【作者】 杨茜；

【导师】 颜炜群；

【作者基本信息】 吉林大学 ， 医学生物工程学， 2014， 博士

【摘要】 受到贝壳类海洋生物分泌的黏附性蛋白质的结构的启发，有许多学者报道了聚多巴胺的粘附机理和性能。结果表明，多巴胺（3,4-二羟基-苯丙氨酸）以及其他具有类似儿茶酚结构的化合物可沉积在不同材料基体的表面，包括有机、无机和金属材料。利用聚多巴胺层表面的基团进行进一步反应可以在材料表面形成其他的功能层，包括利用自组装形成单分子层、通过长链分子反应形成聚合物层，通过无电沉积形成金属层，以及通过大分子接枝形成的生物活性层。明胶是胶原的水解产物，其主要由组成相同且分子量分布较宽的氨基酸构成，分子量一般在几万至十几万。明胶分子结构上含有大量的羟基及少量的羧基和氨基，具有极强的亲水性。因此，明胶分子中的羧基可以与多巴胺的氨基发生缩合反应。明胶具有其他合成材料无法比拟的生物相容性、可降解性以及生物活性,如:低抗原性、促进细胞粘附等。血管内皮生长因子(VEGF)作为一种强有力的血管生长介质，不仅能特异性地作用于血管内皮细胞，刺激细胞分裂、增生和移行，而且在血管成熟和重塑中起着重要作用，从而达到治疗性血管新生的目的。为了检验固定的生长因子的长期性能，在无机材料表面上共价结合生长因子的方法是令人满意的。然而，共价结合的作用力很强而且是不可逆的，这种绑定的方式常常降低了生长因子的生物活性。适当的条件下，可解离的生长因子可以促进信号传导，并释放到组织中可以修复受损组织。因此，我们可以利用蛋白质工程的方法固定生长因子，此方法的亲和力比普通的物理吸附作用力强，而且是可逆性的。这种固定方法主要是依靠生长因子结合黏附性的多肽来实现的。本研究利用多巴胺基团中的氨基与重组人明胶分子的羧基发生了缩合反应，形成具有黏附性重组人明胶；通过在材料表面的固定效果和细胞学评价，证明黏附性重组人明胶可以稳定的固定在金属材料表面，有利于细胞的黏附和增殖；本研究首次利用直链淀粉树脂纯化柱和羟基磷灰石纯化柱提纯重组人血管内皮因子，并将重组人血管内皮生长因子和黏附性多肽DOPA在转肽酶A的作用下连接；通过在材料表面的固定效果和细胞学评价，证明了黏附性重组人血管内皮因子改性后的材料表面具有良好的细胞相容性，无论在游离状态下还是固定状态下的黏附性重组人血管内皮生长因子都可以促进细胞增殖。更多还原

【Abstract】 More and more researchers focus on the high performance of biomaterials inthese years. The surface modification is considered to be an effective way to meet thenew demand of clinical treatment, besides design and preparation of new biomaterialswith good comprehensive properties. The biocompatibility of the resulting metallicmaterials was evaluated.Currently, the modification method of metallic surface including physisorption,chemical bonding and protein engineering. Compared with the other modificationmethod of metallic surface, protein engineering has prominent features:1. both idealinterface performance, but also the nature of the material itself does not affect;2.apply a surface modification of the complex material in a variety of shapes;3. affinitybinding is stronger than normal physisorption and is also reversible;4. can not reducethe bioactivity of bound biological macromolecules.Recently, inspired by and studied on the composition of adhesive proteins inmussels, the adhesive mechanisms and adhesive behaviors of polydopamine havebeen reported. The results indicate that dopamine (or3,4-dihydroxy-phenylalanine)and other catechol compounds perform well as binding agents for coating varioussubstrates, including organic and inorganic. The oxidative polymerization ofdopamine and the spontaneous deposition of polydopamine on various surfacesprovide a powerful route for surface modification and functionalization. Secondaryreactions with the polydopamine layer can be used to create a variety of ad-layers,including self-assembled monolayers, through the deposition of long-chain molecularbuilding blocks, metal films by electroless metallization and bioactive surface viagrafting of macromolecules.The gelatin is collagen hydrolyzate, and its main by the same composition andmolecular weight distribution of a wide amino acids, and generally has a molecularweight in the ten thousands. Gelatin on the molecular structure contains a largenumber of hydroxyl groups and a small amount of carboxyl group and an amino group, with a strong hydrophilic. The carboxyl group in the gelatin molecule canoccur with the amino group of the dopamine. Compare with other synthetic material,gelatin has significant biocompatibility, biodegradability and biological activity, suchas: low antigenicity, promoting cell proliferation and differentiation.The present study was to conjugating dopamine with recombinant human gelatinto further enhance the biological activity of the material surface of the metal substrateto investigate the impact of modified methods to its surface-modified biocompatiblematerial for tissue engineering.As a potential specific endothelial cell (EC) mitogen, Vascular EndothelialGrowth Factor(VEGF) promotes the division, proliferation and migration of ECs，andalso plays an important role in the maturing and remodeling of blood vessels. Toexamine the long-term performance of growth factors, the chemical covalent bondingof growth factors to the surfaces of inorganic materials is desirable. However, strongand irreversible immobilization of growth factors to surfaces often diminishes theirbiological functionality. The dissociation of the growth factors from the scaffold mayalso be necessary for signal transduction. Therefore, the reversible immobilization ofproteins on inorganic surfaces was developed with protein engineering using adhesivepeptide binding motifs.The present study was to conjugating VEGF with adhesive peptide DOPA whichsynthesized by solid-phase synthesis to further enhance the biological activity of thematerial surface of the metal substrate to investigate the impact of itssurface-modified biocompatible biological material for tissue engineering.1. Preparation and characterization of adhesive recombinant human gelatinThrough the activation of carboxyl group, the condensation reaction, dialysis andfreeze-dried after a series of processes, the adhesive gelatin prepared, the adhesivegelatin chemical composition was visually observed as a brown flocculent solid, softand sticky.Respectively, using1H-NMR, mass spectroscopy, circular dichroism andultraviolet spectroscopy of four methods to characterized the molecular structure ofthe adhesive gelatin.The NMR spectrum of D-rhG showed the benzene ring peaks included in thedopamine at6-7ppm. It indicated dopamine was introduced in human gelatin successfully.The molecular weight of unmodified gelatin was89,612Da and of modifiedgelatin was90,634Da. The difference in the weights (1,022Da) between the modifiedand unmodified gelatins corresponded to about6dopamine molecules (152Da).CD spectra showed no significant conformational changes due to dopaminemodification，because small amount of dopamine groups was introduced in humangelatin, slightly conformational change was occurred in adhesive gelatin.Turbidity measurements were performed using a V-550spectrophotometer at awavelength of500nm. Although the color of unmodified gelatin was the same at allpH values, the color of modified gelatin was pH-dependent. At higher pH values, thecolor was dark yellow. The solution turbidity of D-rhG also depended on pH, withmore turbidity at higher pH values. It indicated polydopamine solution was occurredunder alkali condition. Although the amount of ligated dopamine is limited, but itaffects the activity of adhesive recombinant human gelatin.2. Characterization of modified material surfaceThe present study was to conjugating dopamine with recombinant human gelatinto modified titanium surface. Using X-ray photoelectron spectroscopy, quartz crystalmicrobalance and ellipsometer three methods to characterized the modified surface.Bare titanium plate exhibited mainly titanium and oxygen peaks. After treatmentwith gelatin, a new nitrogen peak appeared and the carbon peak intensity increased,while the titanium and oxygen peaks were reduced. These results demonstrate thesurface coverage of titanium by gelatins. The relative magnitudes indicate that D-rhGcovered more of the surface than rhG, and this coverage was pH-dependent.The mass of bound rhG or D-rhG was measured by QCM. Although the bindingof rhG was almost the same as that of D-rhG at acidic pH, it was significantly lessthan that of D-rhG at neutral and basic pH values.This result was also confrmed by thickness measurements using ellipsometry.The thickness ranged from5to30nm. The thickness of D-rhG layers formed at pH8.5was about4times as thick as that formed at pH4.5, and was about6times asthick as rhG formed at pH8.5. 3. Effect of modified material surface on cell behaviorThe effect of modified titanium surface on cell behavior was including cellattachment and cell growth.Cell attachment result shows that bound gelatin enhanced cell attachment, withhigher attachment on surfaces with more bound gelatin. In addition, the increase incell binding was greater for D-rhG-than rhG-treated surfaces at the same surfaceconcentration. This indicates that dopamine conjugation enhances the attachment ofboth modified gelatin and cells.Cell growth result shows the increase in cell growth with culture ongelatin-treated titanium in the presence of CBD-VEGF. Even without CBD-VEGF,D-rhG increased cell growth. The presence of CBD-VEGF significantly enhanced cellgrowth even further. In particular, surfaces treated with D-rhG at high pH lead toremarkably cell growth. As a result of the increase in growth factor binding, dopamineconjugation may enhance cell attachment and growth.4. VEGF purificationFusion protein MBP-VEGF was expressed in E.coli to obtain high levelexpressed MBP-VEGF. Utilizing amylose resin column and hydroxyapatite column topurified VEGF and SDS-PAGE to detect the result of purification. The result shows:only one band stained on the SDS-PAGE gel. And according to the known molecularweight, we can confirm to obtain the purified VEGF.5. Preparation and detection of adhesive VEGF（VEGF-DOPA）GG-XKXKX-Flag and GG-YKYKY-Flag were synthesized by solid-phasesynthesis. VEGF and a short peptide DOPA and Tyrosine were ligated by theenzymatic reaction of sortase A, which exchange the terminal amino acids of twopolypeptides. To examine the activity of VEGF and VEGF-DOPA by anti-hVEGFantibody. To detected DOPA was ligated or not by anti-FLAG antibody. The resultsindicated: western blot result is as same as SDS-PAGE result to further examine thepurification. And anti-FLAG antibody detection result showed that a band appeared atthe position about18k Da, according to the known molecular weight of VEGF (15836Da) and peptide DOPA (1978Da) to confirm that peptide DOPA was introduced inVEGF, and obtained the adhesive VEGF. 6. Characterization of modified material surfaceUsing X-ray photoelectron spectroscopy and immunological method tocharacterized the unmodified and modified surface. After treatment with VEGF,VEGF-Y and VEGF-DOPA, the carbon and nitrogen peak intensity increasedgradually, while the chrome peak intensity was reduced. The result indicated thebinding effect of DOPA is better than tyrosine.7. Effect of modified material surface on cell behaviorThe bioactivity of VEGF, VEGF-Y and VEGF-DOPA under soluble andimmobilized condition on cell behavior was examined by cell growth. The cell growthresults indicated that all the soluble VEGF, VEGF-Y and VEGF-DOPA promoted cellgrowth compare with control. And the cell growth was enhanced even further underhigh concentration of VEGF, VEGF-Y and VEGF-DOPA solutions. The cell growthon the cr. surface was enhanced after VEGF, VEGF-Y and VEGF-DOPA treatmentcompare with control. The bound VEGF-DOPA has higher bioactivity than the boundVEGF and VEGF-Y, whereas the activity of soluble VEGF-DOPA was slightly lowerthan soluble VEGF.Conclusions: In this study, the amino group in dopamine and the recombinanthuman gelatin molecules carboxyl group occurrence of a condensation reaction, ashaving a new adhesive recombinant human gelatin—D-rhG is formed; through theanchoring effect of the surface of the material and cytologic evaluation proved D-rhGstable fixed to the surface of the metallic material, is conducive to cell adhesion andproliferation; This is the first using amylose resin column and hydroxyapatite columnto purified vascular endothelial growth factor（VEGF）. VEGF and peptide DOPAwere ligated by the enzymatic reaction of sortase A; through the immobilized effect ofthe surface of the material and cytologic evaluation proved the surface treated withVEGF-DOPA has cell compatibility; VEGF-DOPA promoted the cell growth undersoluble and immobilized condition.更多还原