外力作为信号诱导基因的选择性剪接与力生长因子表达

许多种类的细胞都响应力信号,人们将这些细胞称为力效应细胞(mechanocyte).应力可引起细胞在基因水平或表达水平的调控,其中胰岛素样生长因子Ⅰ(insulin-like growth factor-Ⅰ,IGFⅠ)是力学敏感因子.对骨骼肌的长期拉伸实验发现,IGF-Ⅰ不仅表达量受到拉伸刺激的调控,而且存在多种变异体形式,其中一种对力刺激敏感,只在拉伸作用下产生,命名为力生长因子(mechano growth factor,MGF).进一步研究发现,MGF能激活卫星细胞、促进成肌细胞增殖,在治疗肌损失、预防心肌损伤和修复神经损伤等方面有重要的作用.机械拉伸也可以使成骨细胞表达MGF,研究表明,对成骨细胞施加应变为15%的周期性拉伸刺激,细胞的IGFⅠ表达量增加,同时表达MGF剪接变异体.对MGF的深入研究可望在疾病治疗和组织工程修复领域取得广泛的应用.     

IGF-1剪接变构体:力生长因子

力刺激与基因表达之间的联系是生理学研究中一个新的重要领域。从基因表达水平研究力学信号的影响有着特殊的重要意义,生物有机体的存在是基因表达的结果,而力在生命活动的整个过程发挥着作用。在骨骼肌中,力刺激诱导胰岛素样生长因子-1(IGF-1)发生选择性剪接,产生能够激活卫星细胞而使细胞增殖的力生长因子(mechano growth factor,MGF),以及能促使细胞分化而形成肌管的肌肉型IGF-1(IGF-1Ea),这两种自分泌的局部生长因子在肌肉修复与再生中起着重要作用。     

MGF对人软骨终板干细胞增殖和迁移的影响

力生长因子(mechano growth factor,MGF)在多种组织均有表达,能促进力效应细胞的增殖和迁移。为研究MGF对人软骨终板干细胞(cartilage endplate derived stem cells,CESCs)增殖和迁移的影响,该研究采用CCK-8检测法、Transwell实验、蛋白免疫印迹法(Western blot)分别对CESCs的增殖、迁移以及Erk磷酸化表达水平进行检测。结果显示,MGF能促进CESCs的增殖和迁移,且效应具有剂量依赖性,当浓度为4.5 ng/m L时MGF促增殖和迁移的效应最大(P0.05)。给予细胞外调节蛋白激酶(extracellular regulated protein kinases,Erk)抑制剂PD98059时,MGF促增殖效应显著下降(P0.001);给予胰岛素样生长因子-1受体(insulin-like growth factor-1 receptor,IGF-1R)抑制剂PQ401时,MGF促迁移效应也显著下降(P0.001)。Western blot检测结果显示,给予MGF后,CESCs的Erk磷酸化表达水平显著升高(P0.001),但IGF-1R抑制剂PQ401能抑制Erk磷酸化的表达(P0.001)。该研究表明,MGF具有促CESCs增殖和迁移的效应,该效应依赖于Erk的磷酸化表达,且IGF-1R在MGF促迁移的效应中扮演重要角色。     

力生长因子及其E肽对成骨细胞分化的影响

力生长因子(mechano growth factor,MGF)是新近发现的一种生长因子,由Igf-1基因剪接变异产生,拉伸刺激会促使成骨细胞表达力生长因子.比较分析了MGF及其羧基端E肽(MGF-Ct24E)对成骨细胞前体细胞MC3T3-E1分化的影响.结果显示:MGF和MGF-Ct24E具有显著激活胞外信号调节激酶1/2(Erk1/2)的作用,并降低了成骨细胞碱性磷酸酶、Ⅰ型胶原的表达,促进了骨桥蛋白(OPN)的表达,减少了核心绑定因子(corebinding factor1,Cbfα-1)的核转运量,对成骨细胞的分化具有延迟效应,这种效应通过抑制剂PD98059抑制Erk1/2的活化得到逆转;MGF还能显著激活磷脂酰肌醇3-激酶信号通路中的蛋白激酶B(Akt),该活化作用对成骨细胞分化是必需的,钙沉积分析显示长期培养下的细胞MGF促进了矿化节结的形成.这些结果说明,MGF-Ct24E对成骨细胞的分化具有抑制作用,这种作用与Erk1/2的活化有关,MGF因为包含E肽和IGF-1部分,能分别激活Erk1/2和Akt,因此对成骨细胞分化表现出双重效用,在成骨细胞分化早期,具有一定的延迟效应,而在分化晚期对成骨...     

IGF-1在治疗骨骼肌损伤中的应用潜能及其相关机制

胰岛素样生长因子-1(IGF-1)作为一种生长因子,在骨骼肌损伤后治疗过程中发挥重要的作用。局部注射外源性IGF-1或通过转基因技术使损伤处骨骼肌细胞过表达IGF-1,均能促进损伤骨骼肌再生。IGF-1促进损伤骨骼肌修复的机制可能与如下因素有关:激活骨骼肌卫星细胞,促进成肌细胞增殖与分化,促进蛋白质合成并抑制蛋白分解;抑制骨骼肌炎症反应,并调节巨噬细胞极化;抑制细胞表达胶原蛋白,减少骨骼肌纤维化;作为一种潜在的神经营养因子和生血管因子,促进损伤后的神经和血管再生。因此,IGF-1在骨骼肌损伤后的治疗中具有重要的应用前景。     

生长激素基因的克隆和表达及其对贵州地方黄牛骨骼肌细胞增殖的影响

本研究旨在探究生长激素(Growth hormone,GH)对贵州地方黄牛骨骼肌细胞增殖的表达调控,探明超表达GH基因对骨骼肌细胞增殖的影响。首先利用反转录PCR扩增黄牛GH基因的蛋白质编码区(Coding sequence,CDS),将其克隆至pUCM-T载体,并连接转化构建超表达载体pEGFP-N3-GH。同时使用实时荧光定量PCR检测GH基因在贵州地方黄牛骨骼肌相关组织(腰大肌与背最长肌)中的表达情况,然后培养牛原代骨骼肌细胞并进行鉴定,并将GH基因超表达载体导入细胞以研究GH基因对牛骨骼肌细胞增殖以及骨骼肌生长发育相关因子胰岛素样生长因子-1(Insulinlikegrowthfactor1,IGF-1)与胰岛素样生长因子-2(Insulinlikegrowth factor 2,IGF-2)基因表达的影响。实时荧光定量PCR结果显示,GH基因在贵州地方黄牛腰大肌中的表达量均高于背最长肌,其中在关岭牛和威宁牛腰大肌中的表达量显著高于背最长肌(P0.05)。细胞转染及增殖结果表明,相比于pEGFP-N3,pEGFP-N3-GH能极显著提高GH与IGF-1、IGF-2基因在骨骼肌细胞中的表达量,且在被检测的4个时期(6 h、12 h、24 h、48 h),超表达GH基因组也能够极显著地提高骨骼肌细胞的增殖速率(P0.01)。结果提示,GH基因可促进贵州地方黄牛骨骼肌细胞的增殖,对其具有正向的调控作用,这为进一步探究GH基因对贵州地方黄牛生长发育的影响机制奠定基础。     

Src蛋白激酶活性的调节机制

Src蛋白激酶在人类多种肿瘤细胞中被激活并在肿瘤发生、发展过程中起重要作用.Src活性的调节主要包括共价修饰、异构调节,但基因突变和其他一些方式也可以调节其活性.Src共价修饰主要是磷酸化,Tyr530、Tyr419、Thr34、Thr46、Ser72、Tyr138和Tyr213等都是Src的磷酸化位点,其中Tyr530位点和Tyr419位点是Src最重要的磷酸化位点.异构调节包括SH3、SH2等区域结合的调节,分别涉及黏着斑激酶(focal adhesion kinase,FAK)、孕酮受体(progesterone receptor,PR)、雌激素受体(estrogen receptor,ER)、雄激素受体(androgen receptor,AR)、P130Cas、血小板源生长因子(the platelet-derived growth factor,PDGF)、血小板衍生生长因子受体(platelet-derived growth factor receptor,PDGFR)、表皮生长因子受体(epidermal growth factor receptor,EGFR,HER1/erb B1)、人类表皮生长因子受体2(human epidermal growth factor receptor-2,ERBB2/HER2/NEU)、胰岛素样生长因子1受体(insulin-like growth factor-1 receptor,IGF-1R)、纤维母细胞生长因子受体1(fibroblast growth factor receptor,FGFR1)、肝细胞生长因子受体(hepatocyte growth factor receptor c-Met)、人类1型T细胞白血病病毒编码的辅助蛋白p13、HIV-1毒力因子Nef和Sin.本文就Src蛋白激酶的调节机制作一简要综述.     

EGF、bFGF和PHGF对大鼠骨骼肌卫星细胞增殖的影响

用MTT、流式细胞技术、溴脱氧核甘尿嘧啶(bromodeoxyuridine,BrdU)掺入法及免疫细胞化学检测增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的方法探讨了表皮生长因子(epidermal growth factor,EGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)及促肝细胞生长因子(hepatocyte growth-promoting factor,PHGF)对大鼠骨骼肌卫星细胞增殖的影响。结果表明bFGF、PHGF对骨骼肌卫星细胞有较强的促增殖作用,且两者之间无差别,bFGF最佳作用浓度为5μg／L,PHGF最佳作用浓度为10μg／ml。与其他生长因子相比,PHGF价格低廉易于获取,适宜推广。EGF增殖作用不明显。     

拉伸刺激下MGF在成骨细胞中的表达及亚细胞定位分析

力生长因子（mechano-growth factor,MGF）是在成肌细胞中发现的一种对拉伸刺激敏感的生长因子,该生长因子也可能在其他的力效应细胞中由力学刺激产生.通过设计细胞拉伸装置,对成骨细胞施加不同时段的周期性动态拉伸刺激.定量分析mRNA和蛋白质表达显示,周期性拉伸刺激下成骨细胞中的MGF mRNA和蛋白质水平快速提高,mRNA的水平在加载6h达到最高峰,与静态对照组相比提高5倍,而MGF的蛋白表达需要加载12h达到最高,与对照相比提高了5.2倍,随后二者分别降低,在24h达到本底水平.荧光免疫细胞化学技术检验MGF在细胞中的分布发现,MGF具有核分布的特点.因此得出结论,周期性拉伸刺激能刺激成骨细胞快速表达,MGF核分布的特点暗示MGF可能是一种自分泌作用因子.     

IGF系统的生物学功能及其与肿瘤的关系

胰岛素样生长因子（insulin-like growth factor,IGF)是体内重要的生长因子。它对组织细胞的增殖。分化,凋亡,机体的生长发育及肿瘤的发生发展起重要的调节作用。IGF系统的组成包括;IGF-Ⅰ,IGF-Ⅱ,IGF-ⅠR,IGF-ⅡR,IGFBP家族,IGF促增殖,促生长的活性主要由IGF-IR介导;IGF-ⅡR的主要作用是清除游离IGF-Ⅱ,调节IGF-Ⅱ的水平,IGFBP1-6是IGFs活性的调节因子。近年的研究表明它们也有不依赖IGF的生物活性。     

Stimulation of mechano-growth factor expression by myofibrillar proteins in murine myoblasts and myotubes

Mechano-growth factor (MGF) is a product of alternative splicing of the insulin-like growth factor 1 (IGF-1) mRNA. MGF is known to stimulate myoblast proliferation and to protect neurons and cardiomyocytes from apoptosis. MGF expression is dramatically increased in response to mechanical stimuli and tissue damage. The mechanisms of induction of MGF expression are as yet imperfectly understood. There is certain evidence that some protein factors able to stimulate MGF synthesis in normal myoblasts are released from damaged muscle. This study was undertaken to explore the nature of these protein inductors of MGF expression and to investigate the mechanism of their action. We report here that myofibrillar fraction of skeletal muscle homogenate activated MGF expression in murine myoblasts and myotubes in culture. The expression of another splice form of IGF-1 gene, IGF-1Ea, was also stimulated by myofibrils. Three myofibrillar proteins able to stimulate MGF synthesis were isolated. These proteins were identified by MALDI and immunoblotting as myomesin, myosin-binding protein C, and titin. The activation of MGF expression was associated with the increase of cAMP level in the cells. Inhibitor of adenylyl cyclase dideoxyadenosine arrested stimulation of MGF synthesis by all three myofibrillar proteins.     

Insulin-Like Growth Factor I (IGF-1) Ec/Mechano Growth Factor – A Splice Variant of IGF-1 within the Growth Plate

Human insulin-like growth factor 1 Ec (IGF-1Ec), also called mechano growth factor (MGF), is a splice variant of insulin-like growth factor 1 (IGF-1), which has been shown in vitro as well as in vivo to induce growth and hypertrophy in mechanically stimulated or damaged muscle. Growth, hypertrophy and responses to mechanical stimulation are important reactions of cartilaginous tissues, especially those in growth plates. Therefore, we wanted to ascertain if MGF is expressed in growth plate cartilage and if it influences proliferation of chondrocytes, as it does in musculoskeletal tissues. MGF expression was analyzed in growth plate and control tissue samples from piglets aged 3 to 6 weeks. Furthermore, growth plate chondrocyte cell culture was used to evaluate the effects of the MGF peptide on proliferation. We showed that MGF is expressed in considerable amounts in the tissues evaluated. We found the MGF peptide to be primarily located in the cytoplasm, and in some instances, it was also found in the nucleus of the cells. Addition of MGF peptides was not associated with growth plate chondrocyte proliferation.     

The E-domain region of mechano-growth factor inhibits cellular apoptosis and preserves cardiac function during myocardial infarction

Insulin-like growth factor-1 (IGF-1) isoforms are expressed via alternative splicing. Expression of the minor isoform IGF-1Eb [also known as mechano-growth factor (MGF)] is responsive to cell stress. Since IGF-1 isoforms differ in their E-domain regions, we are interested in determining the biological function of the MGF E-domain. To do so, a synthetic peptide analog was used to gain mechanistic insight into the actions of the E-domain. Treatment of H9c2 cells indicated a rapid cellular uptake mechanism that did not involve IGF-1 receptor activation but resulted in a nuclear localization. Peptide treatment inhibited the intrinsic apoptotic pathway in H9c2 cells subjected to cell stress with sorbitol by preventing the collapse of the mitochondrial membrane potential and inhibition of caspase-3 activation. Therefore, we administered the peptide at the time of myocardial infarction (MI) in mice. At 2 weeks post-MI cardiac function, gene expression and cell death were assayed. A significant decline in both systolic and diastolic function was evident in untreated mice based on PV loop analysis. Delivery of the E-peptide ameliorated the decline in function and resulted in significant preservation of cardiac contractility. Associated with these changes were an inhibition of pathologic hypertrophy and significantly fewer apoptotic nuclei in the viable myocardium of E-peptide-treated mice post-MI. We conclude that administration of the MGF E-domain peptide may provide a means of modulating local tissue IGF-1 autocrine/paracrine actions to preserve cardiac function, prevent cell death, and pathologic remodeling in the heart.     

Mechano growth factor (MGF) promotes proliferation and inhibits differentiation of porcine satellite cells (PSCs) by down-regulation of key myogenic transcriptional factors

Porcine satellite cells represent an ideal model system for studying the cellular and molecular basis regulating myogenic stem cell proliferation and differentiation and for exploring the experimental conditions for myoblast transplantation. Here, we investigated the effects of mechano growth factor (MGF), a spliced variant of the IGF-1 gene, on porcine satellite cells. We show that MGF potently stimulated proliferation while inhibited differentiation of porcine satellite cells. MGF-treatment acutely down-regulates the expression of myogenic determination factor (MyoD) and the cyclin-dependent kinase inhibitor p21. MGF-treatment also markedly reduced the overall expression of cyclin B1 and key factors of the myogenic regulatory and myocyte enhancer families, including Myogenein and MEF2A. Taken together, the gene expression data from MGF-treated porcine satellite cells are in favor of a molecular model in which MGF inhibits porcine satellite cell differentiation by down-regulating either the activity or expression of MyoD, which, in turn, suppresses the expression of key genes required for cell cycle progression and differentiation, such as p21, Myogenin, and MEF2. Overall, our findings are in support of the previous suggestion that MGF may be used in vivo and in vitro to promote proliferation of myogenic stem cells to prevent and treat age-related muscle degenerative diseases.     

3-D in vitro model of early skeletal muscle development

An understanding of the mechanical and mechano-molecular responses that occur during the differentiation of mouse C2C12 [corrected] myoblasts in 3-D culture is critical for understanding growth, which is important for progress towards producing a tissue-engineered muscle construct. We have established the main differences in force generation between skeletal myoblasts, dermal fibroblasts, and smooth muscle cells in a 3-D culture model in which cells contract a collagen gel construct. This model was developed to provide a reproducible 3-D muscle organoid in which differences in force generation could be measured, as the skeletal myoblasts fused to form myotubes within a collagen gel. Maintenance of the 3-D culture under sustained uni-axial tension, was found to promote fusion of myoblasts to form aligned multi-nucleate myotubes. Gene expression of both Insulin Like Growth Factor (IGF-1 Ea) and an isoform of IGF-1 Ea, Mechano-growth factor (IGF-1 Eb, also termed MGF), was monitored in this differentiating collagen construct over the time course of fusion and maturation (0-7 days). This identified a transient surge in both IGF-1 and MGF expression on day 3 of the developing construct. This peak of IGF-1 and MGF expression, just prior to differentiation, was consistent with the idea that IGF-1 stimulates differentiation through a Myogenin pathway [Florini et al., 1991: Mol. Endocrinol. 5:718-724]. MGF gene expression was increased 77-fold on day 3, compared to a 36-fold increase with IGF-1 on day 3. This indicates an important role for MGF in either differentiation or, more likely, a response to mechanical or tensional cues.     

IGF-1基因产物的结构和功能多样化

胰岛素样生长因子-1(IGF-1)基因包含6个外显子,具有转录和翻译产物多样化的特点,原因在于存在多个转录起始位点的选择性应用,转录产物的选择性剪接,以及不同多聚腺苷酸化位点的使用.长期以来人们普遍关注由外显子3和4编码的循环型IGF-1在生长发育中的作用,最近对肌肉、神经等组织自分泌/旁分泌的局部型IGF-1研究发现,选择性剪接产生的IGF-1变体具有外显子5和6编码的延伸肽(E肽),并表现出特殊的生物学功能,如IGF-1Ea、IGF-1Eb(MGF)及其E肽在骨骼肌、心肌、神经等组织中表现出促进生长和损伤修复的功能,这些特殊功能可能通过细胞表面的一种特殊E肽受体介导.     

Insulin-like growth factor 1 regulation of proliferation and differentiation of <Emphasis Type="Italic">Xenopus laevis</Emphasis> myogenic cells in vitro

To understand the mechanism of muscle remodeling during Xenopus laevis metamorphosis, we examined the in vitro effect of insulin-like growth factor 1 (IGF-1) on growth and differentiation of three different-fate myogenic cell populations: tadpole tail, tadpole dorsal, and young adult leg muscle. IGF-1 promoted growth and differentiation of both tail and leg myogenic cells only under conditions where these cells could proliferate. Inhibition of cell proliferation by DNA synthesis inhibitor cytosine arabinoside completely canceled the IGF-1’s cell differentiation promotion, suggesting the possibility that IGF-1’s differentiation-promotion effect is an indirect effect via IGF-1’s cell proliferation promotion. IGF-1 promoted differentiation dose dependently with maximum effect at 100–500 ng/ml. RT-PCR analysis revealed the upregulation (11-fold) of ifg1 mRNA expression in developing limbs, suggesting that IGF-1 plays a role in promoting muscle differentiation during limb development. The combined effect of triiodo-l-thyronine (T₃) and IGF-1 was also examined. In adult leg cells, IGF-1 promoted growth and differentiation irrespective of the presence of T₃. In larval tail cells, cell count was 76% lower in the presence of T₃, and IGF-1 did not promote proliferation and differentiation in T₃-containing medium. In larval dorsal cells, cell count was also lower in the presence of T₃, but IGF-1 enhanced proliferation and differentiation in T₃-containing medium. This result is likely due to the presence among dorsal cells of both adult and larval types (1:1). Thus, IGF-1 affects only adult-type myogenic cells in the presence of T₃ and helps accelerate dorsal muscle remodeling during metamorphosis.     

Effects of mechanical stretching on caspase and IGF-1 expression during the proliferation process of myoblasts

It has been reported that the synthesis, degradation, and metabolism of muscle proteins in myoblasts, as well as the proliferation and differentiation of cells, are influenced by various related to extracellular signaling molecules, such as neural transmitters, growth factors, and hormones, when muscle tissue has been exposed to mechanical stimulation. However, reports regarding the expression of growth factors during mechanical stimulation of myoblasts are few, and many questions remain unanswered. We examined the mRNA expression of insulin-like growth factor 1 (IGF-1) in myoblasts subjected to mechanical stretching in vitro. In addition, apoptosis caused by intracellular stress has been reported to occur during muscle development at the embryonic stage. To clarify the expression of intracellular stress factors, we here investigated related gene expression. Expression of IGF-1 increased in the early stage of cell stretching, followed by a decrease in the late stage. This suggests that mechanical stimulation resulted in an immediate increase in IGF-1 expression, followed by a decrease as cells acclimated to the inducing environment. Caspase was significantly expressed in a stretch group at 12 hours after the beginning of mechanical stimulation, compared with a control group. This suggests that cellular proliferation is also regulated by intracellular stress factors involving the endoplasmic reticulum, mitochondria, and other organelles during the process of muscle proliferation and differentiation.     

力生长因子在大肠杆菌中的表达及活性分析

MGF(Mechano-growth factor)是一种IGF-1变体形式, 研究发现该因子具有应力敏感性, 并且具有促进肌肉肥大、再生以及神经损伤修复的功能。通过RT-PCR从拉伸刺激的人成骨细胞中克隆MGF cDNA序列, 并去除5'端9 bp的序列, 使N端缺少对肠激酶(Enterokinase, EK)具有抑制作用的脯氨酸, 将截短型MGF (des(1-3) MGF) cDNA序列克隆入pET32a(+)质粒, 构建重组表达质粒。重组质粒转化E. coli BL21(DE3), 在30oC培养下以可溶形式表达融合蛋白Trx/ des(1-3)MGF, 采用离子交换层析和Ni2+金属亲和层析, 获得纯度95%以上的融合蛋白。再对融合蛋白EK酶切, rpHPLC分离获得纯度达98%的des(1-3)MGF, SDS-PAGE电泳及质谱分析蛋白分子量与理论值相符。生物活性实验显示, 所制备的des(1-3)MGF比des(1-3)IGF-1更显著的促进MC3T3-E1细胞的增值和迁移。     

Stimulation of mechano-growth factor expression by second messengers

The effect of second messengers on the expression of mechano-growth factor (MGF) synthesis by myoblasts and differentiated myotubes in culture was investigated. cAMP stimulates MGF expression both in murine and human cells. CNG- and HCN-channel blockers slightly activated MGF synthesis, while an activator of Epac protein had no effect. It is assumed that cAMP activates MGF synthesis via protein kinase A. Phorbol ester (PMA) activates MGF synthesis in human myoblasts and myotubes only. The expression of another splice form of IGF-1 gene, IGF-1Ea, was also stimulated in human cells by db-cAMP and PMA and in murine cells by db-cAMP only. Stimulation of MGF expression in human cells by db-cAMP and PMA demonstrated different time dependences but showed additivity when the compounds were applied in a combination. Inhibitors specific to protein kinase A did not affect PMA-mediated activation, while inhibitors specific to protein kinase C did not affect db-cAMP-mediated process. Ca²⁺ ionophore and ROS inductor strongly inhibited synthesis of the growth factor. PGE2 known as physiological stimulator of cAMP synthesis was shown to stimulate MGF expression both in murine and human cells. Implication of protein kinase A and protein kinase C in MGF synthesis stimulation and a cross-talk between two signaling systems is discussed.