AMPK对BMP信号通路以及异位骨化的抑制作用
发布时间：2018-07-31 12:01
【摘要】：第一部分引言异位骨化(Heterotopic ossification,HO)是指在软组织中病理形成异常的骨组织。一般发生在两个单独的患者群体中：具有严重创伤包括大面积烧伤、肌肉骨骼损伤、矫形手术甚至脊髓损伤的患者;和进行性肌肉骨化症(Fibrodysplasia ossificans progressiva,FOP)等遗传性疾病患者中。FOP由I型骨形态发生蛋白(Bone morphogenetic proteins,BMP)受体ALK2(Activin receptor-like kinase 2,活化素1型受体)中的永久性活化突变引起。这些病理性异位骨形成的临床后遗症,包括非愈合性创伤、慢性疼痛和关节不动性。对于FOP,患者由于胸廓顺应性的损失导致呼吸困难而死亡。BMPs以及其转导的信号通路在异位骨化形成中具有重要作用。BMPs是异位骨化的主要诱导者;软组织创伤后刺激BMPs分泌和激活BMP信号通路,促进软组织形成HO。此外,大约95%的FOP患者发生BMPI型受体(ALK2)突变(R206H),引起BMP信号通路持续性激活,表现为渐近性异位骨化的特性。异位骨化是一个非常普遍和严重的健康问题,目前临床上使用的方法一般是减少疼痛,修复以及预防。大多依赖于抗炎药物的使用,但其效果充其量是部分有效。而且大量的副作用限制了NSAIDs的使用。一个有效的、理想的策略是特异性的针对BMP信号通路成分,抑制异位骨化,另一个有创新性的策略是鉴定现有临床上治疗其他药物,重新发现其治疗效果来满足临床需求。众多研究表明AMP-激活蛋白激酶(AMP-activated protein kinase,AMPK)在骨代谢中发挥重要的生理作用,AMPK激活剂二甲双胍是2型糖尿病一线药物,已经在临床上广泛使用超过半个世纪。我们课题组前期已经发现二甲双胍能够有效性的抑制TGFβ家族信号转导和EMT。继续延伸此研究,探索AMPK对BMP信号通路以及异位骨化的作用。因此,我们首先研究了AMPK对FOP成纤维细胞中BMP信号通路的抑制作用以及分子机制;然后将FOP成纤维细胞诱导成多能干细胞(iPS cells),进行成骨分化诱导,进一步检测了AMPK对成骨分化的抑制作用;为了探索不同细胞的反应性,继续检测AMPK对MC3T3-E1细胞BMP信号通路的影响和作用机制,研究不同AMPK激活剂(单独使用或者联合使用)对成骨分化的影响,确定剂量效应和协同效应;最后建立小鼠异位骨化模型以及检测AMPK激活剂对体内异位骨化的抑制作用。我们的研究结果明确AMPK对BMP信号通路以及成骨分化和异位骨化的抑制作用,也为现有临床使用的AMPK激活剂如二甲双胍、阿司匹林用于预防、治疗FOP疾病和异位骨化提供科学依据。第二部分AMPK对FOP成纤维细胞中BMP信号通路的抑制作用目的：检测AMPK激活是否抑制BMP信号通路转导以及探索AMPK可能的作用机制。方法：DNA测序检测FOP成纤维细胞是否发生ALK2 R206H突变;不同AMPK激活剂处理FOP细胞,Western blot检测AMPK对BMP信号通路成分表达的影响;构建LKB1、AMPKa1a2稳定敲除MEF细胞株,观察不同细胞株对BMP和二甲双胍的反应性;感染持续性激活AMPK腺病毒激活AMPK和显示负性AMPK腺病毒失活AMPK,确定二甲双胍对BMP的信号通路作用是通过AMPK介导的;构建荧光素酶互补结合实验系统,观察AMPK对Smad6与Smurf1、ALK2与Smad1相互作用的影响;转染Smad6 si RNA和Smurf1 si RNA以及用蛋白酶体抑制剂MG132处理细胞,探索AMPK对BMP信号通路抑制作用的分子机制。结果：DNA测序确认FOP成纤维细胞发生ALK2 R206H突变;不同AMPK激活剂激活AMPK并且抑制Smad1/5的磷酸化;感染持续性激活AMPK突变体抑制BMP信号转导,而感染AMPK显性负性突变体后,取消了二甲双胍对Smad1/5磷酸化的抑制作用;稳定敲除LKB1和AMPKa1a2表达后,二甲双胍不能抑制BMP6诱导的Smad1/5的磷酸化。AMPK激活抑制ALK2的表达但上调Smad6和Smurf1的表达;AMPK激活后促进Smad6和Smurf1的结合,但阻遏ALK2和Smad1的相互作用;Smad6和Smurf1基因沉默后,消除了AMPK对BMP信号通路的抑制作用。此外,结果还显示蛋白酶体抑制MG132能够消除AMPK促进ALK2降解的作用。结论：AMPK激活剂或者使用持续性激活AMPK突变体激活AMPK抑制Smad1/5的磷酸化;二甲双胍对Smad1/5的磷酸化抑制是通过AMPK介导的;AMPK激活抑制FOP成纤维细胞中BMP信号通路转导,AMPK激活后上调Smad6和Smurf1表达、增强两个分子之间的相互作用,随后引起ALK2降解增加,从而抑制BMP信号转导。第三部分AMPK抑制来源于FOP成纤维细胞的iPS的成骨分化目的：研究AMPK激活对来源于FOP成纤维细胞的iPS的成骨分化影响。方法：建立、鉴定来源于FOP成纤维细胞的诱导多能干细胞(FOP-iPS);诱导iPS细胞向成骨细胞分化,通过茜素红染色和Western blot检测成骨分化标记物表达来观察ALK2突变(FOP iPS)和野生型ALK2(Control iPS)的iPS诱导成骨细胞分化能力的区别;采用茜素红染色检测AMPK激活剂(二甲双胍、AICAR)对iPS细胞矿化的影响。结果：成功构建来源于FOP成纤维细胞的iPS细胞(FOP iPS);与对照iPS相比较,FOP iPS茜素红染色程度更强,成骨分化标记物Run X2、Osx和OPN表达水平更高;AMPK激活剂二甲双胍、AICAR抑制iPS诱导的细胞矿化。结论：携带ALK2突变的FOP iPS细胞诱导成骨细胞分化的能力更强;AMPK抑制来源于FOP成纤维细胞的iPS的成骨分化。第四部分AMPK对小鼠前成骨细胞MC3T3-E1细胞中BMP信号通路的抑制作用目的：探讨AMPK对MC3T3-E1细胞中BMP信号通路的影响以及探索可能的分子作用机制。方法：AMPK激活剂二甲双胍处理MC3T3-E1细胞不同时间或者不同浓度,Western blot检测AMPK对BMP信号通路成分表达的影响,观察AMPK对Smad6、Smurf1和ALK2等表达的改变;感染持续性激活AMPK腺病毒激活AMPK,观察AMPK对BMP信号通路的直接效应;转染Smad6 si RNA,沉默Smad6表达,探索AMPK对BMP信号通路抑制作用的分子机制。结果：二甲双胍和持续性激活AMPK突变体激活AMPK抑制BMP6诱导的Smad1/5的磷酸化;AMPK激活后上调Smad6表达但不改变Smurf1和ALK2的表达;Smad6基因沉默后,二甲双胍不能抑制BMP6诱导的Smad1/5的磷酸化,消除了AMPK对BMP信号通路的抑制作用。结论：AMPK激活抑制MC3T3-E1细胞中BMP信号通路转导,AMPK激活后通过上调Smad6表达,抑制BMP信号转导,Smad6是AMPK主要靶目标。第五部分AMPK抑制MC3T3-E1细胞成骨分化目的：探讨AMPK对MC3T3-E1细胞成骨分化的影响。方法：MC3T3-E1细胞培养在诱导成骨分化培养基中诱导细胞向成骨细胞分化,分别采用Western bolt和q PCR检测不同时间段(0、1、3、7、14、21天)AMPK活性和成骨细胞分化标记物OPN、Osx和Runx2的改变;观察AMPK活性和成骨分化之间的关系;感染持续性激活AMPK腺病毒激活AMPK和显性负性AMPK腺病毒失活AMPK,观察AMPK对碱性磷酸酶活性的影响;碱性磷酸酶染色检测不同AMPK激活剂(单独使用或者联合使用)对成骨分化早期阶段的影响;茜素红染色检测不同AMPK激活剂对成骨分化晚期阶段-细胞矿化的影响。结果：AMPK活性随着成骨细胞分化程度越高其活性逐渐降低,而成骨细胞分化标记物OPN和Osx在第14天呈现最高表达,Runx2在成骨分化期间表达未发生明显改变;AMPK激活剂包括二甲双胍、阿司匹林、姜黄素和布洛芬等抑制碱性磷酸酶活性,并呈现浓度依赖性;感染AMPK持续性活性突变体也显示同样效应,然而感染AMPK显性负性突变体失活AMPK活性和功能后,取消二甲双胍对碱性磷酸酶活性的抑制作用;二甲双胍和布洛芬联用对碱性磷酸酶活性抑制具有超叠加效应,而二甲双胍和姜黄素联用显示叠加效应。二甲双胍和阿司匹林抑制MC3T3-E1细胞矿化,并呈现浓度依赖性。结论：AMPK在成骨细胞分化过程中发挥重要作用,成骨分化程度越高,AMPK活性越低;AMPK激活抑制MC3T3-E1成骨细胞分化,包括早期阶段和晚期阶段。二甲双胍和布洛芬联合应用具有超叠加效应可能通过不同机制抑制碱性磷酸酶活性。二甲双胍和姜黄素联用具有叠加效应可能通过相似机制抑制碱性磷酸酶活性。第六部分AMPK对小鼠体内异位骨化形成的影响目的：初步探讨AMPK对小鼠体内异位骨化形成的影响。方法：建立创伤-烧伤异位骨化小鼠模型,同时采用二甲双胍干预(饮用水含有0.5mg/ml的二甲双胍,N=5),连续8周后,采用X-RAY扫描检测异位骨的形成;取异位骨化组织,固定,脱钙,进行HE染色观察组织学改变,阿尔新蓝(Alcian Blue)染色检测二甲双胍对软骨内形成改变;q PCR检测成骨细胞分化标记物(包括Osc、BSP、Run X2)以及Smad6、Smurf1的m RNA表达的改变。结果：通过创伤-烧伤方法成功建立小鼠异位骨化模型,术后8周,进行X-RAY检查可见明显的异位骨化形成;采用二甲双胍干预后,与对照组小鼠比较,小鼠体内异位骨化形成减少,HE检测未见软骨细胞、成骨细胞样和骨陷窝结构,大部分为腱性纤维、结缔组织,阿尔新蓝染色可见少量染色;成骨细胞标记物BSP、Osc和Runx2的m RNA表达明显减少,而Smad6和Smurf1的表达水平上升。结论：跟腱切断-烧伤法能够有效诱导小鼠异位骨化形成,二甲双胍可以阻止小鼠体内异位骨化的形成。
[Abstract]:In the first part, Heterotopic ossification (HO) refers to the pathological bone tissue in the soft tissue. It usually occurs in two individual patient groups: Patients with severe trauma including large area burns, musculoskeletal injuries, orthopedics and even spinal injuries; and progressive muscle ossification (Fibrodysplasi A ossificans progressiva, FOP) in patients with hereditary diseases,.FOP is caused by a permanent activation mutation of the I type bone morphogenetic protein (Bone morphogenetic proteins, BMP) receptor ALK2 (Activin = 2, activin 1 receptor). The clinical sequelae of these pathological ectopic bone formation, including non healing trauma, chronic Pain and joint immobility. For FOP, the patient died of respiratory distress due to loss of thoracic compliance resulting in respiratory distress and.BMPs and its transduction pathway plays an important role in ectopic ossification..BMPs is the main inducer of ectopic ossification; soft tissue stimulates BMPs secretion and activates BMP signaling pathway to promote the formation of HO. in soft tissue. In addition, about 95% of FOP patients have BMPI receptor (ALK2) mutation (R206H), which causes the persistent activation of the BMP signaling pathway, showing the characteristics of asymptotically heterotopic ossification. Heterotopic ossification is a very common and serious health problem. The current clinical use is generally to reduce pain, repair and prevent it. Most of them depend on the anti inflammatory drugs. The effect is partly effective at best, and a large number of side effects restrict the use of NSAIDs. An effective, ideal strategy is specific BMP signaling pathway components, inhibits heterotopic ossification, and the other innovative strategy is to identify the existing clinical treatment of other drugs and rediscover the effect of treatment to be full. Many studies have shown that AMP- activated protein kinase (AMP-activated protein kinase, AMPK) plays an important physiological role in bone metabolism. Metformin, a AMPK activator, is a first-line drug of type 2 diabetes. It has been widely used in the clinic for more than half a century. We have found that metformin is effective in the early period of our group. Inhibition of TGF beta family signal transduction and EMT. continue to extend this study to explore the effect of AMPK on BMP signaling pathways and heterotopic ossification. Therefore, we first studied the inhibition and molecular mechanism of AMPK on the BMP signaling pathway in FOP fibroblasts, and then induced FOP fibroblasts into pluripotent stem cells (iPS cells) for osteogenesis. Differentiation induction, further detects the inhibitory effect of AMPK on osteogenic differentiation; in order to explore the reactivity of different cells, continue to detect the effect and mechanism of AMPK on the MC3T3-E1 cell BMP signaling pathway, study the effect of different AMPK activators (alone or combined use) on osteogenic differentiation, determine the dose effect and synergistic effect; finally, determine the dose effect and synergistic effect. To establish a model of heterotopic ossification in mice and to detect the inhibitory effect of AMPK activator on heterotopic ossification in vivo. Our results confirm the inhibitory effect of AMPK on BMP signaling pathway and osteogenic differentiation and ectopic ossification, and also for the current clinical use of AMPK activators such as metformin, aspirin for the prevention and treatment of FOP disease and ectopic bone. To provide scientific basis. Second the inhibitory effect of AMPK on the BMP signaling pathway in FOP fibroblasts: to detect whether AMPK activation inhibits BMP signaling pathway transduction and explore the possible mechanism of AMPK. Method: DNA sequencing to detect the occurrence of ALK2 R206H mutation in FOP fibroblasts and the treatment of FOP cells by different AMPK activators. RN blot detected the effect of AMPK on the expression of BMP signaling pathway components; LKB1, AMPKa1a2 stably knocked out MEF cell lines, and observed the response of different cell lines to BMP and Metformin; infection continued to activate AMPK adenovirus activation AMPK and negative AMPK adenovirus inactivation AMPK. To explore the effect of AMPK on the interaction between Smad6 and Smurf1, ALK2 and Smad1, and the transfection of Smad6 Si RNA and Smurf1 Si RNA as well as proteasome inhibitor MG132 cells. ALK2 R206H mutation was generated; different AMPK activators activated AMPK and inhibited the phosphorylation of Smad1/5; infection sustained activation AMPK mutants inhibited BMP signal transduction, and after infection of AMPK dominant negative mutants, the inhibition of metformin on Smad1/5 phosphorylation was cancelled. After stable knockout LKB1 and AMPKa1a2 expression, metformin could not inhibit BMP6 lure. The activation of phosphorylated.AMPK in Smad1/5 inhibited the expression of ALK2 but up regulation of the expression of Smad6 and Smurf1; AMPK activation promoted the binding of Smad6 and Smurf1, but inhibited the interaction of ALK2 and Smad1, and the inhibition effect on the signaling pathway was eliminated after the silence of Smad6 and Smurf1 genes. In addition to the effect of AMPK on promoting the degradation of ALK2. Conclusion: AMPK activator or the use of persistent activated AMPK mutants to activate AMPK to inhibit the phosphorylation of Smad1/5; the inhibition of phosphorylation of Smad1/5 by metformin is mediated by AMPK; AMPK activation inhibits BMP signal transduction in FOP fibroblasts, and up regulation and expression after activation of AMPK activation, enhancement The interaction between the two molecules then causes an increase in ALK2 degradation, which inhibits BMP signal transduction. The third part AMPK inhibits the osteogenic differentiation of iPS derived from FOP fibroblasts: the study of the effect of AMPK activation on the osteogenic differentiation of iPS derived from FOP fibroblasts. Lead pluripotent stem cells (FOP-iPS); induce iPS cells to differentiate into osteoblasts. The differentiation ability of ALK2 mutation (FOP iPS) and iPS induced ALK2 (Control iPS) induced osteoblast differentiation was observed by alizarin red staining and Western blot detection of osteoblast differentiation markers. The AMPK activator was detected by alizarin red staining. The effect on the mineralization of iPS cells. Results: a successful construction of iPS cells (FOP iPS) derived from FOP fibroblasts. Compared with the control iPS, the FOP iPS alizarin red staining was stronger, the osteogenic differentiation marker Run X2, Osx and OPN expressed higher levels. The ability of FOP iPS cells to induce osteoblast differentiation is stronger; AMPK inhibits the osteogenic differentiation of iPS derived from FOP fibroblasts. Fourth the inhibitory effect of part AMPK on BMP signaling pathway in the MC3T3-E1 cells of mouse anterior osteoblasts: To explore the effect of AMPK on BMP signaling in MC3T3-E1 cells and to explore possible molecular roles Mechanism. Methods: AMPK activator metformin treated MC3T3-E1 cells at different time or different concentrations. Western blot detected the effect of AMPK on the expression of BMP signaling pathway components, observed the changes in the expression of Smad6, Smurf1 and ALK2, and continued activation of AMPK adenovirus activation AMPK, and observed the direct effect of AMPK on the signaling pathway. Smad6 Si RNA, silencing Smad6 expression and exploring the molecular mechanism of AMPK inhibiting the BMP signaling pathway. Results: metformin and persistent activated AMPK mutants activate AMPK to inhibit the phosphorylation of BMP6 induced Smad1/5; AMPK activation after activation of Smad6 expression but not changes in the expression of AMPK The phosphorylation of Smad1/5 induced by BMP6 eliminates the inhibitory effect of AMPK on the BMP signaling pathway. Conclusion: AMPK activation inhibits BMP signaling pathway in MC3T3-E1 cells, and AMPK is activated by up regulation of Smad6 expression and inhibits BMP signal transduction. Smad6 is the main target of AMPK. The fifth part inhibits the osteogenic differentiation of the cells. The effect of PK on osteogenic differentiation of MC3T3-E1 cells. Methods: MC3T3-E1 cells were cultured in the induction of osteogenic differentiation medium to induce cells to differentiate into osteoblasts. Western bolt and Q PCR were used to detect AMPK activity and osteoblast differentiation marker OPN, Osx and Runx2, respectively, with Western bolt and Q PCR. The relationship between differentiation, infection continued activation of AMPK adenovirus activation AMPK and dominant negative AMPK adenovirus inactivation AMPK, observed the effect of AMPK on alkaline phosphatase activity; alkaline phosphatase staining was used to detect the effect of different AMPK activators (alone or combined use) on the early stage of osteogenic differentiation; alizarin red staining was used to detect different AMPK excitation. Results: the activity of AMPK activity gradually decreased with the higher degree of osteoblast differentiation, while the expression of osteoblast differentiation markers OPN and Osx showed the highest expression on the fourteenth day, and the expression of Runx2 was not significantly changed during the osteogenic differentiation; AMPK activator included metformin and asin. The activity of alkaline phosphatase in forest, curcumin and Bloven inhibited the activity of alkaline phosphatase, and showed a concentration dependence; the same effect was also shown in the persistent active mutant of AMPK. However, after the AMPK dominant negative mutant inactivated AMPK activity and function, the inhibitory effect of metformin on alkaline phosphatase activity was abolished; metformin and Bloven combined with the alkali. The activity inhibition of sex phosphatase has superposition effect, while metformin and curcumin display superposition effect. Metformin and aspirin inhibit the mineralization of MC3T3-E1 cells and show a concentration dependence. Conclusion: AMPK plays an important role in the process of osteoblast differentiation, the higher the osteogenesis is, the lower the AMPK activity is, the AMPK activation inhibits MC. 3T3-E1 osteoblast differentiation, including early and late stages. Metformin and ibuprofen combined use of supersuperposition effects may inhibit alkaline phosphatase activity through different mechanisms. Metformin and curcumin have superposition effects that may inhibit alkaline phosphatase activity through similar mechanisms. Sixth part AMPK is in vivo in mice The effect of ectopic ossification on the formation of heterotopic ossification in mice: a preliminary study of the effect of AMPK on the formation of heterotopic ossification in mice. Methods: to establish a mouse model of ectopic ossification of trauma and burn, and the intervention of metformin (drinking water containing 0.5mg/ml, metformin, N=5). After 8 weeks, the formation of ectopic bone was detected by X-RAY scan, and the ectopic ossification group was taken. Weave, fix, decalcified, observe histological changes by HE staining, Alcian Blue staining to detect the formation of metformin in cartilage; Q PCR detection of osteoblast differentiation markers (including Osc, BSP, Run X2) and Smad6, Smurf1 m expression changes. Results: a mouse heterotopic ossification model was successfully established by the method of trauma burn. Type, 8 weeks after operation, X-RAY examination showed obvious heterotopic ossification; after the use of metformin, the formation of heterotopic ossification in the mice was reduced, and HE detected no chondrocytes, osteoblast like and bone lacunae structure, most of the tendinous fiber, connective tissue, coloration of Alpine blue, and osteogenesis. The expression of M RNA of cell markers BSP, Osc and Runx2 decreased significantly, while the expression level of Smad6 and Smurf1 increased. Conclusion: the Achilles tendon cut burn method can effectively induce heterotopic ossification in mice, and metformin can prevent the formation of heterotopic ossification in mice.
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R597
本文编号：2155524
[Abstract]:In the first part, Heterotopic ossification (HO) refers to the pathological bone tissue in the soft tissue. It usually occurs in two individual patient groups: Patients with severe trauma including large area burns, musculoskeletal injuries, orthopedics and even spinal injuries; and progressive muscle ossification (Fibrodysplasi A ossificans progressiva, FOP) in patients with hereditary diseases,.FOP is caused by a permanent activation mutation of the I type bone morphogenetic protein (Bone morphogenetic proteins, BMP) receptor ALK2 (Activin = 2, activin 1 receptor). The clinical sequelae of these pathological ectopic bone formation, including non healing trauma, chronic Pain and joint immobility. For FOP, the patient died of respiratory distress due to loss of thoracic compliance resulting in respiratory distress and.BMPs and its transduction pathway plays an important role in ectopic ossification..BMPs is the main inducer of ectopic ossification; soft tissue stimulates BMPs secretion and activates BMP signaling pathway to promote the formation of HO. in soft tissue. In addition, about 95% of FOP patients have BMPI receptor (ALK2) mutation (R206H), which causes the persistent activation of the BMP signaling pathway, showing the characteristics of asymptotically heterotopic ossification. Heterotopic ossification is a very common and serious health problem. The current clinical use is generally to reduce pain, repair and prevent it. Most of them depend on the anti inflammatory drugs. The effect is partly effective at best, and a large number of side effects restrict the use of NSAIDs. An effective, ideal strategy is specific BMP signaling pathway components, inhibits heterotopic ossification, and the other innovative strategy is to identify the existing clinical treatment of other drugs and rediscover the effect of treatment to be full. Many studies have shown that AMP- activated protein kinase (AMP-activated protein kinase, AMPK) plays an important physiological role in bone metabolism. Metformin, a AMPK activator, is a first-line drug of type 2 diabetes. It has been widely used in the clinic for more than half a century. We have found that metformin is effective in the early period of our group. Inhibition of TGF beta family signal transduction and EMT. continue to extend this study to explore the effect of AMPK on BMP signaling pathways and heterotopic ossification. Therefore, we first studied the inhibition and molecular mechanism of AMPK on the BMP signaling pathway in FOP fibroblasts, and then induced FOP fibroblasts into pluripotent stem cells (iPS cells) for osteogenesis. Differentiation induction, further detects the inhibitory effect of AMPK on osteogenic differentiation; in order to explore the reactivity of different cells, continue to detect the effect and mechanism of AMPK on the MC3T3-E1 cell BMP signaling pathway, study the effect of different AMPK activators (alone or combined use) on osteogenic differentiation, determine the dose effect and synergistic effect; finally, determine the dose effect and synergistic effect. To establish a model of heterotopic ossification in mice and to detect the inhibitory effect of AMPK activator on heterotopic ossification in vivo. Our results confirm the inhibitory effect of AMPK on BMP signaling pathway and osteogenic differentiation and ectopic ossification, and also for the current clinical use of AMPK activators such as metformin, aspirin for the prevention and treatment of FOP disease and ectopic bone. To provide scientific basis. Second the inhibitory effect of AMPK on the BMP signaling pathway in FOP fibroblasts: to detect whether AMPK activation inhibits BMP signaling pathway transduction and explore the possible mechanism of AMPK. Method: DNA sequencing to detect the occurrence of ALK2 R206H mutation in FOP fibroblasts and the treatment of FOP cells by different AMPK activators. RN blot detected the effect of AMPK on the expression of BMP signaling pathway components; LKB1, AMPKa1a2 stably knocked out MEF cell lines, and observed the response of different cell lines to BMP and Metformin; infection continued to activate AMPK adenovirus activation AMPK and negative AMPK adenovirus inactivation AMPK. To explore the effect of AMPK on the interaction between Smad6 and Smurf1, ALK2 and Smad1, and the transfection of Smad6 Si RNA and Smurf1 Si RNA as well as proteasome inhibitor MG132 cells. ALK2 R206H mutation was generated; different AMPK activators activated AMPK and inhibited the phosphorylation of Smad1/5; infection sustained activation AMPK mutants inhibited BMP signal transduction, and after infection of AMPK dominant negative mutants, the inhibition of metformin on Smad1/5 phosphorylation was cancelled. After stable knockout LKB1 and AMPKa1a2 expression, metformin could not inhibit BMP6 lure. The activation of phosphorylated.AMPK in Smad1/5 inhibited the expression of ALK2 but up regulation of the expression of Smad6 and Smurf1; AMPK activation promoted the binding of Smad6 and Smurf1, but inhibited the interaction of ALK2 and Smad1, and the inhibition effect on the signaling pathway was eliminated after the silence of Smad6 and Smurf1 genes. In addition to the effect of AMPK on promoting the degradation of ALK2. Conclusion: AMPK activator or the use of persistent activated AMPK mutants to activate AMPK to inhibit the phosphorylation of Smad1/5; the inhibition of phosphorylation of Smad1/5 by metformin is mediated by AMPK; AMPK activation inhibits BMP signal transduction in FOP fibroblasts, and up regulation and expression after activation of AMPK activation, enhancement The interaction between the two molecules then causes an increase in ALK2 degradation, which inhibits BMP signal transduction. The third part AMPK inhibits the osteogenic differentiation of iPS derived from FOP fibroblasts: the study of the effect of AMPK activation on the osteogenic differentiation of iPS derived from FOP fibroblasts. Lead pluripotent stem cells (FOP-iPS); induce iPS cells to differentiate into osteoblasts. The differentiation ability of ALK2 mutation (FOP iPS) and iPS induced ALK2 (Control iPS) induced osteoblast differentiation was observed by alizarin red staining and Western blot detection of osteoblast differentiation markers. The AMPK activator was detected by alizarin red staining. The effect on the mineralization of iPS cells. Results: a successful construction of iPS cells (FOP iPS) derived from FOP fibroblasts. Compared with the control iPS, the FOP iPS alizarin red staining was stronger, the osteogenic differentiation marker Run X2, Osx and OPN expressed higher levels. The ability of FOP iPS cells to induce osteoblast differentiation is stronger; AMPK inhibits the osteogenic differentiation of iPS derived from FOP fibroblasts. Fourth the inhibitory effect of part AMPK on BMP signaling pathway in the MC3T3-E1 cells of mouse anterior osteoblasts: To explore the effect of AMPK on BMP signaling in MC3T3-E1 cells and to explore possible molecular roles Mechanism. Methods: AMPK activator metformin treated MC3T3-E1 cells at different time or different concentrations. Western blot detected the effect of AMPK on the expression of BMP signaling pathway components, observed the changes in the expression of Smad6, Smurf1 and ALK2, and continued activation of AMPK adenovirus activation AMPK, and observed the direct effect of AMPK on the signaling pathway. Smad6 Si RNA, silencing Smad6 expression and exploring the molecular mechanism of AMPK inhibiting the BMP signaling pathway. Results: metformin and persistent activated AMPK mutants activate AMPK to inhibit the phosphorylation of BMP6 induced Smad1/5; AMPK activation after activation of Smad6 expression but not changes in the expression of AMPK The phosphorylation of Smad1/5 induced by BMP6 eliminates the inhibitory effect of AMPK on the BMP signaling pathway. Conclusion: AMPK activation inhibits BMP signaling pathway in MC3T3-E1 cells, and AMPK is activated by up regulation of Smad6 expression and inhibits BMP signal transduction. Smad6 is the main target of AMPK. The fifth part inhibits the osteogenic differentiation of the cells. The effect of PK on osteogenic differentiation of MC3T3-E1 cells. Methods: MC3T3-E1 cells were cultured in the induction of osteogenic differentiation medium to induce cells to differentiate into osteoblasts. Western bolt and Q PCR were used to detect AMPK activity and osteoblast differentiation marker OPN, Osx and Runx2, respectively, with Western bolt and Q PCR. The relationship between differentiation, infection continued activation of AMPK adenovirus activation AMPK and dominant negative AMPK adenovirus inactivation AMPK, observed the effect of AMPK on alkaline phosphatase activity; alkaline phosphatase staining was used to detect the effect of different AMPK activators (alone or combined use) on the early stage of osteogenic differentiation; alizarin red staining was used to detect different AMPK excitation. Results: the activity of AMPK activity gradually decreased with the higher degree of osteoblast differentiation, while the expression of osteoblast differentiation markers OPN and Osx showed the highest expression on the fourteenth day, and the expression of Runx2 was not significantly changed during the osteogenic differentiation; AMPK activator included metformin and asin. The activity of alkaline phosphatase in forest, curcumin and Bloven inhibited the activity of alkaline phosphatase, and showed a concentration dependence; the same effect was also shown in the persistent active mutant of AMPK. However, after the AMPK dominant negative mutant inactivated AMPK activity and function, the inhibitory effect of metformin on alkaline phosphatase activity was abolished; metformin and Bloven combined with the alkali. The activity inhibition of sex phosphatase has superposition effect, while metformin and curcumin display superposition effect. Metformin and aspirin inhibit the mineralization of MC3T3-E1 cells and show a concentration dependence. Conclusion: AMPK plays an important role in the process of osteoblast differentiation, the higher the osteogenesis is, the lower the AMPK activity is, the AMPK activation inhibits MC. 3T3-E1 osteoblast differentiation, including early and late stages. Metformin and ibuprofen combined use of supersuperposition effects may inhibit alkaline phosphatase activity through different mechanisms. Metformin and curcumin have superposition effects that may inhibit alkaline phosphatase activity through similar mechanisms. Sixth part AMPK is in vivo in mice The effect of ectopic ossification on the formation of heterotopic ossification in mice: a preliminary study of the effect of AMPK on the formation of heterotopic ossification in mice. Methods: to establish a mouse model of ectopic ossification of trauma and burn, and the intervention of metformin (drinking water containing 0.5mg/ml, metformin, N=5). After 8 weeks, the formation of ectopic bone was detected by X-RAY scan, and the ectopic ossification group was taken. Weave, fix, decalcified, observe histological changes by HE staining, Alcian Blue staining to detect the formation of metformin in cartilage; Q PCR detection of osteoblast differentiation markers (including Osc, BSP, Run X2) and Smad6, Smurf1 m expression changes. Results: a mouse heterotopic ossification model was successfully established by the method of trauma burn. Type, 8 weeks after operation, X-RAY examination showed obvious heterotopic ossification; after the use of metformin, the formation of heterotopic ossification in the mice was reduced, and HE detected no chondrocytes, osteoblast like and bone lacunae structure, most of the tendinous fiber, connective tissue, coloration of Alpine blue, and osteogenesis. The expression of M RNA of cell markers BSP, Osc and Runx2 decreased significantly, while the expression level of Smad6 and Smurf1 increased. Conclusion: the Achilles tendon cut burn method can effectively induce heterotopic ossification in mice, and metformin can prevent the formation of heterotopic ossification in mice.
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R597
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