胶原模拟多肽及其对成纤维细胞粘附作用的研究

发布时间：2018-06-18 11:45

本文选题：胶原 + 胶原模拟多肽　；参考：《北京协和医学院》2012年硕士论文

【摘要】：胶原是胞外基质的主要成分,具有独特的三螺旋结构。它不仅对组织和器官起支持和保护作用,还参与介导许多生物学过程。这些生物学功能是通过细胞粘附分子与胶原特征结构域的特异性结合来实现的。深入研究胶原特征结构与细胞的相互作用,是胶原作为生物活性材料应用以及开发人工类胶原生物材料的重要基础。胶原模拟多肽(CMP)可以模拟胶原的特征结构,为研究胶原的结构和功能提供了一个新方法。本研究设计合成了包含类胶原重复序列(Pro-Hyp-Gly)n的CMP21、含整合素α2β1识别序列Gly-Phe-Hyp-Gly-Glu-Arg (GFOGER)的CMP9以及同时包含以上两种序列的CMP27。研究了Ⅰ型胶原和胶原模拟多肽的三螺旋结构及其热稳定性,观察了胶原模拟多肽对成纤维细胞的粘附作用,阐明了两种序列对细胞粘附的不同影响作用。主要研究工作如下： 1.胶原及胶原模拟多肽三螺旋结构及热稳定性研究通过圆二色谱(CD)表征了胶原及胶原模拟多肽的三螺旋结构及其热稳定性,CD谱结果显示,仅有整合素识别位点序列的CMP9不具有三螺旋结构,而CMP21和CMP27能自发形成类似胶原的三螺旋结构。热稳定性研究证实,胶原溶液经温度诱导后发生“螺旋—解旋”的热变性过程,该过程不可逆；CMP21和CMP27溶液温度升高后构象连续发生变化,在一定条件下其热变性过程实现可逆恢复,发生“螺旋—解旋—复旋”的变化过程。 2.胶原及胶原模拟多肽细胞毒性评价用CCK-8法考察CMP及胶原溶液对L929细胞的毒性作用。结果显示,CMP溶液对已贴壁成纤维细胞生长无抑制作用,未发生明显的细胞毒性反应,其中CMP27组对L929细胞生长有一定的促进作用。 3.物理吸附胶原模拟多肽对成纤维细胞粘附的影响通过物理吸附固定CMP,利用细胞粘附、竞争抑制、荧光染色等实验证实,三种包被CMP的基底均能在一定程度上促进细胞粘附、生长,具有良好的细胞粘附率和细胞附着形态,其中CMP27具有更好的促粘附效果,可观察到大量的应力纤维及伪足,粘附效果与Ⅰ型胶原相近。由此认为,胶原三螺旋结构对细胞粘附有促进作用,GFOGER序列对细胞伪足生成及应力纤维形成有支持作用。研究结果初步证实,胶原三螺旋结构与整合素识别位点序列共同作用促进成纤维细胞粘附。 4.壳聚糖膜共价偶联胶原模拟多肽对成纤维细胞粘附的影响以3-马来酰亚胺基苯甲酸琥珀酰亚胺酯(MBS)为连接臂,将壳聚糖膜与CMP共价偶联。利用核磁共振(1H-NMR)与X-射线光电子能谱(XPS)对样品进行了表征,证实多肽与MB-壳聚糖膜共价偶联成功。细胞粘附等实验说明,MB-壳聚糖浓度对成纤维细胞粘附有一定影响,当MB-壳聚糖浓度为3ng/mm2和6ng/mm2时,粘附效果较好。MB-壳聚糖膜偶联CMP后对L929细胞粘附的促进作用明显增强,偶联CMP27的效果较好,可以观察到长势良好的应力纤维与微绒毛,其粘附效果与吸附胶原的基底近似。以上结果进一步证实,胶原三螺旋结构及整合素识别位点序列共同作用促进成纤维细胞粘附。综上所述,包含胶原三螺旋结构与整合素识别位点序列的CMP可以有效促进成纤维细胞粘附,可作为表面修饰基团促进α2β1,整合素介导的细胞粘附。本研究可为设计以多肽为基础的生物活性材料提供新的研究思路,也为开发以及应用人工类胶原生物材料提供研究基础。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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