Variations in skeletal morphology have often been used to interpret an organism’s overall activity level when direct observation is not possible. Although skeletal change in response to exercise is well documented, the skeleton’s response to mechanical loading is modulated by several factors (e.g. age, hormones, sex). Additionally, variation in skeletal morphology is partially a result of genetic variation, which is rarely accounted for in inferences of locomotor activity from skeletal remains. However, blood flow to long bones serves as a proxy for bone metabolic activity, which can be used to infer locomotor activity. Long bones receive blood from three sources, with the nutrient artery supplying the bulk of total blood volume in mammals (50-70%). The size of the nutrient artery can be estimated from the dimensions of the nutrient canal, which is present long after the vascular tissue has degenerated. The literature on nutrient canals is sparse, with most studies consisting of anatomical descriptions from surgical proceedings, and only a few studies investigating the links between nutrient canals and physiology or behavior. Moreover, no study to date has accurately modelled the size and shape of the nutrient canal. For this study, mice from an artificial selection experiment for high voluntary wheel-running behavior were used. High Runner mice from the experiment are known to differ in both metabolic and locomotor activity, with mice from HR lines having increased VO2max and increased voluntary wheel-running behavior when compared to controls. 137 femora from mice of the 11th generation of this selection experiment were µCT scanned. Three-dimensional reconstructions of nutrient canals were measured for minimum cross-sectional area (an index of blood flow). Nutrient canals varied far more in number and shape than prior descriptions would indicate. Canals adopted non-linear shape and pathing as they traversed from the periosteum to the medullary cavity, occasionally even branching within the cortical bone. Additionally, mice from both HR and control lines had more than four nutrient canals per femur. Mice from HR lines had significantly larger nutrient canal area than controls, which was not the result of an increase in the number of nutrient canals, but rather an increase in their average size. This study demonstrates that mice with an evolutionary history of increased locomotor activity and metabolic rate have a concomitant increase in the size of their nutrient canals.

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Dab2是一個細胞內接頭蛋白和腫瘤抑制因子。在小鼠胚胎中，應用免疫熒光染色技術，從E8.5-E11.0 Dab2發現表達於肌節的生皮肌節中。從E8.5 E9.5，Dab2表達於生皮肌節的中部。在E10.5，Dab2表達於生皮肌節的腹外側唇部，與肌肉發育的早期標誌基因Pax3和 Myf5共定位。從E11.5-E14.5，Dab2表達於四肢與軀體的肌肉中,Dab2在出生後小鼠肌肉中的表達逐漸減弱。此外，因為肌肉正常發育需要很多細胞信號的調節並且Dab2已經發現調節MAPK, TGF-β和 Wnt信號轉導通路。這些發現預示了Dab2在肌肉發育和分化中可能具有重要作用。 / 為了進一步研究它在肌肉發育中的作用，非洲爪蟾的胚胎和C2C12 肌原細胞在此研究中分別被用作體內和體外的研究模型。原位雜交結果揭示非洲爪蟾的Dab2基因表達於其胚胎的肌節中，並與肌肉發育的標誌基因XPax3, XMyoD, XMef2c和 XMyos共定位於此。用morpholino敲低XDab2 在非洲爪蟾胚胎中的表達，下調了許多肌肉發育標誌基因的表達，例如：XPax3, XMyf5, XMef2c, XMyoS 和XAC100。與此同時，免疫熒光技術也檢測到MHC(MF20)和12/101在肌節中的表達下調。 / 來源於小鼠肌肉衛星細胞的C2C12肌原細胞系被用作體外模型來檢測Dab2基因在骨骼肌發育和分化中的作用。在C2C12肌原細胞被誘導分化形成肌管的過程中，Dab2基因在RNA和蛋白水平的表達被誘導性的升高。Dab2基因超表達能夠加速肌原細胞的融合，從而增加肌小管的形成。利用miRNA敲低Dab2基因的表達能夠減緩肌原細胞的融合，從而減少肌小管的形成。利用慢病毒shRNA技術我們得到了2個Dab2穩定敲低細胞系，命名為克隆5-2和克隆5-7。這兩個克隆具有減少或抑制減少或抑制肌小管形成的特點。蛋白免疫印跡實驗表明，磷酸化p38 MAPK的表達在這兩個克隆中被抑制。在克隆5-2中超表達Dab2基因能夠恢復肌小管的形成。這個研究表明Dab2基因在肌小管的形成過程中具有至關重要的作用。 / 利用Affymetrix微陣列技術，我們檢測並分析了在克隆5-2和對照細胞中差異表達的基因。235個探針（155個基因）的顯示出超過2倍的差異表達。在這155個基因中，127個基因下調表達，28個基因上調表達。熒光定量PCR結果顯示出與微陣列結果相一致的結果。這些差異表達基因的功能發現與肌肉系統的發育和功能具有顯著地聯系。它影響了與肌肉收縮，橫紋肌的收縮，肌前體細胞的分化和肌肉發育相關功能的基因。基因網絡分析結果揭示，在克隆5-2中Mef2c基因的下調表達可能是一個導致肌細胞分化抑制的原因。 Mef2c基因在克隆5-2中超表達能夠拯救肌細胞的分化。 / 總括來說，體內和體外實驗共同表明Dab2基因是一個肌肉發育和分化的正調控基因。 / Dab2 is an intracellular adaptor protein and a tumor suppressor. In mouse embryos, Dab2 was found to be expressed in the dermomyotome of somites from E8.5 to E11.0 using immunofluorescence staining, with expression first detected in the medial aspect of the dermomyotome at E8.5 and then co-localized with the early muscle markers Pax3 and Myf5 at the ventrolateral lip of the dermomyotome at E10.5. From E11.5 to E14.5, Dab2 was expressed in muscle masses of limb buds and the trunk. Dab2 expression in skeletal muscles was gradually decreased after birth. These observations suggested potential roles of Dab2 in the skeletal muscle myogenesis. In addition, since the normal development of skeletal muscles requires proper signal transduction, and Dab2 has been known to be involved in the MAPK, TGF-β and Wnt signaling pathways, Dab2 may therefore be important for the muscle development. / To determine the role of Dab2 in the skeletal muscle development, Xenopus laevis embryos and C2C12 myoblasts were employed as in vivo and in vitro models, respectively. In situ hybridization results showed that XDab2 was expressed in somites of Xenopus embryos and co-localized with the muscle markers XPax3, XMyoD, XMef2c and XMyos. Knockdown of XDab2 expression with antisense morpholinos down regulated the expression of several muscle markers in somites including XPax3, XMyf5, XMef2c, XMyoS and XAC100. Down-regulation of MHC and 12/101 were also observed in whole mount preparations and transverse sections of XDab2 morpholino-injected embryos after immunohistochemical staining. / The C2C12 cell line derived from mouse muscle satellite cells was then employed as an in vitro model to determine the role of Dab2 during early muscle development. When C2C12 myoblasts were induced to differentiate into myotubes, Dab2 expression was simultaneously increased at RNA and protein levels. Dab2 over-expression after transfection with Dab2 plasmids resulted in enhanced myoblast fusion and increased numbers of myotubes. Conversely, suppression of Dab2 expression with miRNAs resulted in reduced myoblast fusion and decreased numbers of myotubes. Lentiviral shRNA-mediated Dab2 stable knockdown reduced myotube formation in 2 representative stable clones, clone 5-2 and clone 5-7. Western blot analysis showed that expression of phospho-p38 MAPK was down-regulated in clone 5-2 and 5-7. Dab2 re-expression through plasmid-mediated transient transfection in clone 5-2 could partially restore the myotube formation. These observations therefore suggested that Dab2 plays essential roles in the formation of myotubes. / Comprehensive profiling of differentially expressed genes was performed with the Affymetrix microarray analysis between the Dab2-knockdown clone 5-2 and the C2C12 parental cell line. As compared to the parental cells, the clone 5-2 showed significant changes in the expression of 235 probe sets representing 155 genes (p<0.05) with 2 folds or greater changes. Among the 155 genes, 127 were down-regulated, while 28 up-regulated. qRT-PCR results were found to be consistent with the microarray results. Functions of the differentially expressed genes were found to be significantly associated with the development and functions of the muscular system. Knockdown of Dab2 affected the genes involved in muscle contraction, the contraction of striated muscle, differentiation of muscle precursor cells, and the development of skeletal muscle fibers. A network analysis and a gene expression study revealed that Mef2c down-regulation was related to the inhibition of myogenic differentiation in the clone 5-2. Furthermore, forced expression of Mef2c in the clone 5-2 could rescue the myogenic differentiation. / In conclusion, these results indicated that Dab2 is positive regulator of the skeletal muscle development and differentiation both in vivo and in vitro. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Shang, Na. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 211-227). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgements --- p.vi / Table of contents --- p.vii / Abbreviation --- p.xiii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Characterizations of the Dab2 gene --- p.1 / Chapter 1.2 --- The role of Dab2 in Wnt/ beta-catenin signaling --- p.2 / Chapter 1.3 --- The role of Dab2 in TGF beta signaling --- p.3 / Chapter 1.4 --- The role of Dab2 in Ras-MAPK signaling --- p.4 / Chapter 1.5 --- The role of Dab2 in protein trafficking and endocytosis --- p.5 / Chapter 1.6 --- Dab2 expression and its functions. --- p.7 / Chapter 1.7 --- Somite and skeletal muscle development --- p.8 / Chapter 1.8 --- The formation of the somite and its structure --- p.9 / Chapter 1.9 --- The formation of dermomyotome and its function --- p.10 / Chapter 1.10 --- The formation of myotome and its function --- p.11 / Chapter 1.11 --- The formation of muscle fibers and musculatures --- p.12 / Chapter 1.12 --- The formation of satellite cells and its function in skeletal muscle differentiation --- p.12 / Chapter 1.13 --- The gene expression during skeletal muscle development and differentiation --- p.13 / Chapter 1.14 --- Dab2 genetically modified mice --- p.16 / Chapter 1.15 --- Objectives of this research --- p.17 / Chapter Figures and legends --- p.21 / Chapter Chapter 2 --- Expression of Dab2 in the mouse somites and skeletal muscles --- p.32 / Chapter 2.1 --- Introduction --- p.32 / Chapter 2.2 --- Materials and Methods --- p.34 / Chapter 2.2.1 --- Mouse embryos and tissue isolation --- p.34 / Chapter 2.2.2 --- Histological preparation of embryos and tissues --- p.34 / Chapter 2.2.3 --- Immunostaining using Tyramide signal amplification kits --- p.35 / Chapter 2.3 --- Results --- p.36 / Chapter 2.3.1 --- Dab2 expression in somites of the mouse embryos --- p.36 / Chapter 2.3.2 --- Dab2 expression in skeletal muscles of embryonic and postnatal mice --- p.36 / Chapter 2.3.3 --- Co-localization of Dab2 and Pax3 immunoreactivities with double immunofluorescence staining --- p.37 / Chapter 2.3.4 --- Co-localization of Dab2 and Myf5 immunoreactivities with double immunofluorescence staining --- p.38 / Chapter 2.3.5 --- Co-localization of Dab2 and Myogenin immunoreactivities with double immunofluorescence staining --- p.38 / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.5 --- Summary --- p.42 / Chapter Table 2.1 --- p.44 / Chapter Figures and Legends --- p.45 / Chapter Chapter 3 --- Dab2 is a positive regulator of skeletal muscle development in Xenopus embryos --- p.58 / Chapter 3.1 --- Introduction --- p.58 / Chapter 3.2 --- Materials and Methods --- p.61 / Chapter 3.2.1 --- RNA extraction --- p.61 / Chapter 3.2.2 --- Reverse-transcription polymerase chain reaction (RT-PCR) --- p.61 / Chapter 3.2.3 --- Gene cloning and sequencing analysis --- p.61 / Chapter 3.2.4 --- Transformation --- p.62 / Chapter 3.2.5 --- Plasmid mini and midi-preparation --- p.62 / Chapter 3.2.6 --- Frogs and embryos handling --- p.63 / Chapter 3.2.7 --- Synthesis of mRNA for microinjection --- p.64 / Chapter 3.2.8 --- Microinjection --- p.64 / Chapter 3.2.9 --- Synthesis of DIG-labeled anti-sense RNA probe --- p.65 / Chapter 3.2.10 --- Whole mount in situ hybridization (WMISH) and whole mount immunohistochemical localization --- p.65 / Chapter 3.3 --- Results --- p.67 / Chapter 3.3.1 --- Cloning of Xenopus Dab2 long isoform and the sequence analysis --- p.67 / Chapter 3.3.2 --- Phylogenetic analysis --- p.67 / Chapter 3.3.3 --- RT-PCR analysis of Xenopus Dab2 (XDab2) expression --- p.68 / Chapter 3.3.4 --- Xenopus Dab2 spatial and temporal expression examined by WMISH analysis --- p.68 / Chapter 3.3.5 --- Dab2 expression in somites and its colocalization with myogenic transcription factors --- p.69 / Chapter 3.3.6 --- XDab2 knockdown led to down-regulation of myogenic transcription factors and muscle markers at the RNA level --- p.70 / Chapter 3.3.7 --- XDab2 knockdown led to down-regulation of muscle markers at the protein level --- p.70 / Chapter 3.3.8 --- XDab2 overexpression led to up-regulation of XPax3, XMyf5 and XMyoS --- p.71 / Chapter 3.4 --- Discussion --- p.72 / Chapter 3.5 --- Summary --- p.77 / Chapter Table 3.1 --- p.78 / Chapter Figures and Legends --- p.79 / Chapter Chapter 4 --- Potential roles of Dab2 in C2C12 myoblast differentiation --- p.99 / Chapter 4.1 --- Introduction --- p.99 / Chapter 4.2 --- Materials and Methods --- p.101 / Chapter 4.2.1 --- Cell culture and differentiation in vitro --- p.101 / Chapter 4.2.2 --- Cell sample preparation --- p.102 / Chapter 4.2.3 --- Real-time PCR --- p.102 / Chapter 4.2.4 --- SDS-PAGE --- p.103 / Chapter 4.2.5 --- Western blotting and immunodetection --- p.104 / Chapter 4.2.6 --- Plasmids used for transient over-expression --- p.105 / Chapter 4.2.7 --- Generation of miRNAs targeting at Dab2 --- p.105 / Chapter 4.2.8 --- C2C12 differentiation after transfection --- p.106 / Chapter 4.2.9 --- Immunohistochemical staining for myotubes --- p.106 / Chapter 4.2.10 --- Lentiviral shRNA mediated Dab2 stable knockdown --- p.107 / Chapter 4.2.10.1 --- shRNA Lentiviral Transduction Particles and sequence information --- p.107 / Chapter 4.2.10.2 --- Optimization of puromycin treatment on C2C12 myoblasts --- p.107 / Chapter 4.2.10.3 --- Determination of the optimal MOI for C2C12 --- p.108 / Chapter 4.2.10.4 --- Lentivirus transduction method --- p.109 / Chapter 4.2.10.5 --- Stable cell line generation --- p.109 / Chapter 4.2.11 --- Rescue experiments --- p.109 / Chapter 4.2.12 --- Serum starvation and FGF treatment --- p.110 / Chapter 4.2.13 --- Microarray and data analysis --- p.110 / Chapter 4.3 --- Results --- p.113 / Chapter 4.3.1 --- Expression of Dab2 during myogenesis --- p.113 / Chapter 4.3.2 --- Generation of miRNAs targeting at Dab2 --- p.113 / Chapter 4.3.3 --- Improvement of the transfection efficiency --- p.114 / Chapter 4.3.4 --- Knockdown efficiencies of the 4 miRNAs --- p.114 / Chapter 4.3.5 --- Down-regulation of Dab2 expression by transient transfection inhibited C2C12 differentiation --- p.115 / Chapter 4.3.6 --- Up-regulation of Dab2 expression by transient transfection enhanced myogenic differentiation --- p.116 / Chapter 4.3.7 --- Lentivirus-mediated Dab2 stable knockdown inhibited myotube formation --- p.117 / Chapter 4.3.8 --- Re-expression of Dab2 partially restored myogenic differentiation in the clone 5-2 --- p.120 / Chapter 4.3.9 --- Dab2 knockdown affected the MAPK signaling pathway --- p.122 / Chapter 4.3.10 --- Transcriptome and network analysis revealed changes of gene expression patterns in the C2C12 cell line after Dab2 knockdown --- p.123 / Chapter 4.3.11 --- Mef2c down-regulation was related to the inhibition of the myotube formation in the clone 5-2 --- p.126 / Chapter 4.4 --- Discussion --- p.128 / Chapter 4.4.1 --- Dab2 expression was found to be induced upon differentiation and down-regulated after myotube formation --- p.128 / Chapter 4.4.2 --- Dab2 was found to be a positive regulator of C2C12 differentiation --- p.129 / Chapter 4.4.3 --- Dab2 knockdown affected the MAPK signaling pathway --- p.131 / Chapter 4.4.4 --- Potential roles of Dab2 in myogenic differentiation revealed by transcriptome and network analysis --- p.133 / Chapter 4.4.5 --- Mef2c down-regulation may be involved in the inhibition of myogenic differentiation after Dab2 knockdown --- p.135 / Chapter 4.5 --- Summary --- p.138 / Chapter Table 4.1 --- p.141 / Chapter Table 4.2 --- p.142 / Chapter Table 4.3 --- p.143 / Chapter Table 4.4 --- p.144 / Chapter Table 4.5 --- p.147 / Chapter Table 4.6 --- p.148 / Chapter Table 4.7 --- p.149 / Chapter Figures and Legends --- p.150 / Chapter Chapter 5 --- Conclusions and discussion --- p.192 / Chapter 5.1 --- Dab2 expression in somites and skeletal muscles of mouse embryos --- p.192 / Chapter 5.1 --- Dab2 as a positive regulator for skeletal muscle development in Xenopus embryos in vivo --- p.194 / Chapter 5.3 --- Dab2 as a positive regulator of skeletal muscle development in vitro --- p.196 / Chapter 5.3.1 --- Dab2 was found to be a positive regulator of C2C12 differentiation --- p.196 / Chapter 5.3.2 --- Dab2 knockdown affected the MAPK signaling pathway --- p.198 / Chapter 5.3.3 --- Potential functions of Dab2 revealed by transcriptomeand network analysis --- p.200 / Chapter 5.3.4 --- Mef2c down-regulation was closely related to the inhibition of myogenic differentiation upon Dab2 knockdown --- p.202 / Appendix I --- p.204 / Appendix II --- p.205 / References --- p.211

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

隨著全球人口老化、普遍的久坐生活方式及外太空技術的迅速發展，肌肉廢用已成為日益嚴峻及影響廣泛的公共健康問題。 肌肉廢用可引起肌肉萎縮及肌肉收縮功能衰退，最終影響患者的日常活動能力及生活的獨立性。 此外，重新使用廢用肌肉可引起肌纖維破壞及肌肉功能進一步損失，使得肌肉萎縮問題更加惡化。 低幅高頻振動治療屬於非入侵性的生物物理治療方法，通過給予溫和的全身性機械刺激達到治療目的，被証實可有效強化肌肉功能及刺激肌纖維肥大，為進行有關低幅高頻振動應用於廢用性肌肉萎縮治療的復康研究提供了充足證據。 本研究科研假說為低幅高頻振動治療能通過調節肌纖維形態及激活具生肌能力的肌衛星細胞，以改善廢用性萎縮肌肉收縮功能及促進其康復。 本研究共分為三個部分第1部分是對大鼠懸尾模型引發後肢廢用性肌肉萎縮進行驗證（TS模型）第2部分是研究低幅高頻振動治療對肌肉收縮功能的作用第3部分是振動治療對肌纖維型態及肌肉衛星細胞的影響。 / 第1部分的研究中，十二隻6月齡雄性SD大鼠被隨機分成懸尾組 (TS, n=6)及對照組 (Nor, n=6)。 在懸尾二十八天後，大鼠的比目魚肌被收取並進行體外肌肉功能檢測。 結果顯示懸尾組的肌肉質量及肌纖維橫切面積均顯著下降 (p<0.001)，證明懸尾模型能導致廢用性肌肉萎縮。 功能檢測顯示肌肉收縮功能下降，包括抽搐峰力及最大強直力下降（p=0.011及 p<0.001）。 因此，大鼠懸尾模型可用於研究低幅高頻振動治療對肌肉重用康復過程的作用，即本研究的第2及第3部分。 / 為了驗證本研究的科研假說，七十二隻雄性SD大鼠懸尾28天誘導比目魚肌萎縮後被隨機分為振動治療組 (Vib, n=36) 及重用對照組 (Ctrl, n=36)，並於懸尾後的第7、14 及21天取比目魚肌作進一步實驗 (n=6/組/時間點)。 治療組的大鼠於懸尾後接受每星期5天、每天20分鐘的低幅高頻振動治療 (振幅: 0.6g、頻率: 35Hz)直至對應的實驗時間點，而對照組大鼠則如常在籠中活動，其餘條件均相同。 / 第2部分實驗是通過體外肌肉功能檢測系統，分析低幅高頻振動治療對廢用萎縮後重用肌肉收縮功能的作用。 實驗結果顯示，相對於在第7天時的最大強直力，對照組重用肌肉在21天的康復期間肌力增長32% 。 振動治療組中，相對於振動治療7天時的最大強直力，振動治療14天已能夠使重用肌肉得到相近 (34.6%) 力量增長（p=0.033）。 由於兩組在第七天時的最大強直力量並沒有明顯差別，故結果可證明振動治療能提高肌強直力的恢復速度。具體肌肉強直力量(以肌纖維橫切面積常化的肌肉強直力量) 亦能夠證明有關發現。 振動治療組比目魚肌的具體強直力在振動治療14天後大於同期對照組力量 (p=0.001)。 振動治療組的具體強直力在14天時已達到最高並相近於21天時的水平，但對照組於14天至21天時仍有著明顯的上升趨勢，顯示振動治療組的肌肉完全康復速度比對照組的快。 / 第3部分實驗是探討低幅高頻振動治療，對廢用性萎縮肌肉重用過程中的肌纖維肥大及肌衛星細胞激活的作用。 為標記重用過程中激活的肌衛星細胞，本部分的每一隻大鼠處死前14天在皮下植入一顆5-溴氧尿嘧啶核苷 (BrdU) 的緩釋顆粒 (0.22亳克BrdU/ 千克/ 天)。 大鼠比目魚肌在相應實驗時間點離體迅速冷凍後進行低溫橫截切片(厚度: 7微米)。 以肌球蛋白三磷酸腺苷酶染色方法把肌肉切片染色後，即可進行肌纖維的種類及形態學分析。 第21天時，振動治療組的快肌IIB 型纖維橫切面積大於對照組，表明振動治療能刺激快肌IIB 型纖維肥大 (p=0.031)。 此外，快肌IIB 型肌纖維橫切面積與肌強直力是呈正相關的，說明低幅高頻振動治療所加快的肌強直力量康復可能是通過刺激快肌IIB 型肌纖維肥大所致。 振動治療引發的慢肌纖維轉型至快肌纖維的潛能可能被重用所致的快至慢轉型所掩蓋。 從另一角度說明，肌肉重用所導致的快肌纖維轉型至慢肌纖維過程，不受振動治療的影響。 激活的肌衛星細胞則通過5-溴氧尿嘧啶核苷標記方法，以免疫熒光技術進行顯微檢測及分析。 振動治療組肌衛星細胞普遍多於對照組，刺激效果在快肌IIA 型肌纖維中表現更為明顯。 對照組廢用肌肉重用過程中，快肌IIA 型肌纖維中激活的肌衛星細胞數量呈下降趨勢，治療組的卻比對照組高並呈持續上升趨勢。 肌衛星細胞的數量亦發現與肌纖維橫切面積呈正相關。 肌衛星細胞的其中一項主要功能為肌肉修補及康復，有關實驗結果表明，振動治療可能通過激活更多衛星細胞以提高肌肉功能及刺激肌纖維肥大。 / 綜上所述，本研究探討了低震高頻振動治療對廢用性萎縮肌肉的收縮功能、康復過程及其機理的影響。 較佳的肌肉力量產生能力及較大面積的快肌IIB 型纖維，表明了振動治療可促進廢用性萎縮肌肉康復。根據快肌IIA型纖維衛星細胞數量以及活化的衛星細胞同肌纖維橫切面積之間的相關性研究結果，可以推測震動治療促進肌肉修復的可能機理是促進肌衛星細胞的活化。 本研究為低幅高頻振動治療的進一步臨床實驗及未來在快肌纖維相關的肌肉老化問題研究，提供了可靠及充分的依據。 / Muscle disuse becomes a public health issue due to increasing aged population, prevalent sedentary lifestyles and rapidly growing outer space development. It results in muscle atrophy, contractile function loss and ultimately affects the daily life activities. The pathological conditions are even worse off during reloading because of the resulting muscle fiber damages and further functional deterioration. Low-magnitude high-frequency vibration (LMHFV), a biophysical modality providing a mild, non-invasive and systemic mechanical stimulation, has been reported to improve muscle functions and stimulate muscle hypertrophy. In this study, we hypothesized that LMHFV improved the functional outcomes and recovery of disuse-induced atrophied muscle through modulating muscle fiber morphology and activating myogenic satellite cells. The study was divided into three parts: Part 1 for validation of the tail suspension hindlimbs unloading animal model (TS model); Part 2 for testifying the LMHFV effect on functional outcomes; Part 3 for the effects on fiber morphology and satellite cells. / In Part 1 study, twelve 6-month-old male Sprague Dawley (SD) rats were randomly assigned to tail suspension control group (TS, n=6) and normal control group (Nor, n=6). Rats in TS group were tail-suspended for 28 days and of the harvested soleus muscle (Sol) was subjected to the in vitro muscle functional assessment. Muscle atrophy in TS group was confirmed by the significant decrease of Sol muscle mass (Mm) and fiber cross-sectional area (FCSA) (both p<0.001). Functionally, weakening of contractile forces including peak of twitch force (Pt) and maximum tetanic force (Po) were observed in TS (p=0.011 and p<0.001 respectively). The established animal model was used to study the effects of LMHFV on muscle reloading recovery in Part 2 and 3. / To testify the hypothesis, a total of 72 male SD rats with Sol atrophy induced by 28-day TS were used for Part two (n=36) and Part three studies (n=36). In each part of the studies, the rats were randomized into LMHFV treatment group (Vib) and reloading control group (Ctrl), from which Sol were harvested at Day 7, 14 and 21 post-TS (n=6/group/timepoint). The LMHFV treatment (0.6g, 35Hz) was applied to Vib group 20min per day and 5 days per week until the endpoint while Ctrl rats were allowed free-cage movement. / In Part 2 study, the effects of LMHFV on contractile functional outcomes of reloading muscle following TS were evaluated by in vitro muscle functional test. In Ctrl group, 32% increase of Po was found at day 21 when compared with that at day 7. A similar recovery level was already achieved in Vib group by 14 days of treatment; when compared with Vib-Day7, a 34.6% increase of Po was found at day 14 (p=0.033). Specific Po (Po normalized by FCSA) in Vib was significantly larger than Ctrl at day 14 (p=0.001). Plateau of specific Po was observed at day 14 in Vib group while significant increase was observed in Ctrl group from day 14 to day 21. These findings suggested the facilitated recovery of force generating capacity in Sol by LMHFV treatment. / In Part 3 study, the effects of LMHFV on muscle fiber hypertrophy and fiber type transition during reloading as well as on muscle satellite cells (SC) activation were assessed. In order to label activated SC, a bromodeoxyuridine (BrdU) time release pellet (0.22mg BrdU/ kg body mass/ day) was subcutaneously implanted to every rat 14 days before execution. In order to evaluate the fiber morphology and fiber type transition, Sol were harvested at corresponding endpoints and cryosectioned (cross-sections at 7μm) for ATPase staining. The bromodeoxyuridine (BrdU)-labeled activated SCs were revealed on the cryosections by immunofluorescence method. Results showed that fast-twitch type IIB muscle fiber hypertrophy was stimulated by LMHFV with type IIB fiber cross-sectional area (FCSA) in Vib group significantly larger than Ctrl at day 21 (p= 0.031). Interestingly, the type IIB FCSA was positively correlated with the Po measured, which suggested the possible contribution of stimulated type IIB muscle fiber hypertrophy for improving contractile force in Vib. The potential slow-to-fast fiber type transition induced by LMHFV might be masked by reloading-induced fast-to-slow transition in Sol. In other words, the normal fiber type transition in Sol during reloading was not affected by LMHFV. In SC activation assessment, more BrdU-labeled SCs were observed in Vib group. Particularly in fast twitch type IIA muscle fibers, the SC counts were increasing throughout the treatment period. It suggested the specific stimulatory effect of LMHFV on activation of fast twitch fiber SCs. Since SC activation is important for muscle recovery, the current finding suggested the possible contribution of increased SC activation to muscle fiber hypertrophy in response to LMHFV treatment. It was in fact evident from the positive association between SC counts and muscle FCSA found in this study. / In conclusion, LMHFV was beneficial to muscle disuse recovery, as indicated from higher force generating capacity and larger fast twitch type IIB fiber FCSA. The possible mechanism was to stimulate myogenic SC activation for muscle repair, as evident from the elevated fast twitch type IIA fiber SC counts and the association of activated SC counts to fiber FCSA. This study suggests the beneficial effects of LMHFV on muscle disuse rehabilitation and also justifies the future clinical trials on rehabilitation of bed-rest patients. The profound effects of LMHFV specifically on fast-twitch fibers provided solid basis for further study on treating the loss of fast-twitch type II fibers in muscle aging (i.e. sarcopenia). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Sun, Keng Ting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 113-130). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / Thesis/Assessment Committee --- p.ii / Abstract --- p.iii / 論文摘要 --- p.vii / Publications --- p.x / Acknowledgement --- p.xi / List of Abbreviations --- p.xiii / Figure Index --- p.xvi / Table Index --- p.xvii / Chapter Chapter 1 --- Introduction and Literature Review --- p.1 / Chapter 1.1 --- Skeletal Muscle --- p.1 / Chapter 1.1.1 --- Muscle Structure and Organization --- p.1 / Chapter 1.1.2 --- Muscle Diversity --- p.5 / Chapter 1.1.3 --- Muscle Contraction and Relaxation --- p.7 / Chapter 1.1.4 --- Muscle Plasticity --- p.10 / Chapter 1.1.5 --- Muscle Mechanosensitivity and Mechanotransduction --- p.13 / Chapter 1.1.6 --- Muscle Satellite Cells --- p.16 / Chapter 1.2 --- Muscle Disuse and Rehabilitation --- p.17 / Chapter 1.2.1 --- Epidemiology and Impact of Muscle Disuse --- p.17 / Chapter 1.2.2 --- Effects of Disuse on Muscle Structure and Contractile Function --- p.18 / Chapter 1.2.3 --- Rehabilitation of Disused Muscle --- p.21 / Chapter 1.2.4 --- Countermeasures for Muscle Disuse --- p.22 / Chapter 1.2.5 --- Muscle Disuse Animal Models - Tail Suspension Hindlimbs Unloading Model --- p.23 / Chapter 1.3 --- Low-Magnitude High-Frequency Vibration Intervention --- p.24 / Chapter 1.3.1 --- Stimulatory Effects of Vibration on Muscle --- p.25 / Chapter 1.4 --- Hypothesis and Objectives --- p.27 / Chapter Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1 --- Study Design --- p.30 / Chapter 2.1.1 --- Part 1: Validation of Tail-Suspension Model --- p.32 / Chapter 2.1.2 --- Part 2: Effect of LMHFV on Functional Recovery after Tail Suspension --- p.32 / Chapter 2.1.3 --- Part 3: Effect of LMHFV on Muscle Recovery in Cellular and Histological Aspects --- p.33 / Chapter 2.2 --- Tail Suspension- Hind Limbs Unloading Model and Reloading --- p.33 / Chapter 2.3 --- Low-Magnitude High-Frequency Vibration (LMHFV) Treatment --- p.36 / Chapter 2.4 --- Part 1 and Part 2 Studies --- p.39 / Chapter 2.4.1 --- Isolation of Soleus Muscle for Functional Assessment --- p.39 / Chapter 2.4.2 --- In vitro Muscle Functional Test --- p.42 / Chapter 2.5 --- Part 3 Study --- p.50 / Chapter 2.5.1 --- Implantation of Bromodeoxyuridine (BrdU) Pellet for Satellite Cell Labeling --- p.50 / Chapter 2.5.2 --- Preparation of Soleus Muscle (Sol) for Histological and Cellular Studies --- p.51 / Chapter 2.5.3 --- Preparation of Muscle Cryosections --- p.51 / Chapter 2.5.4 --- Muscle Fiber Typing - ATPase Staining Assay --- p.54 / Chapter 2.5.5 --- Activated Satellite Cell Profiling - Immunofluorescence Imaging --- p.55 / Chapter 2.6 --- Statistical Analysis --- p.57 / Chapter Chapter 3 --- Results --- p.58 / Chapter 3.1 --- Part 1: Validation of Tail Suspension Model --- p.58 / Chapter 3.1.1 --- Morphological Assessment --- p.58 / Chapter 3.1.2 --- Functional Assessment --- p.58 / Chapter 3.2 --- Part 2: Effects of LMHFV on Functional Recovery from Tail Suspension --- p.61 / Chapter 3.2.1 --- Morphological Assessment --- p.61 / Chapter 3.2.2 --- Functional Assessment --- p.63 / Chapter 3.3 --- Part 3: Effects of LMHFV on Muscle Recovery in Cellular and Histological Aspects --- p.72 / Chapter 3.3.1 --- Muscle Fiber Typing - ATPase Staining --- p.72 / Chapter 3.3.2 --- Satellite Cells Proliferation --- p.78 / Chapter 3.4 --- Correlation of outcomes from part II and part III studies --- p.83 / Chapter Chapter 4 --- Discussion --- p.90 / Chapter 4.1 --- Beneficial Effects of LMHFV in Disuse-induced Atrophied Soleus Muscle Recovery --- p.92 / Chapter 4.2 --- Facilitated Recovery of Force Generating Capacity by LMHFV Treatment --- p.93 / Chapter 4.3 --- Biphasic Effects of LMHFV in Muscle Contraction and Relaxation Time --- p.95 / Chapter 4.4 --- Specific Hypertrophy on Type IIB Muscle Fiber Stimulated by LMHFV --- p.96 / Chapter 4.5 --- Physiological Fiber Type Adaptation Maintained in LMHFV Treatment --- p.98 / Chapter 4.6 --- Promoted SC Activation by LMHFV and its Possible Roles in Histological Improvement --- p.99 / Chapter 4.6.1 --- Possible Regulatory Mechanisms of LMHFV in Promoting SC Activation --- p.100 / Chapter 4.7 --- Disuse-induced Muscle Atrophy Successfully Developed by Tail Suspension Model --- p.102 / Chapter 4.8 --- Limitations --- p.103 / Chapter 4.8.1 --- In vitro measurement of isolated muscle contractile functions --- p.103 / Chapter 4.8.2 --- Overestimation of satellite cell (SC) counts --- p.104 / Chapter 4.9 --- Future studies --- p.105 / Chapter 4.9.1 --- Effects of LMHFV on Fast muscle --- p.106 / Chapter 4.9.2 --- Parameters of LMHFV treatment --- p.107 / Chapter 4.9.3 --- Clinical Trials --- p.107 / Chapter Chapter 5 --- Conclusions --- p.110 / Bibliography --- p.113

O treinamento físico aeróbio (TFA) foi estabelecido como uma conduta importante capaz de alterar a musculatura esquelética humana. Os microRNAs (miRs) surgiram como importantes reguladores de processos biológicos, modulando a expressão de genes pós-transcricionalmente. Os myomiRs são miRs específico do músculo esquelético, em especial o miR-206, que é necessário para uma eficiente regeneração das fibras musculares esqueléticas. No entanto, a expressão do miR-206 em resposta ao TFA, não é completamente comprendida. O objetivo do presente estudo foi determinar os padrões de expressão dos myomiRs na musculatura esquelética humana. Doze voluntários saudáveis foram biopsiados pré e pós-treinamento físico. As expressões gênicas e proteicas envolvidas na miogênese foram observadas, incluindo; PAX-7, MYF5, MYOD, MRF4, MYOG, CD31 e FSTL. Além disso, a freqüência cardíaca (FC), pressão arterial média (PAM), consumo máximo de oxigênio (VO2max), fluxo sanguineo no antebraço (FSA) e condutância vascular no antebraço (CVA), foram avaliados. Ademais, os myomiRs foram analisados por PCR em tempo real. O treinamento físico aeróbio foi realizado durante 16 semanas. Todas as variáveis foram reavaliadas após o treinamento. Os indivíduos apresentaram um aumento nas expressões dos myomiRs, em especial do miRs-206 de 93%. Estas alterações foram acompanhadas por aumento nas expressões dos genes; PAX-7, MYOD, MYF5, MFR4, MYOG e FSTL, respectivamente. No entanto, quando analisamos as expressões proteicas, houve redução na FSTL e PAX-7, de 24%, 29%, respectivamente. Além disso, em MYOD, CD31, MYOG e MHC houve aumentos de 21%, 41%, 79% e 94%, respectivamente. Ademais, houve uma diminuição na frequência cardíaca de reposuso de 12,5% e aumentos no VO2pico, FSA e CVA de 14,1%, 68%, 63%, respectivamente. Estes resultados sugerem que em indivíduos saudáveis o miRs-206 é altamente expresso após o treinamento físico aeróbio, dessa forma, modulando localmente processos miogênicos regenerativos em homens saudáveis / Endurance training (ET) has been established as an important phenotype capable of altering the human skeletal muscle. MicroRNAs (miRs) have emerged as important regulators of numerous biological processes by modulating gene expression at the post-transcriptional level. The myomiRs are particulars miRs of muscles, in special skeletal muscle-specific miR-206 that is required for efficient regeneration muscle fiber. However, the expression of myomiRs and in special miR-206 in response to ET in human skeletal muscle is not completely understood. Twelve healthy volunteers were biopsied pre and post period endurance training. Most of the biological processes involved in the transcriptional regulation were observed, including PAX-7, MYF5, MYOD, MRF4, MYOG, CD31 and FSTL, analyzed by real time PCR. Moreover, heart rate (HR), mean blood pressure (MBP), maximal exercise capacity (VO2peak) forearm blood flow (FBF) and forearm vascular conductance (FVC) were evaluated. The myomiRs levels analyzed by real-time PCR. Endurance training was performed for 16 weeks. All variables were re-assessed following completion of the training period. After endurance training, the individuals showed an increase in myomiRs, in special of 93% in human skeletal muscle in miRNA-206 levels. These alterations were accompanied by increase in PAX-7, MYOD, MYF5, MFR4, MYOG and FSTL gene expression, respectively. However, when analyzed by western blot comparing pre and post period there were reduction in FSTL of 24% and PAX-7 of 29% in protein levels, but in MYOD, CD31, MYOG and MHC there were increase of 21%, 41%, 79% and 94% in protein levels, respectively. In addition, there was a decrease in hear rate of 12.5% and increases in VO2peak of 14.1%, FBF of 68% and FVC of 63%.These results suggest that in healthy individuals the miR-206 is highly expressed after endurance training, thus modulating locally important parts in myogenic processes in humans

O autor estudou as propriedades biomecânicas dos tendões dos músculos tibial anterior e tibial posterior congelados a -20°C e a -86°C extraídos de cadáveres humanos frescos. Foram realizados ensaios mecânicos de tração até a ruptura e determinadas as seguintes propriedades: resistência máxima, coeficiente de rigidez, módulo de elasticidade e alongamento máximo relativo. Os dados obtidos foram comparados aos existentes na literatura relativos ao ligamento cruzado anterior, ligamento da patela e aos tendões dos músculos grácil e semitendíneo / The author studied the mechanical properties of the anterior and posterior tibialis muscle tendons frozen at -20°C and -86°C obtained from fresh-frozen human cadavers. The tendons were submitted to axial traction until failure and the following properties were determined: ultimate load, stiffness, modulus of elasticity and relative strain. Data obtained were compared to those from the literature related to the anterior cruciate ligament, patellar tendon, gracilis and semitendinous tendons

O autor estudou as propriedades biomecânicas dos tendões dos músculos tibial anterior e tibial posterior congelados a -20°C e a -86°C extraídos de cadáveres humanos frescos. Foram realizados ensaios mecânicos de tração até a ruptura e determinadas as seguintes propriedades: resistência máxima, coeficiente de rigidez, módulo de elasticidade e alongamento máximo relativo. Os dados obtidos foram comparados aos existentes na literatura relativos ao ligamento cruzado anterior, ligamento da patela e aos tendões dos músculos grácil e semitendíneo / The author studied the mechanical properties of the anterior and posterior tibialis muscle tendons frozen at -20°C and -86°C obtained from fresh-frozen human cadavers. The tendons were submitted to axial traction until failure and the following properties were determined: ultimate load, stiffness, modulus of elasticity and relative strain. Data obtained were compared to those from the literature related to the anterior cruciate ligament, patellar tendon, gracilis and semitendinous tendons

O treinamento físico aeróbio (TFA) foi estabelecido como uma conduta importante capaz de alterar a musculatura esquelética humana. Os microRNAs (miRs) surgiram como importantes reguladores de processos biológicos, modulando a expressão de genes pós-transcricionalmente. Os myomiRs são miRs específico do músculo esquelético, em especial o miR-206, que é necessário para uma eficiente regeneração das fibras musculares esqueléticas. No entanto, a expressão do miR-206 em resposta ao TFA, não é completamente comprendida. O objetivo do presente estudo foi determinar os padrões de expressão dos myomiRs na musculatura esquelética humana. Doze voluntários saudáveis foram biopsiados pré e pós-treinamento físico. As expressões gênicas e proteicas envolvidas na miogênese foram observadas, incluindo; PAX-7, MYF5, MYOD, MRF4, MYOG, CD31 e FSTL. Além disso, a freqüência cardíaca (FC), pressão arterial média (PAM), consumo máximo de oxigênio (VO2max), fluxo sanguineo no antebraço (FSA) e condutância vascular no antebraço (CVA), foram avaliados. Ademais, os myomiRs foram analisados por PCR em tempo real. O treinamento físico aeróbio foi realizado durante 16 semanas. Todas as variáveis foram reavaliadas após o treinamento. Os indivíduos apresentaram um aumento nas expressões dos myomiRs, em especial do miRs-206 de 93%. Estas alterações foram acompanhadas por aumento nas expressões dos genes; PAX-7, MYOD, MYF5, MFR4, MYOG e FSTL, respectivamente. No entanto, quando analisamos as expressões proteicas, houve redução na FSTL e PAX-7, de 24%, 29%, respectivamente. Além disso, em MYOD, CD31, MYOG e MHC houve aumentos de 21%, 41%, 79% e 94%, respectivamente. Ademais, houve uma diminuição na frequência cardíaca de reposuso de 12,5% e aumentos no VO2pico, FSA e CVA de 14,1%, 68%, 63%, respectivamente. Estes resultados sugerem que em indivíduos saudáveis o miRs-206 é altamente expresso após o treinamento físico aeróbio, dessa forma, modulando localmente processos miogênicos regenerativos em homens saudáveis / Endurance training (ET) has been established as an important phenotype capable of altering the human skeletal muscle. MicroRNAs (miRs) have emerged as important regulators of numerous biological processes by modulating gene expression at the post-transcriptional level. The myomiRs are particulars miRs of muscles, in special skeletal muscle-specific miR-206 that is required for efficient regeneration muscle fiber. However, the expression of myomiRs and in special miR-206 in response to ET in human skeletal muscle is not completely understood. Twelve healthy volunteers were biopsied pre and post period endurance training. Most of the biological processes involved in the transcriptional regulation were observed, including PAX-7, MYF5, MYOD, MRF4, MYOG, CD31 and FSTL, analyzed by real time PCR. Moreover, heart rate (HR), mean blood pressure (MBP), maximal exercise capacity (VO2peak) forearm blood flow (FBF) and forearm vascular conductance (FVC) were evaluated. The myomiRs levels analyzed by real-time PCR. Endurance training was performed for 16 weeks. All variables were re-assessed following completion of the training period. After endurance training, the individuals showed an increase in myomiRs, in special of 93% in human skeletal muscle in miRNA-206 levels. These alterations were accompanied by increase in PAX-7, MYOD, MYF5, MFR4, MYOG and FSTL gene expression, respectively. However, when analyzed by western blot comparing pre and post period there were reduction in FSTL of 24% and PAX-7 of 29% in protein levels, but in MYOD, CD31, MYOG and MHC there were increase of 21%, 41%, 79% and 94% in protein levels, respectively. In addition, there was a decrease in hear rate of 12.5% and increases in VO2peak of 14.1%, FBF of 68% and FVC of 63%.These results suggest that in healthy individuals the miR-206 is highly expressed after endurance training, thus modulating locally important parts in myogenic processes in humans