縫隙連接蛋白磷酸化介導(dǎo)鏡像痛敏的時(shí)空表達(dá)研究
發(fā)布時(shí)間：2019-05-30 07:43
【摘要】：疼痛是當(dāng)前嚴(yán)重困擾人類健康的生物醫(yī)學(xué)難題之一。疼痛誘發(fā)因素多樣化,如機(jī)體組織損傷、炎癥、情緒失控、運(yùn)動(dòng)感覺障礙、遺傳突變、腫瘤等均可改變痛覺感受、傳遞、整合的個(gè)體自我保護(hù)和內(nèi)在適應(yīng)的可塑性,所呈現(xiàn)的持續(xù)性、慢性化、個(gè)性化在多層面制約著臨床預(yù)防、診斷、治療策略的有效選擇。疼痛表癥多樣化,如除在軀體損傷部位表現(xiàn)持續(xù)自發(fā)痛、原發(fā)性痛敏和損傷部位周圍區(qū)域出現(xiàn)繼發(fā)性痛敏外,在損傷部位同體節(jié)的對(duì)側(cè)對(duì)稱部位亦出現(xiàn)所謂“鏡像痛敏”。然而,迅猛發(fā)展、高潮迭起的疼痛調(diào)控研究仍缺乏對(duì)諸如鏡像痛敏等復(fù)雜表癥發(fā)生、發(fā)展和維持機(jī)制的系統(tǒng)理解�？p隙連接通道及半通道介導(dǎo)神經(jīng)與膠質(zhì)的胞間通訊互作,且具有蛋白亞型、細(xì)胞表達(dá)分布、磷酸化功能調(diào)控的多樣性,尤其在疼痛等疾病調(diào)控的相關(guān)性機(jī)制是前沿?zé)狳c(diǎn)。本論文工作基于蝎毒素多肽類特異性鈉通道激活劑——Bm K I誘發(fā)鏡像痛敏動(dòng)物模型,采用行為藥理學(xué)、實(shí)時(shí)熒光定量PCR、免疫組化和免疫印跡等方法探究外周和中樞縫隙連接蛋白亞型及其磷酸化動(dòng)態(tài)調(diào)控與鏡像痛敏發(fā)生、發(fā)展和維持的相關(guān)分子與細(xì)胞機(jī)制。主要結(jié)果如下:1.脊髓和背根神經(jīng)節(jié)連接蛋白亞型的差異性表達(dá)分布采用實(shí)時(shí)熒光定量PCR方法發(fā)現(xiàn),大鼠脊髓和背根神經(jīng)節(jié)(DRG)中連接蛋白亞型表達(dá)分布差異較大。脊髓中主要表達(dá)Cx47、Cx45、Cx43、Cx36、Cx32、Cx30、Cx29和Cx26;DRG中主要表達(dá)Cx45、Cx43、Cx39、Cx37、Cx36、Cx32、Cx31.1、Cx31、Cx30、Cx29和Cx26。且脊髓和DRG中同一連接蛋白表達(dá)豐度存在差異。提示,縫隙連接蛋白的表達(dá)和功能調(diào)控具細(xì)胞表達(dá)差異性。2.Connexins以開放和表達(dá)量改變參與鏡像痛敏的發(fā)生、發(fā)展和維持預(yù)先鞘內(nèi)注射縫隙連接通道開放抑制劑CBX、GA或Cx43特異擬似肽Gap26均可劑量依賴性顯著抑制大鼠單側(cè)后足底皮下注射Bm K I誘發(fā)的自發(fā)痛、長時(shí)程雙側(cè)機(jī)械鏡像痛敏。這些行為學(xué)證據(jù)表明,關(guān)閉或抑制縫隙連接通道緩減疼痛發(fā)生、發(fā)展和維持。進(jìn)一步的定量檢測發(fā)現(xiàn),隨疼痛時(shí)程的發(fā)展變化,脊髓雙側(cè)Cx36呈現(xiàn)“n”型,雙側(cè)Cx43呈“v”型,同側(cè)Cx 32呈“n”,而對(duì)側(cè)呈“v”型;DRG中三者均呈現(xiàn)先增后降趨勢。因此,脊髓和DRG雙側(cè)各連接蛋白亞型表達(dá)呈現(xiàn)左右并不完全相同的“時(shí)間窗口”。無論脊髓或DRG,三種Connexins的表達(dá)變化趨勢均可被縫隙連接通道抑制劑CBX不同程度地逆轉(zhuǎn)。結(jié)果表明,縫隙連接蛋白通過開放與關(guān)閉的通道功能或表達(dá)量的變化參與鏡像痛敏發(fā)生、發(fā)展和維持的調(diào)控,而Cx43作用尤為重要。3.Connexins亞型細(xì)胞定位分布與鏡像痛敏發(fā)生、發(fā)展和維持在脊髓,Cx32在脊髓背角均勻分布,均與神經(jīng)元標(biāo)記蛋白Neu N、星形膠質(zhì)細(xì)胞GFAP和小膠質(zhì)細(xì)胞Iba1具不同程度共標(biāo)。Cx36則主要分布在脊髓背角深層,與Neu N共定位在一起,少量與GFAP及Iba1共標(biāo)。Cx43主要與GFAP共標(biāo),少量與Iba1共標(biāo),與Neu N無共標(biāo)。隨著疼痛發(fā)生發(fā)展,脊髓背角淺層Cx43與GFAP共標(biāo)率呈逐漸上升趨勢,而深層長時(shí)程保持高水平增加狀態(tài)。在DRG中,Cx43、Cx36和Cx32陽性與NF200均有不同程度共定位;Cx32與IB4陽性神經(jīng)元共標(biāo),與CGRP神經(jīng)元幾乎不共標(biāo);Cx43和Cx36陽性與IB4和CGRP均有不同程度共標(biāo);Cx43、Cx36和Cx32與衛(wèi)星膠質(zhì)細(xì)胞GFAP和小膠質(zhì)細(xì)胞Iba1均有不同程度共標(biāo)。預(yù)先給予縫隙連接通道抑制劑CBX顯著降低脊髓和DRG connexin與標(biāo)記蛋白共標(biāo)率。結(jié)果表明,脊髓背角和DRG connexins表達(dá)分布的動(dòng)態(tài)差異變化與鏡像痛敏發(fā)生、發(fā)展和維持密切相關(guān),縫隙連接蛋白亞型可作為疼痛相關(guān)細(xì)胞分型的新方式。4.Cx43磷酸化調(diào)控鏡像疼痛的發(fā)生、發(fā)展和維持免疫印跡結(jié)果顯示,疼痛刺激誘致脊髓和DRG中Cx43磷酸化水平顯著變化。在脊髓,同側(cè)p Cx43(Ser262)磷酸化位點(diǎn)呈現(xiàn)“v”型變化,p Cx43(Tyr265)磷酸化位點(diǎn)呈現(xiàn)“n”型變化,p Cx43(Ser279/282)和p Cx43(Ser368)磷酸化位點(diǎn)呈現(xiàn)遞增趨勢;對(duì)側(cè)均呈現(xiàn)遞增趨勢。在DRG,p Cx43(Ser368)磷酸化同側(cè)呈“n”型變化,而對(duì)側(cè)逐漸降低。上述變化趨勢均可被預(yù)先鞘內(nèi)注射縫隙連接抑制劑CBX抑制,但各位點(diǎn)之間仍呈現(xiàn)較大差異。結(jié)果表明,脊髓和DRG中Cx43磷酸化調(diào)控鏡像痛敏發(fā)生、發(fā)展和維持,且疼痛發(fā)生、發(fā)展和維持不同階段參與的磷酸化調(diào)控位點(diǎn)顯著不同。結(jié)論:脊髓和背根神經(jīng)節(jié)connexins和磷酸化在機(jī)體損傷側(cè)和對(duì)側(cè)不對(duì)稱的時(shí)空表達(dá)差異,調(diào)控鏡像痛敏的發(fā)生、發(fā)展和維持。
[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.
【學(xué)位授予單位】：延安大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R402
本文編號(hào)：2488650
[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.
【學(xué)位授予單位】：延安大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R402
【共引文獻(xiàn)】
相關(guān)期刊論文 前2條
1 呂秋蘭;黃安;劉雙梅;梁尚棟;;促炎因子和抗炎因子在神經(jīng)病理痛中的作用[J];神經(jīng)解剖學(xué)雜志;2015年04期
2 劉霞;韓玉瑩;姜戈平;吳楠;杜珂;白占濤;;陜北東亞鉗蝎體重和性別與產(chǎn)毒量的相關(guān)性[J];中國農(nóng)學(xué)通報(bào);2015年35期
相關(guān)博士學(xué)位論文 前1條
1 李卉;與胚胎心肌共培養(yǎng)通過Cx43影響ESC源細(xì)胞間信號(hào)傳導(dǎo)的研究[D];華中科技大學(xué);2013年
相關(guān)碩士學(xué)位論文 前1條
1 王國芹;miRNA-1在離體大鼠心臟缺血后處理中對(duì)縫隙連接蛋白43的作用[D];天津醫(yī)科大學(xué);2014年
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