發(fā)布時(shí)間：2018-07-31 12:01

【摘要】：第一部分引言異位骨化(Heterotopic ossification,HO)是指在軟組織中病理形成異常的骨組織。一般發(fā)生在兩個(gè)單獨(dú)的患者群體中：具有嚴(yán)重創(chuàng)傷包括大面積燒傷、肌肉骨骼損傷、矯形手術(shù)甚至脊髓損傷的患者;和進(jìn)行性肌肉骨化癥(Fibrodysplasia ossificans progressiva,FOP)等遺傳性疾病患者中。FOP由I型骨形態(tài)發(fā)生蛋白(Bone morphogenetic proteins,BMP)受體ALK2(Activin receptor-like kinase 2,活化素1型受體)中的永久性活化突變引起。這些病理性異位骨形成的臨床后遺癥,包括非愈合性創(chuàng)傷、慢性疼痛和關(guān)節(jié)不動(dòng)性。對于FOP,患者由于胸廓順應(yīng)性的損失導(dǎo)致呼吸困難而死亡。BMPs以及其轉(zhuǎn)導(dǎo)的信號通路在異位骨化形成中具有重要作用。BMPs是異位骨化的主要誘導(dǎo)者;軟組織創(chuàng)傷后刺激BMPs分泌和激活BMP信號通路,促進(jìn)軟組織形成HO。此外,大約95%的FOP患者發(fā)生BMPI型受體(ALK2)突變(R206H),引起B(yǎng)MP信號通路持續(xù)性激活,表現(xiàn)為漸近性異位骨化的特性。異位骨化是一個(gè)非常普遍和嚴(yán)重的健康問題,目前臨床上使用的方法一般是減少疼痛,修復(fù)以及預(yù)防。大多依賴于抗炎藥物的使用,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。一個(gè)有效的、理想的策略是特異性的針對BMP信號通路成分,抑制異位骨化,另一個(gè)有創(chuàng)新性的策略是鑒定現(xiàn)有臨床上治療其他藥物,重新發(fā)現(xiàn)其治療效果來滿足臨床需求。眾多研究表明AMP-激活蛋白激酶(AMP-activated protein kinase,AMPK)在骨代謝中發(fā)揮重要的生理作用,AMPK激活劑二甲雙胍是2型糖尿病一線藥物,已經(jīng)在臨床上廣泛使用超過半個(gè)世紀(jì)。我們課題組前期已經(jīng)發(fā)現(xiàn)二甲雙胍能夠有效性的抑制TGFβ家族信號轉(zhuǎn)導(dǎo)和EMT。繼續(xù)延伸此研究,探索AMPK對BMP信號通路以及異位骨化的作用。因此,我們首先研究了AMPK對FOP成纖維細(xì)胞中BMP信號通路的抑制作用以及分子機(jī)制;然后將FOP成纖維細(xì)胞誘導(dǎo)成多能干細(xì)胞(iPS cells),進(jìn)行成骨分化誘導(dǎo),進(jìn)一步檢測了AMPK對成骨分化的抑制作用;為了探索不同細(xì)胞的反應(yīng)性,繼續(xù)檢測AMPK對MC3T3-E1細(xì)胞BMP信號通路的影響和作用機(jī)制,研究不同AMPK激活劑(單獨(dú)使用或者聯(lián)合使用)對成骨分化的影響,確定劑量效應(yīng)和協(xié)同效應(yīng);最后建立小鼠異位骨化模型以及檢測AMPK激活劑對體內(nèi)異位骨化的抑制作用。我們的研究結(jié)果明確AMPK對BMP信號通路以及成骨分化和異位骨化的抑制作用,也為現(xiàn)有臨床使用的AMPK激活劑如二甲雙胍、阿司匹林用于預(yù)防、治療FOP疾病和異位骨化提供科學(xué)依據(jù)。第二部分AMPK對FOP成纖維細(xì)胞中BMP信號通路的抑制作用目的：檢測AMPK激活是否抑制BMP信號通路轉(zhuǎn)導(dǎo)以及探索AMPK可能的作用機(jī)制。方法：DNA測序檢測FOP成纖維細(xì)胞是否發(fā)生ALK2 R206H突變;不同AMPK激活劑處理FOP細(xì)胞,Western blot檢測AMPK對BMP信號通路成分表達(dá)的影響;構(gòu)建LKB1、AMPKa1a2穩(wěn)定敲除MEF細(xì)胞株,觀察不同細(xì)胞株對BMP和二甲雙胍的反應(yīng)性;感染持續(xù)性激活A(yù)MPK腺病毒激活A(yù)MPK和顯示負(fù)性AMPK腺病毒失活A(yù)MPK,確定二甲雙胍對BMP的信號通路作用是通過AMPK介導(dǎo)的;構(gòu)建熒光素酶互補(bǔ)結(jié)合實(shí)驗(yàn)系統(tǒng),觀察AMPK對Smad6與Smurf1、ALK2與Smad1相互作用的影響;轉(zhuǎn)染Smad6 si RNA和Smurf1 si RNA以及用蛋白酶體抑制劑MG132處理細(xì)胞,探索AMPK對BMP信號通路抑制作用的分子機(jī)制。結(jié)果：DNA測序確認(rèn)FOP成纖維細(xì)胞發(fā)生ALK2 R206H突變;不同AMPK激活劑激活A(yù)MPK并且抑制Smad1/5的磷酸化;感染持續(xù)性激活A(yù)MPK突變體抑制BMP信號轉(zhuǎn)導(dǎo),而感染AMPK顯性負(fù)性突變體后,取消了二甲雙胍對Smad1/5磷酸化的抑制作用;穩(wěn)定敲除LKB1和AMPKa1a2表達(dá)后,二甲雙胍不能抑制BMP6誘導(dǎo)的Smad1/5的磷酸化。AMPK激活抑制ALK2的表達(dá)但上調(diào)Smad6和Smurf1的表達(dá);AMPK激活后促進(jìn)Smad6和Smurf1的結(jié)合,但阻遏ALK2和Smad1的相互作用;Smad6和Smurf1基因沉默后,消除了AMPK對BMP信號通路的抑制作用。此外,結(jié)果還顯示蛋白酶體抑制MG132能夠消除AMPK促進(jìn)ALK2降解的作用。結(jié)論：AMPK激活劑或者使用持續(xù)性激活A(yù)MPK突變體激活A(yù)MPK抑制Smad1/5的磷酸化;二甲雙胍對Smad1/5的磷酸化抑制是通過AMPK介導(dǎo)的;AMPK激活抑制FOP成纖維細(xì)胞中BMP信號通路轉(zhuǎn)導(dǎo),AMPK激活后上調(diào)Smad6和Smurf1表達(dá)、增強(qiáng)兩個(gè)分子之間的相互作用,隨后引起ALK2降解增加,從而抑制BMP信號轉(zhuǎn)導(dǎo)。第三部分AMPK抑制來源于FOP成纖維細(xì)胞的iPS的成骨分化目的：研究AMPK激活對來源于FOP成纖維細(xì)胞的iPS的成骨分化影響。方法：建立、鑒定來源于FOP成纖維細(xì)胞的誘導(dǎo)多能干細(xì)胞(FOP-iPS);誘導(dǎo)iPS細(xì)胞向成骨細(xì)胞分化,通過茜素紅染色和Western blot檢測成骨分化標(biāo)記物表達(dá)來觀察ALK2突變(FOP iPS)和野生型ALK2(Control iPS)的iPS誘導(dǎo)成骨細(xì)胞分化能力的區(qū)別;采用茜素紅染色檢測AMPK激活劑(二甲雙胍、AICAR)對iPS細(xì)胞礦化的影響。結(jié)果：成功構(gòu)建來源于FOP成纖維細(xì)胞的iPS細(xì)胞(FOP iPS);與對照iPS相比較,FOP iPS茜素紅染色程度更強(qiáng),成骨分化標(biāo)記物Run X2、Osx和OPN表達(dá)水平更高;AMPK激活劑二甲雙胍、AICAR抑制iPS誘導(dǎo)的細(xì)胞礦化。結(jié)論：攜帶ALK2突變的FOP iPS細(xì)胞誘導(dǎo)成骨細(xì)胞分化的能力更強(qiáng);AMPK抑制來源于FOP成纖維細(xì)胞的iPS的成骨分化。第四部分AMPK對小鼠前成骨細(xì)胞MC3T3-E1細(xì)胞中BMP信號通路的抑制作用目的：探討AMPK對MC3T3-E1細(xì)胞中BMP信號通路的影響以及探索可能的分子作用機(jī)制。方法：AMPK激活劑二甲雙胍處理MC3T3-E1細(xì)胞不同時(shí)間或者不同濃度,Western blot檢測AMPK對BMP信號通路成分表達(dá)的影響,觀察AMPK對Smad6、Smurf1和ALK2等表達(dá)的改變;感染持續(xù)性激活A(yù)MPK腺病毒激活A(yù)MPK,觀察AMPK對BMP信號通路的直接效應(yīng);轉(zhuǎn)染Smad6 si RNA,沉默Smad6表達(dá),探索AMPK對BMP信號通路抑制作用的分子機(jī)制。結(jié)果：二甲雙胍和持續(xù)性激活A(yù)MPK突變體激活A(yù)MPK抑制BMP6誘導(dǎo)的Smad1/5的磷酸化;AMPK激活后上調(diào)Smad6表達(dá)但不改變Smurf1和ALK2的表達(dá);Smad6基因沉默后,二甲雙胍不能抑制BMP6誘導(dǎo)的Smad1/5的磷酸化,消除了AMPK對BMP信號通路的抑制作用。結(jié)論：AMPK激活抑制MC3T3-E1細(xì)胞中BMP信號通路轉(zhuǎn)導(dǎo),AMPK激活后通過上調(diào)Smad6表達(dá),抑制BMP信號轉(zhuǎn)導(dǎo),Smad6是AMPK主要靶目標(biāo)。第五部分AMPK抑制MC3T3-E1細(xì)胞成骨分化目的：探討AMPK對MC3T3-E1細(xì)胞成骨分化的影響。方法：MC3T3-E1細(xì)胞培養(yǎng)在誘導(dǎo)成骨分化培養(yǎng)基中誘導(dǎo)細(xì)胞向成骨細(xì)胞分化,分別采用Western bolt和q PCR檢測不同時(shí)間段(0、1、3、7、14、21天)AMPK活性和成骨細(xì)胞分化標(biāo)記物OPN、Osx和Runx2的改變;觀察AMPK活性和成骨分化之間的關(guān)系;感染持續(xù)性激活A(yù)MPK腺病毒激活A(yù)MPK和顯性負(fù)性AMPK腺病毒失活A(yù)MPK,觀察AMPK對堿性磷酸酶活性的影響;堿性磷酸酶染色檢測不同AMPK激活劑(單獨(dú)使用或者聯(lián)合使用)對成骨分化早期階段的影響;茜素紅染色檢測不同AMPK激活劑對成骨分化晚期階段-細(xì)胞礦化的影響。結(jié)果：AMPK活性隨著成骨細(xì)胞分化程度越高其活性逐漸降低,而成骨細(xì)胞分化標(biāo)記物OPN和Osx在第14天呈現(xiàn)最高表達(dá),Runx2在成骨分化期間表達(dá)未發(fā)生明顯改變;AMPK激活劑包括二甲雙胍、阿司匹林、姜黃素和布洛芬等抑制堿性磷酸酶活性,并呈現(xiàn)濃度依賴性;感染AMPK持續(xù)性活性突變體也顯示同樣效應(yīng),然而感染AMPK顯性負(fù)性突變體失活A(yù)MPK活性和功能后,取消二甲雙胍對堿性磷酸酶活性的抑制作用;二甲雙胍和布洛芬聯(lián)用對堿性磷酸酶活性抑制具有超疊加效應(yīng),而二甲雙胍和姜黃素聯(lián)用顯示疊加效應(yīng)。二甲雙胍和阿司匹林抑制MC3T3-E1細(xì)胞礦化,并呈現(xiàn)濃度依賴性。結(jié)論：AMPK在成骨細(xì)胞分化過程中發(fā)揮重要作用,成骨分化程度越高,AMPK活性越低;AMPK激活抑制MC3T3-E1成骨細(xì)胞分化,包括早期階段和晚期階段。二甲雙胍和布洛芬聯(lián)合應(yīng)用具有超疊加效應(yīng)可能通過不同機(jī)制抑制堿性磷酸酶活性。二甲雙胍和姜黃素聯(lián)用具有疊加效應(yīng)可能通過相似機(jī)制抑制堿性磷酸酶活性。第六部分AMPK對小鼠體內(nèi)異位骨化形成的影響目的：初步探討AMPK對小鼠體內(nèi)異位骨化形成的影響。方法：建立創(chuàng)傷-燒傷異位骨化小鼠模型,同時(shí)采用二甲雙胍干預(yù)(飲用水含有0.5mg/ml的二甲雙胍,N=5),連續(xù)8周后,采用X-RAY掃描檢測異位骨的形成;取異位骨化組織,固定,脫鈣,進(jìn)行HE染色觀察組織學(xué)改變,阿爾新藍(lán)(Alcian Blue)染色檢測二甲雙胍對軟骨內(nèi)形成改變;q PCR檢測成骨細(xì)胞分化標(biāo)記物(包括Osc、BSP、Run X2)以及Smad6、Smurf1的m RNA表達(dá)的改變。結(jié)果：通過創(chuàng)傷-燒傷方法成功建立小鼠異位骨化模型,術(shù)后8周,進(jìn)行X-RAY檢查可見明顯的異位骨化形成;采用二甲雙胍干預(yù)后,與對照組小鼠比較,小鼠體內(nèi)異位骨化形成減少,HE檢測未見軟骨細(xì)胞、成骨細(xì)胞樣和骨陷窩結(jié)構(gòu),大部分為腱性纖維、結(jié)締組織,阿爾新藍(lán)染色可見少量染色;成骨細(xì)胞標(biāo)記物BSP、Osc和Runx2的m RNA表達(dá)明顯減少,而Smad6和Smurf1的表達(dá)水平上升。結(jié)論：跟腱切斷-燒傷法能夠有效誘導(dǎo)小鼠異位骨化形成,二甲雙胍可以阻止小鼠體內(nèi)異位骨化的形成。
[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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R597

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