【作者】 辛娟；

【导师】 王远亮；

【作者基本信息】 重庆大学 ， 生物制药工程， 2014， 博士

【摘要】 胰岛素样生长因子I（IGFI）是含有70个氨基酸的单链多肽，为骨基质中含量最丰富的生长因子，对骨代谢有着重要的调节作用。IGFI的E结构域发生选择性剪接产生3种异构体：IGFIEa，IGFIEb（大鼠）和IGFIEc（人类）。其中IGFIEb和IGFIEc一般在肌肉或成骨细胞受到机械刺激或损伤时才得以表达，因此也称作力生长因子（mechano-growth factor，MGF）。分子结构表征发现MGF羧基端E结构域含有24个氨基酸组成的短肽（也称MGF-Ct24E），正是由于MGF-Ct24E才导致MGF功能上与IGFI和IGFIEa不同。MGF自1996年被Goldspink研究团队发现以来，许多研究证实MGF是一种重要的局部组织修复因子。当机体受到机械刺激或组织受损后，MGF得以表达并诱导体内卫星细胞增殖和分化。MGF-Ct24E与MGF功能相似，也具有诱导成肌细胞增殖和神经保护作用，提示MGF-Ct24E一旦从MGF分离出来，其本身固有的生物活性如组织保护、修复和适应等将得以发挥。我们前期研究发现MGF以自分泌或旁分泌的方式积极响应骨细胞（或组织）损伤修复，在兔骨折模型中MGF-Ct24E通过促进成骨细胞增殖和血管新生加速骨折愈合。基于本课题组对MGF-Ct24E的前期研究成果，本研究试图从细胞水平和分子水平探索MGF-Ct24E对骨形成与骨吸收的影响，以便于寻求确切的靶基因和信息通路，为其将来临床应用于骨修复提供可能。本论文主要研究内容和结果如下：（1）MGF-Ct24E对成骨细胞的功能调控及转录组分析：①通过检测MGF-Ct24E对新生大鼠颅骨成骨细胞增殖、周期、分化及矿化的影响，结果表明MGF-Ct24E促进早期成骨细胞增殖延迟终末分化，促进晚期成骨细胞分化和矿化，最终促进骨形成。②利用基因芯片检测MGF-Ct24E对成骨细胞骨形成过程中基因表达的影响，结果表明MGF-Ct24E能有效调节成骨细胞基因的表达，差异表达基因总共2054个，其中上调1179个，下调基因875个；qPCR检测结果显示，所选择的12个差异表达基因（IL1RN、FIGF、CXCL12、ZW10、NOTHCH2、TWIST2、ALPL、IGFBF5、FGFR2、COMP、COL2A1和IGFI）的差异表达情况与基因芯片实验结果基本一致，验证了基因芯片实验结果的可靠性。③应用GoSurfer软件和在线软件DAVID对MGF-Ct24E调控成骨细胞差异表达的基因进行GO分析，发现富集指数最高的差异表达基因定位于细胞内部、具有酶结合功能、参与细胞周期进程。Pathway分析发现这些差异表达基因主要参与调控粘着斑通路、细胞周期通路、肌动蛋白细胞骨架调控通路、缝隙连接通路、MAPK信号通路、黏着连接通路和胰岛素信号通路等67个信号通路。④对差异表达基因GO生物学过程第3~8层次的节点和Term进行仔细筛选，总共得到95个成骨细胞相关基因。GO生物学过程分析发现，这些基因主要富集的功能分类包括成细胞周期、骨细胞分化负调控、骨化负调控、软骨骨化、骨化调控、骨发育、骨化、骨矿化、血管生成、成骨细胞分化、骨骼系统发育、细胞正在调控、细胞增殖、创伤反应等。Pathway分析发现它们主要参与调控黏着斑、肌动蛋白细胞骨架调控通路、细胞外基质受体作用通路、细胞因子及其受体的相互作用通路、MAPK信号通路、黏着连接通路、TGF-β信号通路、白细胞跨内皮迁移信号通路和Wnt信号通路等多9个通路。（2）MGF-Ct24E对破骨细胞的功能调控及转录组分析：①通过检测MGF-Ct24E对破骨细胞增殖、周期、分化以及骨吸收功能的影响，结果显示：MGF-Ct24E不仅抑制破骨前体细胞的增殖，还抑制破骨细胞的形成和分化，且具有浓度依赖性；此外，MGF-Ct24E对破骨细胞的骨吸收功能也存在较为显著的抑制作用。②利用基因芯片检测MGF-Ct24E对破骨细胞分化过程中基因表达的调控，结果表明MGF-Ct24E能有效调节破骨细胞基因的表达，差异表达基因总共1712个，其中上调717个，下调895个；qPCR检测结果显示所选择的8差异表达基因（AKT2、SPP1、NF1、ITGB5、NFATc1、TRAF6、MMP9和CTSK）的差异表达情况与基因芯片实验结果基本一致，验证了基因芯片实验结果的可靠性。③应用GoSurfer软件和在线软件DAVID对MGF-Ct24E调控破骨细胞差异表达的基因进行GO分析，结果显示富集指数最高的基因定位于细胞内部、具有核苷酸结合功能、参与细胞周期进程。Pathway分析发现MGF-Ct24E调控的差异表达基因主要涉及细胞周期、DNA复制、错配修复、核苷酸切除修复、同源重组、碱基切除修复、嘧啶代谢、TGF-β信号通路、 p53信号通路、硫酸乙酰肝素生物合成、孕激素介导的卵母细胞成熟、戊糖磷酸途径、谷胱甘肽代谢通路、Wnt信号通路、嘌呤代谢通路、卵母细胞减数分裂、血管内皮生长因子信号通路、肌动蛋白细胞骨架调控和白细胞跨内皮迁移等20个信号通路。④对差异表达基因GO生物学过程第3~8层次的节点和Term进行仔细筛选，得到79个与破骨细胞密切相关联的差异表达基因，进一步生物学过程GO分析发现，上调基因主要富集的功能包括细胞分化，器官发育，骨骼发育，骨矿化，骨重塑，成骨细胞分化的；下调基因主要富集的功能包括发育过程，多细胞有机体的发育，系统发育、骨化调控、破骨细胞分化和骨骼发育等。Pathway分析显示这79个基因主要参与调控粘着斑、细胞因子及其受体的相互作用、MAPK信号通路、血管内皮生长因子信号通路、肌动蛋白细胞骨架的调控、白细胞跨内皮迁移、趋化因子信号转导通路、紧密连接、TGF-β信号通路和Wnt信号通路等10个信号通路。上述结果提示，MGF-Ct24E通过调控细胞周期进程和调控粘着斑、MAPK信号通路、肌动蛋白细胞骨架调控、TGF-β信号通路、白细胞跨内皮迁移、Wnt信号通路等6条信号通路共同调控体外骨形成和骨吸收。更多还原

【Abstract】 Insulin-like growth factor I (IGFI), a single-chain polypeptide of70amino acids, isan important growth factor of bone matrix and has important roles in regulating bonemetabolism. Alternate splicing within the E domain of the IGFI gene produces threedistinct isoforms: IGFIEa, IGFIEb (in rat), and IGFIEc (in human). IGFIEb and IGFIEcare also known as mechano-growth factor (MGF) because they are often discovered inskeletal muscle or osteoblasts subjected to stretch or injury. MGF was discovered by theresearch group of Goldspink in1996. Since then many studies have confirmed thatMGF is an important local tissue repair factor. Structural characterization of MGFidentified a unique C-terminal E domain contains24amino acids (MGF-Ct24E) thatendows MGF with functions distinct from mature IGF-I and IGF-IEa. Previous studieshave demonstrated that MGF is a key factor involved in mediating mechanicalstimulation or tissue injury induced satellite cell proliferation and differentiation in vivo.Consistent with the function of MGF, MGF-Ct24E induces proliferation of myoblastsand has a strong neuroprotective effect in brain ischemia, suggesting that MGF-Ct24Eitself, once cleaved from the mature protein, has intrinsic biological activities: tissueprotection, repair and adaptation. Our previous studies even found that MGF wasinitially shown to participate in the response to injury in bone cells (or tissues) in anautocrine/paracrine manner, suggesting a potentially positive role in bone remodeling.Subsequently, MGF-Ct24E has been shown to accelerate fracture healing in a rabbitmodel by enhancing osteoblasts proliferation. The mechanisms of MGF-Ct24E functionin the regulation of bone growth, development, and injury repair are, however, far fromunderstood. Only our laboratory studies show that the MGF-Ct24E appears to stimulateproangiogenic activities in human vascular endothelial cells. According to the previousresearch results of our team on MGF-Ct24E, the studies are undertaken to investigatethe roles and mechanism of MGF-Ct24E on bone formation and absorption, in order todevelop therapies for bone repair using MGF-Ct24E. The main works and conclusionsare included as follows:(1) Functional and transcriptomic analysis of the regulation of osteoblasts byMGF-Ct24E.①The roles of MGF-Ct24E in proliferation, differentiation, and mineralization ofrat calvarial osteoblasts were investigated. And the results showed that MGF-Ct24E promotes proliferation while delaying terminal differentiation of osteoblasts in prophase,conceivably to allow the accumulation of more cells to prepare for differentiation andmineralization in more advanced stages of bone formation.②Microarray was carried to determine the molecular mechanisms of boneformation regulated by MGF-Ct24E. The result showed that2054genes in osteoblstswere regulated. Among them,1179genes were found up-regulated while875geneswere down-regulated. Quantitative real-time PCR experiment results showed that theexpression levels of selected twelve differentially expressed genes (IL1RN, FIGF,CXCL12, ZW10, NOTHCH2, TWIST2, ALPL, IGFBF5, FGFR2, COMP, COL2A1andIGFI) were generally in accordance with the microarray data, validating the reliabilityof microarray experiment results.③The GoSurfer software and online software DAVID were applied to GO andKEGG pathway analysis of the screened differentially expressed genes in osteoblastsregulated by MGF-Ct24E. GO analysis of the2054differential expressed genes inbiological process，molecular function and cellular component, the results showed thatthe main enrichment of functional classification were cell cycle process, enzymebinding and intracellular part. KEGG pathway analysis results showed that67pathwayswere modulated by MGF-Ct24E, such as focal adhesion, cell cycle, regulation of actincytoskeleton, gap junction, MAPK signaling pathway, adheres junction, and insulinsignaling pathway, and so on.④Based on GO analysis,95osteogenesis-related genes were screened out from the2054differential expressed genes in osteoblasts. The biological process of GO analysisresults showed that the main enrichment of functional classification of them were asfollowing: cell proliferating, cell cycle, bone development, angiogenesis, regulation ofossification, cartilage ossification, bone mineralization, ossification and so on. KEGGpathway analysis results showed that nine pathways associated with focal adhesion,regulation of actin cytoskeleton, ECM-receptor interaction, cytokine-cytokine receptorinteraction, MAPK signaling pathway, adherens junction, TGF-beta signaling pathway,leukocyte transendothelial migrationk, Wnt signaling pathway and so on weremodulated by MGF-Ct24E.(2) Functional and transcriptomic analysis of the regulation of osteoclasts byMGF-Ct24E.①The roles of MGF-Ct24E in the proliferation, differentiation, and mineralizationof osteoclasts were investigated. The results show that MGF-Ct24E not only inhibits the proliferation, but also inhibits the formation and differentiation of osteoclasts inconcentration-dependent manner. In addition, MGF-Ct24E also shows a significantinhibitory effect on bone absorbing.②Microarray was carried to determine the molecular mechanisms of boneabsorbing regulated by MGF-Ct24E. The result shows that1712genes in osteoclastswere regulated. Among them, about717genes were found up-regulated while895genes were down-regulated. Quantitative real-time PCR experiment results showed thatthe expression levels of selected eight differentially expressed genes (AKT2, SPP1, NF1,ITGB5, NFATc1, TRAF6, MMP9and CTSK) were generally in accordance with themicroarray data, validating the reliability of microarray experiment results.③The GoSurfer software and online software DAVID were applied to GO andKEGG pathway analysis of the screened differentially expressed genes in osteoclastsregulated by MGF-Ct24E. GO analysis of the1712differential expressed genes inbiological process，molecular function and cellular component, the results showed thatthe main enrichment of functional classification were cell cycle process, enzymebinding and intracellular part. KEGG pathway analysis results showed that20pathwayswere modulated by MGF-Ct24E, such as cell cycle，DNA replication，mismatch repair,nucleotide excision repair, homologous recombination, TGF-beta signaling pathway,p53signaling pathway, VEGF signaling pathway, and so on.④Based on GO analysis,79osteoclastogenesis-related genes were screened outfrom the1712differential expressed genes in osteoclasts. The biological process of GOanalysis results showed that the main enrichment of functional classification of themwere as following: cell differentiation, organ development, bone development, bonemineralization, bone remodeling, osteoblast differentiation, developmental process,multicellular organismal development, system development, regulation of ossification,osteoclast differentiation, bone development and so on. KEGG pathway analysis resultsshowed that ten pathways associated with focal adhesion, cytokine-cytokine receptorinteraction, MAPK signaling pathway, VEGF signaling pathway，regulation of actincytoskeleton, leukocyte transendothelial migration, chemokine signaling pathway, tightjunction, TGF-beta signaling pathway, Wnt signaling pathway and so on weremodulated by MGF-Ct24E.In summary, all the results suggest that MGF-Ct24E has a remarkable ability toincrease bone formation and inhibit bone resorption through regulating genes involvedin cell cycle process and six signaling pathways, such as focal adhesion, MAPK signaling pathway, regulation of actin cytoskeleton, TGF-beta signaling pathway,leukocyte transendothelial migration, Wnt signaling pathway and so on.更多还原