本文選題：膠原 + 膠原模擬多肽��； 參考：《北京協(xié)和醫(yī)學(xué)院》2012年碩士論文

【摘要】：膠原是胞外基質(zhì)的主要成分,具有獨(dú)特的三螺旋結(jié)構(gòu)。它不僅對(duì)組織和器官起支持和保護(hù)作用,還參與介導(dǎo)許多生物學(xué)過(guò)程。這些生物學(xué)功能是通過(guò)細(xì)胞粘附分子與膠原特征結(jié)構(gòu)域的特異性結(jié)合來(lái)實(shí)現(xiàn)的。深入研究膠原特征結(jié)構(gòu)與細(xì)胞的相互作用,是膠原作為生物活性材料應(yīng)用以及開發(fā)人工類膠原生物材料的重要基礎(chǔ)。膠原模擬多肽(CMP)可以模擬膠原的特征結(jié)構(gòu),為研究膠原的結(jié)構(gòu)和功能提供了一個(gè)新方法。 本研究設(shè)計(jì)合成了包含類膠原重復(fù)序列(Pro-Hyp-Gly)n的CMP21、含整合素α2β1識(shí)別序列Gly-Phe-Hyp-Gly-Glu-Arg (GFOGER)的CMP9以及同時(shí)包含以上兩種序列的CMP27。研究了Ⅰ型膠原和膠原模擬多肽的三螺旋結(jié)構(gòu)及其熱穩(wěn)定性,觀察了膠原模擬多肽對(duì)成纖維細(xì)胞的粘附作用,闡明了兩種序列對(duì)細(xì)胞粘附的不同影響作用。主要研究工作如下： 1.膠原及膠原模擬多肽三螺旋結(jié)構(gòu)及熱穩(wěn)定性研究 通過(guò)圓二色譜(CD)表征了膠原及膠原模擬多肽的三螺旋結(jié)構(gòu)及其熱穩(wěn)定性,CD譜結(jié)果顯示,僅有整合素識(shí)別位點(diǎn)序列的CMP9不具有三螺旋結(jié)構(gòu),而CMP21和CMP27能自發(fā)形成類似膠原的三螺旋結(jié)構(gòu)。熱穩(wěn)定性研究證實(shí),膠原溶液經(jīng)溫度誘導(dǎo)后發(fā)生“螺旋—解旋”的熱變性過(guò)程,該過(guò)程不可逆；CMP21和CMP27溶液溫度升高后構(gòu)象連續(xù)發(fā)生變化,在一定條件下其熱變性過(guò)程實(shí)現(xiàn)可逆恢復(fù),發(fā)生“螺旋—解旋—復(fù)旋”的變化過(guò)程。 2.膠原及膠原模擬多肽細(xì)胞毒性評(píng)價(jià) 用CCK-8法考察CMP及膠原溶液對(duì)L929細(xì)胞的毒性作用。結(jié)果顯示,CMP溶液對(duì)已貼壁成纖維細(xì)胞生長(zhǎng)無(wú)抑制作用,未發(fā)生明顯的細(xì)胞毒性反應(yīng),其中CMP27組對(duì)L929細(xì)胞生長(zhǎng)有一定的促進(jìn)作用。 3.物理吸附膠原模擬多肽對(duì)成纖維細(xì)胞粘附的影響 通過(guò)物理吸附固定CMP,利用細(xì)胞粘附、競(jìng)爭(zhēng)抑制、熒光染色等實(shí)驗(yàn)證實(shí),三種包被CMP的基底均能在一定程度上促進(jìn)細(xì)胞粘附、生長(zhǎng),具有良好的細(xì)胞粘附率和細(xì)胞附著形態(tài),其中CMP27具有更好的促粘附效果,可觀察到大量的應(yīng)力纖維及偽足,粘附效果與Ⅰ型膠原相近。由此認(rèn)為,膠原三螺旋結(jié)構(gòu)對(duì)細(xì)胞粘附有促進(jìn)作用,GFOGER序列對(duì)細(xì)胞偽足生成及應(yīng)力纖維形成有支持作用。研究結(jié)果初步證實(shí),膠原三螺旋結(jié)構(gòu)與整合素識(shí)別位點(diǎn)序列共同作用促進(jìn)成纖維細(xì)胞粘附。 4.殼聚糖膜共價(jià)偶聯(lián)膠原模擬多肽對(duì)成纖維細(xì)胞粘附的影響 以3-馬來(lái)酰亞胺基苯甲酸琥珀酰亞胺酯(MBS)為連接臂,將殼聚糖膜與CMP共價(jià)偶聯(lián)。利用核磁共振(1H-NMR)與X-射線光電子能譜(XPS)對(duì)樣品進(jìn)行了表征,證實(shí)多肽與MB-殼聚糖膜共價(jià)偶聯(lián)成功。細(xì)胞粘附等實(shí)驗(yàn)說(shuō)明,MB-殼聚糖濃度對(duì)成纖維細(xì)胞粘附有一定影響,當(dāng)MB-殼聚糖濃度為3ng/mm2和6ng/mm2時(shí),粘附效果較好。MB-殼聚糖膜偶聯(lián)CMP后對(duì)L929細(xì)胞粘附的促進(jìn)作用明顯增強(qiáng),偶聯(lián)CMP27的效果較好,可以觀察到長(zhǎng)勢(shì)良好的應(yīng)力纖維與微絨毛,其粘附效果與吸附膠原的基底近似。以上結(jié)果進(jìn)一步證實(shí),膠原三螺旋結(jié)構(gòu)及整合素識(shí)別位點(diǎn)序列共同作用促進(jìn)成纖維細(xì)胞粘附。 綜上所述,包含膠原三螺旋結(jié)構(gòu)與整合素識(shí)別位點(diǎn)序列的CMP可以有效促進(jìn)成纖維細(xì)胞粘附,可作為表面修飾基團(tuán)促進(jìn)α2β1,整合素介導(dǎo)的細(xì)胞粘附。本研究可為設(shè)計(jì)以多肽為基礎(chǔ)的生物活性材料提供新的研究思路,也為開發(fā)以及應(yīng)用人工類膠原生物材料提供研究基礎(chǔ)。
[Abstract]:Collagen is the main component of the extracellular matrix and has a unique three helix structure. It not only supports and protects the tissues and organs, but also mediates many biological processes. These biological functions are realized by the specific binding of cell adhesion molecules to the characteristic domain of collagen. The interaction is an important basis for the application of collagen as bioactive material and the development of artificial collagen biomaterials. The collagen mimic peptide (CMP) can simulate the characteristic structure of collagen and provide a new method for the study of the structure and function of collagen.
In this study, we designed and synthesized CMP21 containing collagen like repeat sequence (Pro-Hyp-Gly) n, CMP9 containing integrin alpha 2 beta 1 recognition sequence Gly-Phe-Hyp-Gly-Glu-Arg (GFOGER), and CMP27. containing the above two sequences, which studied the three spiral structure and thermal stability of type I collagen and collagen mimic peptides, and observed the collagen mimic peptide pairs. The adhesion of fibroblasts elucidated the different effects of the two sequences on cell adhesion. The main research work is as follows:
Helical structure and thermal stability of collagen 1. and collagen mimetic polypeptide three
The three spiral structure and thermal stability of collagen and collagen mimic peptides were characterized by circular two chromatography (CD). The results of CD spectrum showed that the CMP9 with only the sequence of the integrin identification site did not have a three helix structure, while CMP21 and CMP27 could spontaneously form a three helix structure similar to collagen. The process of thermal denaturation of "spiral - spin spin" is irreversible. The conformation changes continuously after the CMP21 and CMP27 solution temperature rises. Under certain conditions, the thermal denaturation process is reversible, and the process of "spiral - spin - complex rotation" occurs.
Evaluation of the toxicity of 2. collagen and collagen mimic polypeptide cells
The toxic effects of CMP and collagen solution on L929 cells were investigated by CCK-8 method. The results showed that CMP solution had no inhibitory effect on the growth of adherent fibroblasts, and no obvious cytotoxic reaction occurred. The CMP27 group had a certain promotion effect on the growth of L929 cells.
3. effect of physical adsorption collagen mimetic polypeptide on adhesion of fibroblasts
By immobiling CMP by physical adsorption and using cell adhesion, competition inhibition, and fluorescence staining, the three kinds of CMP substrates can promote cell adhesion and growth to a certain extent, and have good cell adhesion and cell attachment form, and CMP27 has a better adhesion effect, and a large number of stress fibers and pseudo feet can be observed. The adhesion effect is similar to that of type I collagen. Therefore, it is believed that the three spiral structure of collagen promotes cell adhesion, and the GFOGER sequence supports the formation of cell pseudo foot and the formation of stress fiber. The results preliminarily confirm that the co action of the three spiral structure of collagen and the sequence of the integrin identification site promotes the adhesion of fibroblasts.
Effect of 4. chitosan membrane covalently coupled collagen mimetic polypeptide on adhesion of fibroblasts
With 3- maleimide benzoate succinimide (MBS) as the connecting arm, the chitosan membrane was covalently coupled with CMP. The samples were characterized by nuclear magnetic resonance (1H-NMR) and X- ray photoelectron spectroscopy (XPS). It was proved that the peptide was covalently coupled with the MB- chitosan membrane. Cell adhesion and other experiments showed that the concentration of MB- chitosan was on the fibroblasts. Adhesion has a certain effect. When the concentration of MB- chitosan is 3ng/mm2 and 6ng/mm2, the adhesion effect is better than that of the.MB- chitosan membrane coupling CMP. The effect of coupling CMP27 is better. The effect of coupling CMP27 is better. The adhesion effect of the chitosan membrane can be observed and the adhesion effect is similar to that of the adsorbed collagen. It is further confirmed that collagen three helix structure and integrin recognition site sequence contribute to fibroblast adhesion.
To sum up, the CMP containing the collagen three helix structure and the integrin identification site sequence can effectively promote the adhesion of fibroblasts, which can be used as a surface modifier to promote alpha 2 beta 1, integrin mediated cell adhesion. This study can provide new research ideas for the design of bioactive materials based on polypeptide, and also for development and application. The research basis of collagen biomaterials is provided.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

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