microRNA-146a調(diào)控Toll樣受體4信號通路對神經(jīng)病理性疼痛的影響
發(fā)布時間:2018-07-01 07:55
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)信號轉(zhuǎn)導(dǎo)通路中重要銜接因子,而TLR4信號轉(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的表達變化及TLR4信號轉(zhuǎn)導(dǎo)通路活化情況,研究其對IRAK1和TRAF6參與疼痛相關(guān)TLR4信號轉(zhuǎn)導(dǎo)通路的調(diào)節(jié)作用,并探討干預(yù)miR-146a表達后對bCCl神經(jīng)病理性疼痛大鼠疼痛行為的影響,從而明確miR-146a對神經(jīng)病理性疼痛的調(diào)控機制。研究方法1.檢測bCCI神經(jīng)病理性疼痛大鼠DRG中miR-146a表達及TLR4信號轉(zhuǎn)導(dǎo)通路活化情況雌性SD大鼠,體重200-250g,采用隨機數(shù)字表法隨機分為①正常對照組(Naive) (N=12);②假手術(shù)組(Sham) (N=12):進行大鼠雙側(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3組大鼠機械刺激誘發(fā)痛、熱刺激誘發(fā)痛行為學(xué)指標(biāo)。實時定量PCR法檢測大鼠L4-6 DRG中TLR4和miR-146a及其靶基因IRAK1、TRAF6 的mRNA表達水平;Western-blot法檢測TLR4信號轉(zhuǎn)導(dǎo)通路中TLR4、MyD88、TRIF、IRAK1、TRAF6、NF-κB及p-NF-κB蛋白表達水平變化;免疫組化對TLR4、IRAK1 和 TRAF6定位檢測。ELISA檢測不同時間點三組大鼠DRG中IL-6和TNF-a的表達水平。2.干預(yù)miR-146a表達對bCCI神經(jīng)病理性疼痛大鼠疼痛行為學(xué)影響及其分子機制雌性SD大鼠,體重200-250g,采用隨機數(shù)字表法隨機分為①miR-146a過表達處理組(agomir-146a) (n=6); ②miR-146a低表達處理組(antagomir-146a)(n=6);⑧陰性對照組(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天進行動物行為學(xué)評估,觀察大鼠對機械和熱刺激的反應(yīng)。實時定量PCR法檢測術(shù)后第14天大鼠L4-6 DRG miR-146a及IRAK1、TRAF6的mRNA表達水平;Western-blot法檢測IRAK1、TRAF6蛋白表達水平變化。培養(yǎng)DRG神經(jīng)元原代細(xì)胞,分別孵育agomiR-146a、antagomiR-146a及對照試劑24小時后利用鈣成像技術(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組在不同時間點具有統(tǒng)計學(xué)差異(P0.05)。免疫組化證實IRAK1和TRAF6均在DRG神經(jīng)元細(xì)胞上表達,且IRAK1以在肽能神經(jīng)元上表達為主(52.3%),而TRAF6則較多表達在非肽能神經(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,三組在不同時間點具有統(tǒng)計學(xué)差異(P0.05)。TLR4、MyD88、NF-κB和p-NF-κB蛋白水平升高(P0.05),TRIF蛋白水平無明顯改變(P0.05)。免疫組化證實TLR4在DRG神經(jīng)元細(xì)胞上表達;ELISA結(jié)果提示相比Naive組及Sham組,bCCI組大鼠DRG中IL-6和TNF-α表達水平增加(P0.05)3.與NC組相比,agomiR-146a組bCCI大鼠機械刺激閾值和熱輻射刺激閾值均升高,各組不同時間點差異具體統(tǒng)計學(xué)意義(P0.05)。agomiR-146a組bCCI大鼠L4-6 DRG中miR-146a表達量顯著上調(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)機械刺激和熱刺激閾值無統(tǒng)計學(xué)差異。antagomiR-146a組bCCI大鼠L4-6DRG中miR-146a表達下調(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信號轉(zhuǎn)導(dǎo)通路活化。3.鞘內(nèi)注射agomiR-146a上調(diào)miR-146a表達水平可有效緩解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)位級別】:博士
【學(xué)位授予年份】:2015
【分類號】:R741
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