microRNA-146a调控Toll样受体4信号通路对神经病理性疼痛的影响

发布时间：2018-07-01 07:55

本文选题：神经病理性疼痛 + 双侧坐骨神经慢性压迫　；参考：《北京协和医学院》2015年博士论文

【摘要】：研究目的miR-146a是炎性免疫反应的重要调节因子之一,其两个主要靶基因IL-1相关蛋白激酶1(interleukin-1 receptor-associated kinase 1, IRAK1)和肿瘤坏死因子受体活化因子6(TNF receptor-associated factor 6, TRAF6)是Toll样受体4(toll like receptor 4, TLR4)信号转导通路中重要衔接因子,而TLR4信号转导通路的活化与神经病理性疼痛密切相关。本课题拟检测双侧坐骨神经慢性压迫损伤(bilateral chronic constriction injury, bCCI)大鼠L4-6背根神经节(dorsal root ganglion, DRG)中 microRNA-146a的表达变化及TLR4信号转导通路活化情况,研究其对IRAK1和TRAF6参与疼痛相关TLR4信号转导通路的调节作用,并探讨干预miR-146a表达后对bCCl神经病理性疼痛大鼠疼痛行为的影响,从而明确miR-146a对神经病理性疼痛的调控机制。研究方法1.检测bCCI神经病理性疼痛大鼠DRG中miR-146a表达及TLR4信号转导通路活化情况雌性SD大鼠,体重200-250g,采用随机数字表法随机分为①正常对照组(Naive) (N=12);②假手术组(Sham) (N=12):进行大鼠双侧假手术,仅暴露双侧坐骨神经不结扎；③双侧坐骨神经压迫损伤组(bCCI组)(N=12)：建立大鼠bCCI模型。在建模前1天、后1、3、7、14及21天监测3组大鼠机械刺激诱发痛、热刺激诱发痛行为学指标。实时定量PCR法检测大鼠L4-6 DRG中TLR4和miR-146a及其靶基因IRAK1、TRAF6 的mRNA表达水平；Western-blot法检测TLR4信号转导通路中TLR4、MyD88、TRIF、IRAK1、TRAF6、NF-κB及p-NF-κB蛋白表达水平变化；免疫组化对TLR4、IRAK1 和 TRAF6定位检测。ELISA检测不同时间点三组大鼠DRG中IL-6和TNF-a的表达水平。2.干预miR-146a表达对bCCI神经病理性疼痛大鼠疼痛行为学影响及其分子机制雌性SD大鼠,体重200-250g,采用随机数字表法随机分为①miR-146a过表达处理组(agomir-146a) (n=6); ②miR-146a低表达处理组(antagomir-146a)(n=6)；⑧阴性对照组(negative control, NC) (n=12)。各组均行双侧坐骨神经慢性压迫手术,于手术当天及术后第3、5、7、10天分别经鞘内注射容量为10 μ1的 agomiR-146a或 antagomiR-146a及相应对照试剂(5nmol溶于生理盐水),于术前及术后第1、3、7、14天进行动物行为学评估,观察大鼠对机械和热刺激的反应。实时定量PCR法检测术后第14天大鼠L4-6 DRG miR-146a及IRAK1、TRAF6的mRNA表达水平；Western-blot法检测IRAK1、TRAF6蛋白表达水平变化。培养DRG神经元原代细胞,分别孵育agomiR-146a、antagomiR-146a及对照试剂24小时后利用钙成像技术观察各组细胞LPS刺激后细胞内钙离子内流情况。结果1.与Naive组及Sham组相比,bCCI组大鼠L4-6 DRG中miR-146a在建模后第3天降低,第7天达最低值,第21天恢复基线水平。IRAK1和TRAF6的mRNA和蛋白水平均明显升高,且3组在不同时间点具有统计学差异(P0.05)。免疫组化证实IRAK1和TRAF6均在DRG神经元细胞上表达,且IRAK1以在肽能神经元上表达为主(52.3%),而TRAF6则较多表达在非肽能神经元上(63%)。2.与Naive组及Sham组相比,bCCI组大鼠L4-6 DRG中TLR4 mRNA水平显著升高,三组在不同时间点具有统计学差异(P0.05)。TLR4、MyD88、NF-κB和p-NF-κB蛋白水平升高(P0.05),TRIF蛋白水平无明显改变(P0.05)。免疫组化证实TLR4在DRG神经元细胞上表达；ELISA结果提示相比Naive组及Sham组,bCCI组大鼠DRG中IL-6和TNF-α表达水平增加(P0.05)3.与NC组相比,agomiR-146a组bCCI大鼠机械刺激阈值和热辐射刺激阈值均升高,各组不同时间点差异具体统计学意义(P0.05)。agomiR-146a组bCCI大鼠L4-6 DRG中miR-146a表达量显著上调,IRAK1和TRAF6 mRNA和蛋白水平均显著下降(P0.05)。与NC组相比,antagomiR-146a组bCCI大鼠疼痛行为学机械刺激和热刺激阈值无统计学差异。antagomiR-146a组bCCI大鼠L4-6DRG中miR-146a表达下调,IRAK1和TRAF6 mRNA和蛋白水平均显著增加(P0.05)。原代DRG神经元细胞钙成像结果显示agomiR-146a可有效减少细胞内钙离子内流阳性率(1.59%),而antagomiR-146a组细胞内钙离子增加(12.5%)。结论1.背根神经节中niR-146a表达下调,作用其靶基因IRAK1和TRAF6参与bCCI大鼠神经病理性疼痛。2.bCCI神经病理性疼痛大鼠背根神经节中TLR4-MyD88-NF-κB信号转导通路活化。3.鞘内注射agomiR-146a上调miR-146a表达水平可有效缓解bCCI神经病理性疼痛大鼠疼痛行为学。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R741

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