發(fā)布時(shí)間：2018-05-20 12:51

  本文選題：力生長因子 + 肌腱細(xì)胞　； 參考：《重慶大學(xué)》2015年博士論文

【摘要】：肌腱是連接肌肉與骨的單軸致密纖維結(jié)締組織,傳遞由肌肉產(chǎn)生的力從而帶動(dòng)骨的活動(dòng),在機(jī)體運(yùn)動(dòng)中起著重要作用。但運(yùn)動(dòng)、訓(xùn)練不當(dāng)或重復(fù)牽拉過劇常會(huì)導(dǎo)致肌腱損傷甚至斷裂,對(duì)人的身體健康和生活質(zhì)量產(chǎn)生嚴(yán)重影響。由于肌腱組織血管分布少、新陳代謝率低,因此損傷肌腱的自愈能力很差,修復(fù)過程十分緩慢,并很難恢復(fù)其損傷前的正常水平。如何促進(jìn)損傷肌腱的修復(fù)、提高治療效果并完全恢復(fù)其功能,目前仍然是臨床面臨的重要挑戰(zhàn)。已有研究發(fā)現(xiàn),細(xì)胞因子/生長因子對(duì)損傷肌腱的修復(fù)具有良好的促進(jìn)作用。力生長因子(mechano-growth factor,MGF)是由胰島素樣生長因子1(insulin-like growth factor-I,IGF-1)基因選擇性剪接產(chǎn)生的一種變異體,由于最初研究時(shí)發(fā)現(xiàn)其對(duì)力學(xué)刺激十分敏感,因此被命名為力生長因子。在人體中,IGF-1基因能夠產(chǎn)生3種亞型,IGF-1 Ea,IGF-1 Eb和IGF-1 Ec,在嚙齒動(dòng)物中產(chǎn)生兩種類型,IGF-1Ea和IGF-1 Eb。人的IGF-1 Ec和嚙齒動(dòng)物的IGF-1 Eb被稱為MGF。MGF的主要特點(diǎn)是在IGF-1的羧基端多出1個(gè)延伸肽(extention peptide,E肽),所以具有與成熟IGF-1不同的生物學(xué)作用。研究已經(jīng)證實(shí),MGF在治療肌肉缺損、預(yù)防心肌損傷、修復(fù)受損神經(jīng)、加速血管生成和促進(jìn)骨修復(fù)等方面起著重要作用,但是其在損傷肌腱修復(fù)中的作用及其分子機(jī)制尚不清楚。肌腱細(xì)胞的運(yùn)動(dòng)是肌腱損傷修復(fù)過程中的重要環(huán)節(jié)。肌腱損傷后,正常的肌腱細(xì)胞會(huì)遷移到損傷位點(diǎn),增殖并分泌細(xì)胞外基質(zhì),從而促進(jìn)損傷肌腱修復(fù)。本文以大鼠為動(dòng)物模型,研究了MGF-C25E(一種合成的大鼠MGF E肽)對(duì)肌腱細(xì)胞運(yùn)動(dòng)能力的作用及其分子機(jī)制,并在此基礎(chǔ)上進(jìn)一步研究了MGF-C25E對(duì)損傷肌腱修復(fù)的影響。主要實(shí)驗(yàn)結(jié)果如下:①M(fèi)GF-C25E通過FAK-ERK1/2信號(hào)通路促進(jìn)大鼠肌腱細(xì)胞運(yùn)動(dòng)劃痕法和Transwell法檢測結(jié)果表明,MGF-C25E明顯促進(jìn)肌腱細(xì)胞的遷移和侵襲能力,并在一定濃度范圍內(nèi)呈現(xiàn)濃度相關(guān)性。Western blot檢測結(jié)果發(fā)現(xiàn),MGF-C25E作用肌腱細(xì)胞后,粘著斑激酶(focal adhesion kinase,FAK)和細(xì)胞外信號(hào)調(diào)節(jié)激酶1/2(extracellular signal regulated kinase 1/2,ERK1/2)磷酸化水平明顯增加,抑制FAK或ERK1/2激活后,MGF-C25E促進(jìn)的肌腱細(xì)胞遷移和侵襲受到明顯抑制。此外,FAK抑制劑亦阻斷MGF-C25E誘導(dǎo)的ERK1/2磷酸化。因此,MGF-C25E可能通過FAK-ERK1/2信號(hào)通路促進(jìn)大鼠肌腱細(xì)胞遷移和侵襲。明膠酶譜實(shí)驗(yàn)表明,MGF-C25E促進(jìn)肌腱細(xì)胞基質(zhì)金屬蛋白酶2(matrix metalloproteinase 2,MMP-2)分泌,對(duì)基質(zhì)金屬蛋白酶9(matrix metalloproteinase 9,mmp-9)分泌沒有明顯影響。抑制mgf-c25e激活的mmp-2后,mgf-c25e促進(jìn)的細(xì)胞侵襲受到明顯抑制。此外,通過免疫熒光染色觀察mgf-c25e對(duì)細(xì)胞骨架蛋白f-actin的影響,實(shí)驗(yàn)結(jié)果顯示,mgf-c25e可顯著促進(jìn)肌腱細(xì)胞f-actin重排和偽足增加。抑制fak和erk1/2激活后,mgf-c25e促進(jìn)的偽足增加和f-actin重排受到明顯抑制。進(jìn)一步利用原子力顯微鏡(atomicforcemicroscopy,afm)檢測mgf-c25e作用肌腱細(xì)胞后細(xì)胞楊氏模量的變化,結(jié)果發(fā)現(xiàn)mgf-c25e顯著降低肌腱細(xì)胞的楊氏模量。抑制fak或erk1/2激活后,mgf-c25e降低的細(xì)胞楊氏模量得到明顯恢復(fù)。上述結(jié)果提示,mgf-c25e可能通過fak-erk1/2信號(hào)通路促進(jìn)偽足形成,增加mmp-2分泌和降低細(xì)胞硬度,從而增加肌腱細(xì)胞的運(yùn)動(dòng)能力。②mgf-c25e通過fak-erk1/2信號(hào)通路增加細(xì)胞核硬度促進(jìn)肌腱細(xì)胞遷移利用afm直接測量肌腱細(xì)胞核楊氏模量,實(shí)驗(yàn)結(jié)果顯示,mgf-c25e通過fak-erk1/2信號(hào)通路增大肌腱細(xì)胞核的楊氏模量,使細(xì)胞核硬度增加。mgf-c25e對(duì)細(xì)胞核骨架蛋白lamina/c的表達(dá)沒有明顯影響,但可以促進(jìn)染色質(zhì)濃縮。抑制fak或erk1/2激活后,mgf-c25e促進(jìn)的染色質(zhì)濃縮受到明顯抑制。此外,mgf-c25e促進(jìn)肌腱細(xì)胞dna甲基化,dna甲基化抑制劑(methylthioadenosine,mta)作用后,mgf-c25e促進(jìn)的染色質(zhì)濃縮、細(xì)胞核硬度及細(xì)胞遷移受到明顯抑制。fak和erk1/2的抑制劑亦阻斷mgf-c25e促進(jìn)的dna甲基化。上述結(jié)果表明,mgf-c25e可能通過fak-erk1/2信號(hào)通路促進(jìn)染色質(zhì)濃縮,增加細(xì)胞核硬度,從而提高肌腱細(xì)胞遷移能力。③mgf-c25e降低大鼠肌腱細(xì)胞炎癥因子的表達(dá)通過機(jī)械拉伸或腫瘤壞死因子-α(tumornecrosisfactorα,tnf-α)構(gòu)建大鼠肌腱細(xì)胞損傷模型,利用qrt-pcr檢測i型膠原(collageni)、iii型膠原(collageniii)、環(huán)氧化酶-2(cyclooxygenase2,cox-2)和前列腺素e2(prostaglandine2,pge2)基因的表達(dá)確定肌腱細(xì)胞損傷模型是否構(gòu)建成功。實(shí)驗(yàn)結(jié)果顯示,機(jī)械拉伸顯著降低大鼠肌腱細(xì)胞collageni和collageniiimrna表達(dá),對(duì)炎癥因子cox-2和pge2mrna表達(dá)無明顯作用。tnf-α作用大鼠肌腱細(xì)胞后,炎癥因子cox-2和pge2mrna水平明顯上調(diào),collageni和collageniiimrna水平顯著下調(diào)。機(jī)械過載和tnf-α均能對(duì)大鼠肌腱細(xì)胞產(chǎn)生一定的損害作用。進(jìn)一步在tnf-α誘導(dǎo)的損傷條件下檢測mgf-c25e對(duì)損傷肌腱細(xì)胞的影響。結(jié)果顯示,mgf-c25e降低了tnf-α促進(jìn)的cox-2和pge2mrna表達(dá),對(duì)collageni和collageniiimrna表達(dá)沒有影響,提示mgf-c25e可能對(duì)損傷肌腱細(xì)胞具有一定的保護(hù)作用。④mgf-c25e對(duì)大鼠跟腱損傷修復(fù)的影響通過手術(shù)切割縫合術(shù)構(gòu)建大鼠跟腱損傷模型,利用跟腱功能指數(shù)(achillesfunctional index,AFI)、生物力學(xué)評(píng)價(jià)和組織學(xué)評(píng)價(jià)等確定MGF-C25E對(duì)損傷跟腱修復(fù)的影響。實(shí)驗(yàn)結(jié)果顯示,高濃度MGF-C25E(1000 ng/mL)顯著增加修復(fù)跟腱的AFI和力學(xué)強(qiáng)度。進(jìn)一步研究MGF-C25E對(duì)修復(fù)肌腱組織結(jié)構(gòu)的影響,蘇木素-伊紅(Hematoxylin and eosin,HE)染色結(jié)果顯示高濃度MGF-C25E處理組肌腱較對(duì)照組(生理鹽水處理組)具有更豐富的細(xì)胞外基質(zhì),并且細(xì)胞數(shù)量減少。此外,Masson’s trichrome染色證明高濃度MGF-C25E處理組比對(duì)照組含有更加豐富的膠原纖維。通過組織學(xué)評(píng)分系統(tǒng)進(jìn)行評(píng)分,結(jié)果顯示高濃度MGF-C25E處理組相較于對(duì)照組具有更高的組織學(xué)分?jǐn)?shù)。綜合上述結(jié)果,MGF-C25E可能通過促進(jìn)大鼠肌腱細(xì)胞運(yùn)動(dòng)、降低肌腱細(xì)胞炎癥反應(yīng)和增加細(xì)胞外基質(zhì)合成等促進(jìn)損傷肌腱修復(fù)。本研究結(jié)果為深入認(rèn)識(shí)MGF在損傷肌腱修復(fù)中的作用及其機(jī)制提供了實(shí)驗(yàn)依據(jù),為臨床上應(yīng)用MGF等生長因子修復(fù)損傷肌腱提供了理論指導(dǎo)。
[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.
【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R686.1

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