缝隙连接蛋白磷酸化介导镜像痛敏的时空表达研究

发布时间：2019-05-30 07:43

【摘要】：疼痛是当前严重困扰人类健康的生物医学难题之一。疼痛诱发因素多样化,如机体组织损伤、炎症、情绪失控、运动感觉障碍、遗传突变、肿瘤等均可改变痛觉感受、传递、整合的个体自我保护和内在适应的可塑性,所呈现的持续性、慢性化、个性化在多层面制约着临床预防、诊断、治疗策略的有效选择。疼痛表症多样化,如除在躯体损伤部位表现持续自发痛、原发性痛敏和损伤部位周围区域出现继发性痛敏外,在损伤部位同体节的对侧对称部位亦出现所谓“镜像痛敏”。然而,迅猛发展、高潮迭起的疼痛调控研究仍缺乏对诸如镜像痛敏等复杂表症发生、发展和维持机制的系统理解。缝隙连接通道及半通道介导神经与胶质的胞间通讯互作,且具有蛋白亚型、细胞表达分布、磷酸化功能调控的多样性,尤其在疼痛等疾病调控的相关性机制是前沿热点。本论文工作基于蝎毒素多肽类特异性钠通道激活剂——Bm K I诱发镜像痛敏动物模型,采用行为药理学、实时荧光定量PCR、免疫组化和免疫印迹等方法探究外周和中枢缝隙连接蛋白亚型及其磷酸化动态调控与镜像痛敏发生、发展和维持的相关分子与细胞机制。主要结果如下:1.脊髓和背根神经节连接蛋白亚型的差异性表达分布采用实时荧光定量PCR方法发现,大鼠脊髓和背根神经节(DRG)中连接蛋白亚型表达分布差异较大。脊髓中主要表达Cx47、Cx45、Cx43、Cx36、Cx32、Cx30、Cx29和Cx26;DRG中主要表达Cx45、Cx43、Cx39、Cx37、Cx36、Cx32、Cx31.1、Cx31、Cx30、Cx29和Cx26。且脊髓和DRG中同一连接蛋白表达丰度存在差异。提示,缝隙连接蛋白的表达和功能调控具细胞表达差异性。2.Connexins以开放和表达量改变参与镜像痛敏的发生、发展和维持预先鞘内注射缝隙连接通道开放抑制剂CBX、GA或Cx43特异拟似肽Gap26均可剂量依赖性显著抑制大鼠单侧后足底皮下注射Bm K I诱发的自发痛、长时程双侧机械镜像痛敏。这些行为学证据表明,关闭或抑制缝隙连接通道缓减疼痛发生、发展和维持。进一步的定量检测发现,随疼痛时程的发展变化,脊髓双侧Cx36呈现“n”型,双侧Cx43呈“v”型,同侧Cx 32呈“n”,而对侧呈“v”型;DRG中三者均呈现先增后降趋势。因此,脊髓和DRG双侧各连接蛋白亚型表达呈现左右并不完全相同的“时间窗口”。无论脊髓或DRG,三种Connexins的表达变化趋势均可被缝隙连接通道抑制剂CBX不同程度地逆转。结果表明,缝隙连接蛋白通过开放与关闭的通道功能或表达量的变化参与镜像痛敏发生、发展和维持的调控,而Cx43作用尤为重要。3.Connexins亚型细胞定位分布与镜像痛敏发生、发展和维持在脊髓,Cx32在脊髓背角均匀分布,均与神经元标记蛋白Neu N、星形胶质细胞GFAP和小胶质细胞Iba1具不同程度共标。Cx36则主要分布在脊髓背角深层,与Neu N共定位在一起,少量与GFAP及Iba1共标。Cx43主要与GFAP共标,少量与Iba1共标,与Neu N无共标。随着疼痛发生发展,脊髓背角浅层Cx43与GFAP共标率呈逐渐上升趋势,而深层长时程保持高水平增加状态。在DRG中,Cx43、Cx36和Cx32阳性与NF200均有不同程度共定位;Cx32与IB4阳性神经元共标,与CGRP神经元几乎不共标;Cx43和Cx36阳性与IB4和CGRP均有不同程度共标;Cx43、Cx36和Cx32与卫星胶质细胞GFAP和小胶质细胞Iba1均有不同程度共标。预先给予缝隙连接通道抑制剂CBX显著降低脊髓和DRG connexin与标记蛋白共标率。结果表明,脊髓背角和DRG connexins表达分布的动态差异变化与镜像痛敏发生、发展和维持密切相关,缝隙连接蛋白亚型可作为疼痛相关细胞分型的新方式。4.Cx43磷酸化调控镜像疼痛的发生、发展和维持免疫印迹结果显示,疼痛刺激诱致脊髓和DRG中Cx43磷酸化水平显著变化。在脊髓,同侧p Cx43(Ser262)磷酸化位点呈现“v”型变化,p Cx43(Tyr265)磷酸化位点呈现“n”型变化,p Cx43(Ser279/282)和p Cx43(Ser368)磷酸化位点呈现递增趋势;对侧均呈现递增趋势。在DRG,p Cx43(Ser368)磷酸化同侧呈“n”型变化,而对侧逐渐降低。上述变化趋势均可被预先鞘内注射缝隙连接抑制剂CBX抑制,但各位点之间仍呈现较大差异。结果表明,脊髓和DRG中Cx43磷酸化调控镜像痛敏发生、发展和维持,且疼痛发生、发展和维持不同阶段参与的磷酸化调控位点显著不同。结论:脊髓和背根神经节connexins和磷酸化在机体损伤侧和对侧不对称的时空表达差异,调控镜像痛敏的发生、发展和维持。
[Abstract]:Pain is one of the most serious biomedical challenges that are currently seriously afflicting human health. The pain-inducing factors are diversified, such as body tissue injury, inflammation, emotion out-of-control, movement sense disorder, genetic mutation, tumor, and the like, Individualization restricts the effective choice of clinical prevention, diagnosis and treatment strategy. The so-called "image pain sensitivity" is also present at the opposite side of the body section in the lesion site, except for the secondary pain-sensitive area in the peripheral area of the lesion site, except for the persistent spontaneous pain in the body-damaged area, the primary pain-sensitive and the secondary pain-sensitive area in the surrounding area of the lesion. However, the rapid development of pain control in the high tide still lacks a systematic understanding of the occurrence, development and maintenance of complex surface disorders, such as mirror pain. The gap junction channel and the half-channel mediate the intercell communication of the nerve and the colloid, and has the diversity of protein subtype, cell expression distribution, and the regulation of the phosphorylation function, in particular, the correlation mechanism of the regulation of the diseases such as pain is the leading hot spot. in that work, a mirror pain-sensitive animal model is induced by the scorpion toxin polypeptide-specific sodium channel activator _ Bm K I, and the behavior pharmacology, real-time fluorescence quantitative PCR is adopted, Immunohistochemistry and immunoblotting were used to explore the relationship between the peripheral and central gap junction protein and its phosphorylation, and the related molecular and cellular mechanisms of the development and maintenance. The main results are as follows:1. The differential expression profile of the connexin subtypes in the spinal cord and the dorsal root ganglion was found by real-time fluorescence quantitative PCR, and the expression profile of the connexin in the spinal cord and the dorsal root ganglion (DRG) of the rat was significantly different. The main expression of Cx47, Cx45, Cx43, Cx36, Cx32, Cx30, Cx29 and Cx26 in the spinal cord; Cx45, Cx43, Cx39, Cx37, Cx36, Cx32, Cx31.1, Cx31, Cx30, Cx29, and Cx26 are mainly expressed in the DRG. There was a difference in the expression of the same connexin in the spinal cord and the DRG. 2. The expression of the gap junction protein and the difference of the expression of the functional regulator are shown.2. Connexins changes the occurrence, development and maintenance of the opening inhibitor CBX of the injection-gap junction channel in the in-situ injection in the presence of open and expression. The dose-dependence of GA or Cx43-specific quasi-peptidomimetic Gap26 significantly inhibited the spontaneous pain induced by Bm K I in the single-side sole of the rat, and the long-term bilateral mechanical image pain. These behavioral evidence suggests that the closure or inhibition of the gap junction channel is responsible for the occurrence, development and maintenance of pain. Further quantitative detection showed that the bilateral Cx36 in the spinal cord exhibited the "n" type with the development of the time history of the pain, the bilateral Cx43 was in the "v" type, the same side Cx 32 was in the "n", and the opposite side was of the "v" type, and the three of the DRGs showed a tendency to increase in the first step. Therefore, the expression of the bilateral connexin subtypes in the spinal cord and the DRG appears to be left and right and not exactly the same time window ". Regardless of the spinal cord or DRG, the change of expression of the three Connexins may be reversed to a different extent by the gap junction channel inhibitor CBX. The results showed that the change of the function or expression of the gap junction protein in the open and closed channel was involved in the control of the occurrence, development and maintenance of the image, and the role of the Cx43 was of particular importance.3. The localization and distribution of the Connexins subtype and the occurrence, development and maintenance of the image pain in the spinal cord. The distribution of Cx32 in the dorsal horn of the spinal cord is different from that of the neuronal marker protein Neu N, the astrocytes GFAP and the microglia Iba1. Cx36 is mainly distributed in the deep of the dorsal horn of the spinal cord and is co-located with the Neu N, and a small amount is co-labeled with the GFAP and Iba1. Cx43 is co-labeled with GFAP, and a small amount is co-labeled with Iba1 and is not co-labeled with Neu N. With the development of pain, the co-labeling rate of the shallow Cx43 and GFAP in the dorsal horn of the spinal cord gradually increased, while the deep long time-course remained high. In the DRG, Cx43, Cx36 and Cx32 were co-located with the NF200, and the Cx32 and IB4-positive neurons were co-labeled, and they were almost non-co-labeled with the CGRP neurons; Cx43 and Cx36 positive and IB4 and CGRP had different degrees of co-bid; and Cx43, The Cx36 and Cx32 were co-labeled with both the GFAP and the microglia Iba1 of the satellite. The pre-administration of the gap junction channel inhibitor CBX significantly reduced the co-labeling of the spinal cord and the DRG connexin with the marker protein. The results showed that the change of the expression profile of the dorsal horn of the spinal cord and the expression of the DRG connexins was closely related to the occurrence, development and maintenance of the image pain. The development and maintenance of immunoblotting revealed a significant change in the level of Cx43 phosphorylation in the spinal cord and DRG induced by pain stimulation. At the same ipsilateral p-Cx43 (Ser262) phosphorylation site in the spinal cord, the "v"-type changes were present, and the p-Cx43 (Tyr265) phosphorylation site exhibited a "n"-type change, and the p-Cx43 (Ser279/282) and the p-Cx43 (Ser368) phosphorylation sites showed an increasing trend; both sides exhibited an increasing trend. On the same side of DRG, p-Cx43 (Ser368), the ipsilateral was n-type, while the opposite side was gradually decreased. The above-mentioned change trend can be inhibited by the pre-pre-injection of the gap junction inhibitor CBX, but there is still a large difference between the points. The results showed that the phosphorylation and control of Cx43 in the spinal cord and DRG were sensitive to the occurrence, development and maintenance of pain, and the phosphorylation and control sites involved in the different stages of pain, development and maintenance were significantly different. Conclusion: The changes of the spatial and temporal expression of connexins and phosphorylation of the spinal cord and the dorsal root ganglion on the injured side and the opposite side of the body are different, and the occurrence, development and maintenance of the mirror pain are controlled.
【学位授予单位】：延安大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R402

【共引文献】