)。各組均行雙側(cè)坐骨神經(jīng)慢性壓迫手術(shù),于手術(shù)當(dāng)天及術(shù)后第3、5、7、10天分別經(jīng)鞘內(nèi)注射容量為10 μ1的 agomiR-146a或 antagomiR-146a及相應(yīng)對(duì)照試劑(5nmol溶于生理鹽水),于術(shù)前及術(shù)后第1、3、7、14天進(jìn)行動(dòng)物行為學(xué)評(píng)估,觀察大鼠對(duì)機(jī)械和熱刺激的反應(yīng)。實(shí)時(shí)定量PCR法檢測(cè)術(shù)后第14天大鼠L4-6 DRG miR-146a及IRAK1、TRAF6的mRNA表達(dá)水平；Western-blot法檢測(cè)IRAK1、TRAF6蛋白表達(dá)水平變化。培養(yǎng)DRG神經(jīng)元原代細(xì)胞,分別孵育agomiR-146a、antagomiR-146a及對(duì)照試劑24小時(shí)后利用鈣成像技術(shù)觀察各組細(xì)胞LPS刺激后細(xì)胞內(nèi)鈣離子內(nèi)流情況。結(jié)果1.與Naive組及Sham組相比,bCCI組大鼠L4-6 DRG中miR-146a在建模后第3天降低,第7天達(dá)最低值,第21天恢復(fù)基線水平。IRAK1和TRAF6的mRNA和蛋白水平均明顯升高,且3組在不同時(shí)間點(diǎn)具有統(tǒng)計(jì)學(xué)差異(P0.05)。免疫組化證實(shí)IRAK1和TRAF6均在DRG神經(jīng)元細(xì)胞上表達(dá),且IRAK1以在肽能神經(jīng)元上表達(dá)為主(52.3%),而TRAF6則較多表達(dá)在非肽能神經(jīng)元上(63%)。2.與Naive組及Sham組相比,bCCI組大鼠L4-6 DRG中TLR4 mRNA水平顯著升高,三組在不同時(shí)間點(diǎn)具有統(tǒng)計(jì)學(xué)差異(P0.05)。TLR4、MyD88、NF-κB和p-NF-κB蛋白水平升高(P0.05),TRIF蛋白水平無(wú)明顯改變(P0.05)。免疫組化證實(shí)TLR4在DRG神經(jīng)元細(xì)胞上表達(dá)；ELISA結(jié)果提示相比Naive組及Sham組,bCCI組大鼠DRG中IL-6和TNF-α表達(dá)水平增加(P0.05)3.與NC組相比,agomiR-146a組bCCI大鼠機(jī)械刺激閾值和熱輻射刺激閾值均升高,各組不同時(shí)間點(diǎn)差異具體統(tǒng)計(jì)學(xué)意義(P0.05)。agomiR-146a組bCCI大鼠L4-6 DRG中miR-146a表達(dá)量顯著上調(diào),IRAK1和TRAF6 mRNA和蛋白水平均顯著下降(P0.05)。與NC組相比,antagomiR-146a組bCCI大鼠疼痛行為學(xué)機(jī)械刺激和熱刺激閾值無(wú)統(tǒng)計(jì)學(xué)差異。antagomiR-146a組bCCI大鼠L4-6DRG中miR-146a表達(dá)下調(diào),IRAK1和TRAF6 mRNA和蛋白水平均顯著增加(P0.05)。原代DRG神經(jīng)元細(xì)胞鈣成像結(jié)果顯示agomiR-146a可有效減少細(xì)胞內(nèi)鈣離子內(nèi)流陽(yáng)性率(1.59%),而antagomiR-146a組細(xì)胞內(nèi)鈣離子增加(12.5%)。結(jié)論1.背根神經(jīng)節(jié)中niR-146a表達(dá)下調(diào),作用其靶基因IRAK1和TRAF6參與bCCI大鼠神經(jīng)病理性疼痛。2.bCCI神經(jīng)病理性疼痛大鼠背根神經(jīng)節(jié)中TLR4-MyD88-NF-κB信號(hào)轉(zhuǎn)導(dǎo)通路活化。3.鞘內(nèi)注射agomiR-146a上調(diào)miR-146a表達(dá)水平可有效緩解bCCI神經(jīng)病理性疼痛大鼠疼痛行為學(xué)。
[Abstract]:Objective miR-146a is one of the important regulators of inflammatory immune response, and the two major target genes, IL-1 related protein kinase 1 (interleukin-1 receptor-associated kinase 1, IRAK1) and tumor necrosis factor receptor activator 6 (TNF receptor-associated factor 6, TRAF6), are Toll like receptor 4 (Toll 4, 4,) The activation of TLR4 signaling pathway is closely related to neuropathic pain in the transduction pathway. This topic is to detect the changes in the expression of microRNA-146a in the L4-6 dorsal root ganglion (dorsal root ganglion, DRG) of the bilateral chronic constriction injury (bCCI) rats. The activation of signal transduction pathway, the regulatory role of IRAK1 and TRAF6 involved in the pain related TLR4 signal transduction pathway, and the effect of miR-146a expression on the pain behavior of bCCl neuropathic pain rats, so as to clarify the regulation mechanism of miR-146a on neuropathic pain. Method 1. detection of bCCI neuropathic pain. The expression of miR-146a in DRG and the activation of TLR4 signal transduction pathway in the female SD rats were activated with 200-250g, and were randomly divided into 1 normal control group (Naive) (Naive) (N=12), and (Sham) (N=12): bilateral sciatic nerve non ligature in rats, and the group of bilateral sciatic nerve compression injury group (b). Group CCI (N=12): establish rat bCCI model. 1 days before modeling, after 1,3,7,14 and 21 days, the mechanical stimulation pain of 3 groups of rats and the behavioral indexes of heat stimulation induced pain were monitored. The real-time quantitative PCR method was used to detect TLR4 and miR-146a, IRAK1 of the target gene and TRAF6 mRNA expression level in the L4-6 DRG of rats. Changes in the expression level of LR4, MyD88, TRIF, IRAK1, TRAF6, NF- kappa B and p-NF- kappa B protein; immunohistochemistry on TLR4, IRAK1, and TRAF6 location detection in three groups of rats at different time points Rats, weight 200-250g, were randomly divided into miR-146a overexpression treatment group (agomir-146a) (n=6), (antagomir-146a) miR-146a low expression group (n=6) and negative control group (negative control, NC) (n=12). Both groups were operated on bilateral sciatic nerve chronic compression operation, and the day of operation and postoperative 3,5,7,10 days were respectively. AgomiR-146a or antagomiR-146a and corresponding control reagents (5nmol dissolved in physiological saline) were injected into the intrathecal 10 mu 1. The animal behavioral assessment was performed before and after the operation on day 1,3,7,14. The response to mechanical and thermal stimulation was observed. Real-time quantitative PCR was used to detect L4-6 DRG miR-146a and IRAK1, mRNA table of TRAF6. The expression level of IRAK1 and TRAF6 protein was detected by Western-blot. The primary cells of DRG neurons were cultured, and agomiR-146a, antagomiR-146a and control reagents were incubated for 24 hours respectively. Calcium imaging was used to observe the intracellular calcium influx of each cell after LPS stimulation. The fruit 1. was compared with the Naive group and the Sham group, and L4-6 in the bCCI group L4-6. In DRG, miR-146a was reduced at third days after modeling, seventh Tianda was lowest, the mRNA and protein levels of.IRAK1 and TRAF6 at twenty-first days of recovery were significantly increased, and the 3 groups were statistically different at different time points (P0.05). Immunohistochemistry confirmed that both IRAK1 and TRAF6 were expressed on the fine cell of DRG neurons, and IRAK1 was expressed on peptiderma neurons. Main (52.3%), while TRAF6 was more expressed on non peptidergic neurons (63%).2. and Naive group and Sham group, TLR4 mRNA level in bCCI group L4-6 DRG increased significantly. The three groups had statistical difference at different time points (P0.05).TLR4. The expression of TLR4 on DRG neuron cells was confirmed by histochemistry. ELISA results showed that the level of IL-6 and TNF- alpha in DRG of bCCI group increased (P0.05) 3. compared with that of group Naive and Sham group, and the threshold of mechanical stimulation and the threshold of thermal radiation stimulation were all higher in the agomiR-146a group, and the difference of time points in each group was statistically significant. The expression of miR-146a in L4-6 DRG in group omiR-146a bCCI rats was significantly up-regulated, and IRAK1 and TRAF6 mRNA and protein levels were significantly decreased (P0.05). The level of NA and protein increased significantly (P0.05). The results of primary DRG neuron cell calcium imaging showed that agomiR-146a could effectively reduce the positive rate of intracellular calcium influx (1.59%), while the intracellular calcium ion increased (12.5%) in antagomiR-146a group. Conclusion the niR-146a table in 1. dorsal root ganglia was downregulated, and the target gene IRAK1 and TRAF6 were involved in bCCI rats. Neuropathic pain.2.bCCI neuropathic pain rats TLR4-MyD88-NF- kappa B signal transduction pathway activation.3. intrathecal agomiR-146a up regulation of miR-146a expression level can effectively alleviate the pain behavior of bCCI neuropathic pain rats.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R741

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本文編號(hào)：2087086