【摘要】：尋求新的保護因子或新的治療策略對心肌細胞(cardiac muscle cell,CM)的保護作用及其機制對于心臟疾病的治療和改善心功能具有重要的意義。雖然對心肌細胞肥大和心室重構的研究取得了許多重大的突破性進展,但這眾多的研究成果大多是基于心肌細胞或心室結構本身而開展的,而對心臟神經調控機制對心肌細胞結構和功能的影響尚有待于從更廣和更深的層次開展研究。神經調節(jié)蛋白-1β(neuregulin-1β,NRG-1β)可通過激活其酪氨酸激酶受體ErbB2和ErbB4而對神經元發(fā)揮多種調控作用,NRG-1β很可能具備對心臟神經發(fā)揮特有的調控作用的潛能,到目前為止,還沒有發(fā)現NRG-1β通過調控心臟神經而對心肌發(fā)揮調控作用的報道。有趣的是,NRG-1β還可對心肌細胞直接發(fā)揮重要的調控功能。鑒于心臟神經和心肌細胞均為NRG-1β直接調控的靶組織,這將使NRG-1β對心功能的調節(jié)作用機制更為復雜。本課題將基于以上研究背景開展如下一系列實驗研究:首先,研究NRG-1β對培養(yǎng)的新生大鼠心肌細胞的ErbB2和ErbB4受體及其下游的磷酸肌醇-3-激酶(phosphoinositide-3 kinase,PI3K)/蛋白激酶B(protein kinase B,PKB,Akt)和細胞外信號調節(jié)激酶 1/2(extracellular signal-regulated kinase 1/2,ERK1/2)的表達及其激活的影響作用;其次,研究NRG-1β及其相關的信號通路對心肌細胞重要的形態(tài)指標和功能指標的影響作用;最終,利用具有選擇性交感神經支配的心肌細胞,研究NRG-1β及其相關的信號通路對神經和心肌的雙重調控作用。通過以上系列研究,以便進一步闡明NRG-1β直接調節(jié)心肌細胞或通過調節(jié)其神經支配而調節(jié)心肌細胞的作用及其機制。從而提出不僅通過NRG-1β及其受體信號的活性而直接增強心肌細胞再生能力或功能,而且還可通過NRG-1β及其受體信號系統介導的心臟神經調控作用而進一步增強其對心肌細胞結構和功能的調控作用的新的治療策略。第一部分神經調節(jié)蛋白-1β在心肌細胞激活其受體及其下游信號通路的實驗研究NRG-1β對心肌細胞ErbB2和ErbB4受體及其下游的PI3K/Akt和ERK1/2的激活是其發(fā)揮生物學效應的重要途徑。為了研究NRG-1β對心肌細胞ErbB2和ErbB4受體及其下游的信號分子Akt和ERK1/2表達及其激活的影響作用,本課題利用培養(yǎng)的新生大鼠心肌細胞觀察了 NRG-1β快速孵育和持續(xù)孵育對這些分子表達和激活的影響作用程度。結果顯示,NRG-1β急性孵育不會對心肌細胞表面積產生影響,而NRG-1β持續(xù)孵育,則可增加心肌細胞的表面積,有利于心肌細胞的生長發(fā)育;NRG-1β急性孵育雖然不能促進其受體ErbB2和ErbB4及其下游信號分子Akt和ERK1/2的表達,但可顯著提高其磷酸化水平,表明NRG-1β急性孵育可激活其受體及其相關的下游細胞信號轉導通路;NRG-1β持續(xù)孵育不僅能促進其受體ErbB2和ErbB4及其下游信號分子Akt和ERK1/2的表達,而且還使其磷酸化水平顯著上升,從而改善心肌細胞的生長狀態(tài);NRG-1β的促心肌細胞生長效應可被PI3K抑制劑LY294002或ERK1/2抑制劑PD98059所阻斷,而不能阻斷NRG-1β的誘導其受體ErbB2和ErbB4表達和磷酸化的效應。以上結果表明,NRG-1β急性孵育和NRG-1β持續(xù)孵育對心肌細胞具有不同的生物學效應。NRG-1β急性孵育和NRG-1β持續(xù)孵育對其受體ErbB2和ErbB4及其下游信號分子Akt和ERK1/2表達和激活的不同生物學效應揭示了將來利用NRG-1β治療相應的心臟疾患所可能采取的不同治療策略。第二部分神經調節(jié)蛋白-1β改善心肌細胞結構和功能指標的作用參與粗肌絲構成的肌球蛋白重鏈(myosin heavy chain,MHC)、能夠激活調節(jié)性肌球蛋白輕鏈(myosin regulatory light chain,rMLC)的心肌特異的肌球蛋白輕鏈激酶(cardiac-specific myosin light-chain kinase,cMLCK)是調節(jié)肌節(jié)組裝的重要因子,心肌細胞的主要門控通道L-型鈣通道(L-type calcium channel,LTCC,Cav1.2)是Ca2+進入心肌細胞的主要通道,肌質網鈣離子三磷酸腺苷酶或肌質網鈣離子泵(sarco(endo)plasmic reticulum Ca2+ ATPases or sarco(endo)plasmic reticulum-Ca2+ pump,SERCA2a)通過將胞質內 Ca2+的轉運至肌質網(sarcoplasmic reticulum,SR)內,從而調節(jié)收縮和舒張循環(huán)的協調性。以上指標是影響心肌細胞結構和功能的關鍵因素,為了檢測NRG-1β對這些指標的影響作用及其相關機制,本課題利用培養(yǎng)的新生大鼠心肌細胞,研究了 NRG-1β及其相關的信號通路對心肌細胞的這些重要形態(tài)指標和功能指標的影響作用,并深入分析了 NRG-1β改善心肌細胞內在組分的潛能。結果顯示,NRG-1β可分別促進培養(yǎng)的心肌細胞代表性結構指標MHC、對心肌細胞內的關鍵激酶cMLCK、代表性功能指標L-型鈣通道和SERCA2a的表達,P13K抑制劑LY294002或ERK1/2抑制劑PD98059可抑制NRG-1β引起的促心肌細胞MHC、cMLCK、L-型鈣通道和SERCA2a表達上調的作用。這些結果表明,NRG-1β可有效促進對心肌細胞收縮的基本結構分子和功能分子的表達,從而改善心肌細胞的內在組分構成,并由此而提高心肌細胞功能的潛在能力,這是NRG-1β將來有可能成為改善心功能的有效治療分子的重要實驗依據。第三部分神經調節(jié)蛋白-1β增強心肌細胞去甲腺素能命運的潛能心肌細胞的去甲腎上腺素能命運是保證和維持心肌細胞功能的前提,在此基礎上,心肌細胞的功能調控還有賴于正常交感神經的支配。心肌細胞β-腎上腺素能受體(β-adrenergic receptor,β-AR)的表達是心肌細胞的去甲腎上腺素能命運的重要體現,β-AR的激活是交感神經支配信號傳遞的必由途經。本課題將根據NRG-1β及其相關的信號通路對神經和心肌的雙重調控作用,建立器官型的頸上神經節(jié)(superior cervical ganglion,SCG)組織塊和分散心肌細胞的聯合培養(yǎng)體系,研究有或沒有交感神經支配的培養(yǎng)的心肌細胞,再附加NRG-1β孵育的條件下,心肌細胞去甲腎上腺素能命運潛能的變化。結果顯示,NRG-1β孵育可使心肌細胞β1-AR和β2-AR的表達水平上調,而選擇性交感神經支配使心肌細胞β1-AR和β2-AR表達水平上調的效應更加明顯,NRG-1β孵育可進一步增強交感神經支配信號對心肌細胞β1-AR和β2-AR表達水平上調的促進效應;在心肌細胞與交感神經元聯合培養(yǎng)的過程中,應用NRG-1β孵育能夠通過增加交感神經元突起的數目增強交感神經信號對心肌細胞支配的效能;PI3K抑制劑LY294002或ERK1/2抑制劑PD98059可抑制NRG-1β引起的促心肌細胞β1-AR和β2-AR表達上調作用和促神經元突起生長的效應。這些結果表明,NRG-1β具有進一步增強交感神經支配信號的促心肌細胞去甲腎上腺素能命運的效應,這一效應是通過PI3K/Akt和ERK1/2細胞信號轉導通路介導的。本課題的研究結果對于以NRG-1β及其受體信號系統為主導的具有針對性的治療策略的制定提供了具有指導意義的理論基礎和具有參考價值的實驗依據。
[Abstract]:The protective effect of new protective factors or new therapeutic strategies on cardiac muscle cell (CM) and its mechanism are of great significance for the treatment of heart disease and the improvement of cardiac function. Although many major breakthroughs have been made in the study of cardiac myocyte hypertrophy and ventricular remodeling, many research achievements have been made. Many of them are based on cardiac myocytes or ventricular structure itself, and the effect of the cardiac nerve regulation mechanism on the structure and function of cardiac myocytes remains to be studied from a broader and deeper level. The neuroregulatory protein -1 beta (neuregulin-1 beta, NRG-1 beta) can play a role in neurons by activating its tyrosine kinase receptor ErbB2 and ErbB4. A variety of regulatory functions, NRG-1 beta is likely to have the potential to play a unique regulatory role in the heart nerve. Up to now, there has been no discovery of the regulation of the NRG-1 beta by regulating the heart nerve. It is interesting that NRG-1 beta can also play an important regulatory function on cardiac myocytes. All the cells are the target tissues directly regulated by NRG-1 beta, which will make the regulatory mechanism of NRG-1 beta more complex. This topic will be based on a series of experimental studies based on the above research background. First, the ErbB2 and ErbB4 receptors of NRG-1 beta cultured neonatal rat cardiomyocytes and their downstream inositol -3- kinase (phosphoi) are studied. Nositide-3 kinase, PI3K) / protein kinase B (protein kinase B, PKB, Akt) and the expression of extracellular signal regulated kinase 1/2 (extracellular signal-regulated) and its effect on activation; secondly, the effect of beta and its related signal pathways on the important morphological and functional indexes of cardiac myocytes; Finally, using the cardiomyocytes with selective sympathetic innervation, the dual regulatory role of NRG-1 beta and its related signaling pathway on the nerve and myocardium is studied. Through the above series of studies, the role and mechanism of NRG-1 beta regulating cardiac myocytes directly or regulating the innervation of the cardiac myocytes by regulating their innervation are further elucidated. A new therapeutic strategy is proposed to enhance the ability or function of cardiac myocyte regeneration not only through the activity of NRG-1 beta and its receptor signal, but also through the regulatory role of the cardiac nerve in the NRG-1 beta and its receptor signaling system. Experimental study of white -1 beta activation of its receptor and its downstream signaling pathway in cardiac myocytes: NRG-1 beta activation of ErbB2 and ErbB4 receptors and its downstream PI3K/Akt and ERK1/2 is an important pathway for its biological effects. In order to study the expression of NRG-1 beta on ErbB2 and ErbB4 receptors and the downstream signaling molecules Akt and ERK1/2 expression of NRG-1 beta The effect of NRG-1 beta incubation and continuous incubation on the expression and activation of these molecules was observed by the cultured neonatal rat cardiomyocytes. The results showed that the acute incubation of NRG-1 beta did not affect the surface area of cardiac myocytes, while NRG-1 beta incubation could increase the myocardial cells. The surface area is beneficial to the growth and development of cardiac myocytes; the acute incubation of NRG-1 beta can not promote the expression of its receptor ErbB2 and ErbB4 and their downstream signal molecules Akt and ERK1/2, but can significantly increase their phosphorylation level. It indicates that the acute incubation of NRG-1 beta can activate its receptor and its related downstream cell signal transduction pathway; NRG-1 beta incubates continuously. It can not only promote the expression of its receptor ErbB2 and ErbB4 and their downstream signal molecules Akt and ERK1/2, but also increase their phosphorylation level significantly, thus improving the growth state of cardiac myocytes; the growth effect of NRG-1 beta myocytes can be blocked by PI3K inhibitor LY294002 or ERK1/2 inhibitor PD98059, but can not block the induction of NRG-1 beta. The effects of receptor ErbB2 and ErbB4 expression and phosphorylation show that NRG-1 beta acute incubation and NRG-1 beta incubation have different biological effects on cardiac myocytes with different biological effects of.NRG-1 beta incubation and NRG-1 beta incubation on the different biological effects of ErbB2 and ErbB4 and its downstream signal molecules Akt and ERK1 /2 expression and activation. The different treatment strategies may be taken in the future using NRG-1 beta for the treatment of corresponding cardiac disorders. The role of the second part of the neuromodulation protein -1 beta to improve the structure and function of cardiac myocytes is involved in the myosin heavy chain (myosin heavy chain, MHC), which can stimulate the light chain (myosin regulatory lig) of the myosin (myosin regulatory lig). The myocardial specific myosin light chain kinase (cardiac-specific myosin light-chain kinase, cMLCK) of HT chain, rMLC is an important factor regulating the assembly of myosine, the main gated channel of cardiac myocytes is the L- type calcium channel (L-type calcium channel) is the main channel to enter the cardiac myocytes, and the calcium ion three phosphate gland of the sarcoplasmic reticulum Sarco (Endo) plasmic reticulum Ca2+ ATPases or sarco (Endo) plasmic reticulum-Ca2+ pump, which regulates the coordination of contractile and diastolic circulation by transferring the cytoplasm to the sarcoplasmic reticulum. The above index is the key to the structure and function of the cardiac myocytes. Key factors, in order to detect the effect of NRG-1 beta on these indexes and their related mechanisms, we used the cultured neonatal rat cardiomyocytes to study the effects of NRG-1 beta and related signal pathways on these important morphological and functional indexes of cardiac myocytes, and the improvement of NRG-1 beta to improve the internal group of cardiac myocytes. The results showed that NRG-1 beta could promote the representative structural index MHC of cultured cardiac myocytes, the key kinase cMLCK in the cardiac myocytes, the expression of L- type calcium channel and SERCA2a, the P13K inhibitor LY294002 or ERK1/2 inhibitor PD98059 could inhibit the MHC, cMLCK, and calcium channel induced by NRG-1 beta. These results suggest that NRG-1 beta can effectively promote the expression of the basic structural molecules and functional molecules of cardiac myocyte contraction, thus improving the intrinsic composition of cardiac myocytes and thereby enhancing the potential ability of cardiac myocyte function, which is an effective treatment for NRG-1 beta in the future to improve cardiac function. The third part of the neuromodulation protein -1 beta enhances the fate of noradrenalin in cardiac myocytes, the fate of noradrenalin in cardiac myocytes is the prerequisite to guarantee and maintain the function of cardiac myocytes. On this basis, the function regulation of cardiac myocytes depends on the control of normal sympathetic nerve. The expression of adrenergic receptor (beta -adrenergic receptor, beta -AR) is an important manifestation of the fate of noradrenalin in cardiac myocytes. The activation of beta -AR is a must by the transmission of sympathetic innervation signal. This subject will establish an organotypic neck based on the dual regulatory effect of NRG-1 beta and its related signal pathways on the nerve and myocardium. A joint culture system of the superior cervical ganglion (SCG) tissue and scattered myocardial cells to study the cultured cardiac myocytes with or without sympathetic innervation, and then add NRG-1 beta incubation to change the fate potential of norepinephrine in the cardiomyocytes. The results show that NRG-1 beta incubation can make myocardial cells beta 1-AR. And the expression level of beta 2-AR was up-regulated, and the effect of selective sympathetic innervation on the up regulation of beta 1-AR and beta 2-AR expression was more obvious. NRG-1 beta incubation could further enhance the promotion effect of sympathetic innervation signal on the up regulation of the expression level of beta and beta 2-AR in cardiac myocytes, and Co culture of cardiac myocytes and sympathetic neurons. In the process, NRG-1 beta incubation can enhance the efficacy of sympathetic nerve signal to cardiac myocytes by increasing the number of sympathetic neurites. The PI3K inhibitor LY294002 or ERK1/2 inhibitor PD98059 inhibits the effect of NRG-1 beta induced up-regulated expression of beta 1-AR and beta 2-AR and promoting the growth of neuronal protrusion. The results suggest that NRG-1 beta has the effect of further enhancement of sympathetic innervation signal to the fate of noradrenaline, which is mediated by the PI3K/Akt and ERK1/2 cell signal transduction pathways. The results of this study are targeted at the targeted treatment strategy based on the NRG-1 beta and its receptor signaling system. The formulation provides a theoretical basis and a theoretical basis for reference.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R54

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