力生长因子对大鼠肌腱细胞运动能力及损伤肌腱修复的影响

发布时间：2018-05-20 12:51

本文选题：力生长因子 + 肌腱细胞　；参考：《重庆大学》2015年博士论文

【摘要】：肌腱是连接肌肉与骨的单轴致密纤维结缔组织,传递由肌肉产生的力从而带动骨的活动,在机体运动中起着重要作用。但运动、训练不当或重复牵拉过剧常会导致肌腱损伤甚至断裂,对人的身体健康和生活质量产生严重影响。由于肌腱组织血管分布少、新陈代谢率低,因此损伤肌腱的自愈能力很差,修复过程十分缓慢,并很难恢复其损伤前的正常水平。如何促进损伤肌腱的修复、提高治疗效果并完全恢复其功能,目前仍然是临床面临的重要挑战。已有研究发现,细胞因子/生长因子对损伤肌腱的修复具有良好的促进作用。力生长因子(mechano-growth factor,MGF)是由胰岛素样生长因子1(insulin-like growth factor-I,IGF-1)基因选择性剪接产生的一种变异体,由于最初研究时发现其对力学刺激十分敏感,因此被命名为力生长因子。在人体中,IGF-1基因能够产生3种亚型,IGF-1 Ea,IGF-1 Eb和IGF-1 Ec,在啮齿动物中产生两种类型,IGF-1Ea和IGF-1 Eb。人的IGF-1 Ec和啮齿动物的IGF-1 Eb被称为MGF。MGF的主要特点是在IGF-1的羧基端多出1个延伸肽(extention peptide,E肽),所以具有与成熟IGF-1不同的生物学作用。研究已经证实,MGF在治疗肌肉缺损、预防心肌损伤、修复受损神经、加速血管生成和促进骨修复等方面起着重要作用,但是其在损伤肌腱修复中的作用及其分子机制尚不清楚。肌腱细胞的运动是肌腱损伤修复过程中的重要环节。肌腱损伤后,正常的肌腱细胞会迁移到损伤位点,增殖并分泌细胞外基质,从而促进损伤肌腱修复。本文以大鼠为动物模型,研究了MGF-C25E(一种合成的大鼠MGF E肽)对肌腱细胞运动能力的作用及其分子机制,并在此基础上进一步研究了MGF-C25E对损伤肌腱修复的影响。主要实验结果如下:①MGF-C25E通过FAK-ERK1/2信号通路促进大鼠肌腱细胞运动划痕法和Transwell法检测结果表明,MGF-C25E明显促进肌腱细胞的迁移和侵袭能力,并在一定浓度范围内呈现浓度相关性。Western blot检测结果发现,MGF-C25E作用肌腱细胞后,粘着斑激酶(focal adhesion kinase,FAK)和细胞外信号调节激酶1/2(extracellular signal regulated kinase 1/2,ERK1/2)磷酸化水平明显增加,抑制FAK或ERK1/2激活后,MGF-C25E促进的肌腱细胞迁移和侵袭受到明显抑制。此外,FAK抑制剂亦阻断MGF-C25E诱导的ERK1/2磷酸化。因此,MGF-C25E可能通过FAK-ERK1/2信号通路促进大鼠肌腱细胞迁移和侵袭。明胶酶谱实验表明,MGF-C25E促进肌腱细胞基质金属蛋白酶2(matrix metalloproteinase 2,MMP-2)分泌,对基质金属蛋白酶9(matrix metalloproteinase 9,mmp-9)分泌没有明显影响。抑制mgf-c25e激活的mmp-2后,mgf-c25e促进的细胞侵袭受到明显抑制。此外,通过免疫荧光染色观察mgf-c25e对细胞骨架蛋白f-actin的影响,实验结果显示,mgf-c25e可显著促进肌腱细胞f-actin重排和伪足增加。抑制fak和erk1/2激活后,mgf-c25e促进的伪足增加和f-actin重排受到明显抑制。进一步利用原子力显微镜(atomicforcemicroscopy,afm)检测mgf-c25e作用肌腱细胞后细胞杨氏模量的变化,结果发现mgf-c25e显著降低肌腱细胞的杨氏模量。抑制fak或erk1/2激活后,mgf-c25e降低的细胞杨氏模量得到明显恢复。上述结果提示,mgf-c25e可能通过fak-erk1/2信号通路促进伪足形成,增加mmp-2分泌和降低细胞硬度,从而增加肌腱细胞的运动能力。②mgf-c25e通过fak-erk1/2信号通路增加细胞核硬度促进肌腱细胞迁移利用afm直接测量肌腱细胞核杨氏模量,实验结果显示,mgf-c25e通过fak-erk1/2信号通路增大肌腱细胞核的杨氏模量,使细胞核硬度增加。mgf-c25e对细胞核骨架蛋白lamina/c的表达没有明显影响,但可以促进染色质浓缩。抑制fak或erk1/2激活后,mgf-c25e促进的染色质浓缩受到明显抑制。此外,mgf-c25e促进肌腱细胞dna甲基化,dna甲基化抑制剂(methylthioadenosine,mta)作用后,mgf-c25e促进的染色质浓缩、细胞核硬度及细胞迁移受到明显抑制。fak和erk1/2的抑制剂亦阻断mgf-c25e促进的dna甲基化。上述结果表明,mgf-c25e可能通过fak-erk1/2信号通路促进染色质浓缩,增加细胞核硬度,从而提高肌腱细胞迁移能力。③mgf-c25e降低大鼠肌腱细胞炎症因子的表达通过机械拉伸或肿瘤坏死因子-α(tumornecrosisfactorα,tnf-α)构建大鼠肌腱细胞损伤模型,利用qrt-pcr检测i型胶原(collageni)、iii型胶原(collageniii)、环氧化酶-2(cyclooxygenase2,cox-2)和前列腺素e2(prostaglandine2,pge2)基因的表达确定肌腱细胞损伤模型是否构建成功。实验结果显示,机械拉伸显著降低大鼠肌腱细胞collageni和collageniiimrna表达,对炎症因子cox-2和pge2mrna表达无明显作用。tnf-α作用大鼠肌腱细胞后,炎症因子cox-2和pge2mrna水平明显上调,collageni和collageniiimrna水平显著下调。机械过载和tnf-α均能对大鼠肌腱细胞产生一定的损害作用。进一步在tnf-α诱导的损伤条件下检测mgf-c25e对损伤肌腱细胞的影响。结果显示,mgf-c25e降低了tnf-α促进的cox-2和pge2mrna表达,对collageni和collageniiimrna表达没有影响,提示mgf-c25e可能对损伤肌腱细胞具有一定的保护作用。④mgf-c25e对大鼠跟腱损伤修复的影响通过手术切割缝合术构建大鼠跟腱损伤模型,利用跟腱功能指数(achillesfunctional index,AFI)、生物力学评价和组织学评价等确定MGF-C25E对损伤跟腱修复的影响。实验结果显示,高浓度MGF-C25E(1000 ng/mL)显著增加修复跟腱的AFI和力学强度。进一步研究MGF-C25E对修复肌腱组织结构的影响,苏木素-伊红(Hematoxylin and eosin,HE)染色结果显示高浓度MGF-C25E处理组肌腱较对照组(生理盐水处理组)具有更丰富的细胞外基质,并且细胞数量减少。此外,Masson’s trichrome染色证明高浓度MGF-C25E处理组比对照组含有更加丰富的胶原纤维。通过组织学评分系统进行评分,结果显示高浓度MGF-C25E处理组相较于对照组具有更高的组织学分数。综合上述结果,MGF-C25E可能通过促进大鼠肌腱细胞运动、降低肌腱细胞炎症反应和增加细胞外基质合成等促进损伤肌腱修复。本研究结果为深入认识MGF在损伤肌腱修复中的作用及其机制提供了实验依据,为临床上应用MGF等生长因子修复损伤肌腱提供了理论指导。
[Abstract]:Tendon is a uniaxial dense fibrous connective tissue connected to the muscles and bones, transmitting the force produced by the muscles to drive the activity of the bone, which plays an important role in the movement of the body. But exercise, improper training or repeated pulling and pulling play often lead to tendon injury or even fracture, which have a serious effect on the health and quality of life of the human body. There are few tissue vessels and low metabolism rate, so the self healing ability of the tendon is very poor, the repair process is very slow, and it is difficult to restore the normal level before the injury. It is still an important challenge to promote the repair of the damaged tendon, improve the therapeutic effect and completely restore its function. Mechano-growth factor (MGF) is a variant produced by the selective splicing of the insulin like growth factor 1 (insulin-like growth factor-I, IGF-1) gene. In the human body, the IGF-1 gene produces 3 subtypes, IGF-1 Ea, IGF-1 Eb and IGF-1 Ec, producing two types in rodents, IGF-1Ea and IGF-1 Eb. human IGF-1 Ec and rodents, which are characterized by 1 extensions of the carboxyl terminus. It has been proved that MGF plays an important role in the treatment of muscle defects, preventing myocardial damage, repairing damaged nerves, accelerating angiogenesis and promoting bone repair, but its role in the repair of tendon injury and its molecular mechanism are not clear. The movement of the tendon cells is the repair of tendon injury. After the tendon injury, the normal tendon cells migrate to the damage site, proliferate and secrete the extracellular matrix, thus promoting the injury of the tendon repair. In this paper, rats are used as animal models to study the effect and molecular mechanism of MGF-C25E (a synthetic MGF E peptide) on the muscle tendon movement and its molecular mechanism. The effect of MGF-C25E on the repair of damaged tendon was further studied. The main experimental results were as follows: (1) the results of MGF-C25E through FAK-ERK1/2 signal pathway to promote the movement of tendon cells and the detection results by Transwell method showed that MGF-C25E obviously promoted the migration and invasion ability of the tendon cells, and showed a concentration phase in a certain concentration range. The results of the.Western blot detection showed that the phosphorylation level of the adhesion kinase (focal adhesion kinase, FAK) and the extracellular signal regulated kinase 1/2 (extracellular signal regulated kinase) increased significantly after the action of MGF-C25E on the tendon cells. In addition, FAK inhibitors also block the phosphorylation of ERK1/2 induced by MGF-C25E. Therefore, MGF-C25E may promote the migration and invasion of rat tendon cells through the FAK-ERK1/2 signaling pathway. The gelatinase assay shows that MGF-C25E promotes the secretion of matrix metalloproteinase 2 (matrix metalloproteinase 2, MMP-2) and matrix metalloproteinases in the tendon cells. The secretion of enzyme 9 (matrix metalloproteinase 9, MMP-9) was not significantly affected. After the inhibition of mgf-c25e activated MMP-2, the invasion of mgf-c25e promoted cells was significantly inhibited. In addition, the effect of mgf-c25e on cytoskeleton F-actin was observed by immunofluorescence staining. The experimental results showed that mgf-c25e could significantly promote the rearrangement and artifact of the F-actin of the tendon cells. Increase in foot. After inhibition of FAK and erk1/2 activation, mgf-c25e promoted pseudo foot increase and F-actin rearrangement were significantly inhibited. Further using atomic force microscopy (atomicforcemicroscopy, AFM) to detect the change of Young's modulus of cells after mgf-c25e action tendon cells, the results showed that mgf-c25e significantly reduced the young's modulus of tendon cells and inhibited FAK or er. After the activation of k1/2, the young's modulus of mgf-c25e decreased obviously. These results suggest that mgf-c25e may promote the formation of pseudo foot through the fak-erk1/2 signaling pathway, increase the secretion of MMP-2 and decrease the cell hardness, thereby increasing the motor ability of the tendon cells. 2. The increase of the hardness of the nucleus by the fak-erk1/2 signaling pathway and the enhancement of the tendon to promote the tendon. Cell migration was used to measure the young's modulus of the nucleus of the tendon directly by AFM. The experimental results showed that mgf-c25e increased the young's modulus of the nucleus of the tendon through the fak-erk1/2 signal pathway, which made the nuclear hardness increase.Mgf-c25e had no significant influence on the expression of nuclear cytoskeleton lamina/c, but could promote chromatin concentration and inhibit FAK or erk1/2 excitation. After living, mgf-c25e promoted chromatin concentration was significantly inhibited. In addition, mgf-c25e promoted DNA methylation of tendon cells, DNA methylation inhibitor (methylthioadenosine, MTA), mgf-c25e promoted chromatin concentration, cell nuclear hardness and cell migration under inhibition of.Fak and erk1/2 inhibitors also blocked mgf-c25e promoted DNA armour These results suggest that mgf-c25e may promote chromatin concentration, increase cell nuclear hardness, and enhance the mobility of tendon cells through fak-erk1/2 signaling pathway. (3) mgf-c25e reduces the expression of inflammatory factors in rat tendon cells by mechanical stretching or tumor necrosis factor - alpha (tumornecrosisfactor alpha, tnf- alpha) in the construction of rat tendon fine Cell damage model, using qRT-PCR to detect type I collagen (CollagenI), type III collagen (collageniii), cyclooxygenase -2 (cyclooxygenase2, COX-2) and prostaglandin E2 (prostaglandine2, PGE2) gene expression to determine whether the tendon cell damage model was constructed successfully. Lageniiimrna expression has no obvious effect on the expression of inflammatory factors COX-2 and pge2mrna in the.Tnf- - alpha action of rat tendon cells, the level of inflammatory factors COX-2 and pge2mrna is obviously up - regulated, and the levels of CollagenI and collageniiimrna are significantly down - regulated. Both mechanical overload and tnf- alpha can cause certain damage to the rat tendon cells. Further in tnf- alpha induction The effects of mgf-c25e on damaged tendon cells were detected. The results showed that mgf-c25e decreased the expression of COX-2 and pge2mrna promoted by tnf- alpha, and did not affect the expression of CollagenI and collageniiimrna, suggesting that mgf-c25e may have protective effects on the injured tendon cells. 4. The effect of mgf-c25e on the repair of Achilles tendon injury in rats The effect of MGF-C25E on the repair of injured Achilles tendon was determined by using the Achilles tendon function index (achillesfunctional index, AFI), biomechanical evaluation and histological evaluation. The experimental results showed that the high concentration of MGF-C25E (1000 ng/mL) significantly increased the AFI and mechanical strength of the repair of the Achilles tendon. The effect of MGF-C25E on the repair of tendon tissue was studied. The results of Hematoxylin and eosin (HE) staining showed that the tendon of high concentration MGF-C25E treatment group had more extracellular matrix than the control group (saline treatment group), and the number of cells decreased. In addition, Masson 's trichrome staining proved that the high concentration MGF-C25E treatment was high concentration. The group had more abundant collagen fibers than the control group. The results showed that the high concentration MGF-C25E treatment group had a higher histological score compared with the control group. The results suggested that MGF-C25E may reduce the inflammatory response and increase the extracellular matrix by promoting the exercise of rat tendon cells. The results of this study provide an experimental basis for the in-depth understanding of the role and mechanism of MGF in the repair of damaged tendon, and provide theoretical guidance for the clinical application of MGF like growth factors to repair the injury of tendon.
【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R686.1

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