【作者】 毛峥伟；

【导师】 沈家骢； 高长有；

【作者基本信息】 浙江大学 ， 材料学， 2007， 博士

【摘要】 随着再生医学／组织工程的发展，人们对支架材料的要求也不再局限于生物相容性，而要求支架具有生物活性，能够促进细胞增殖、分化，从而引导组织再生。本文首先尝试了用生长因子构建活性支架。采用层层自组装技术，在TCPS表面成功地构建了(aFGF／heparin)／PEI自组装多层膜。体外成纤维细胞培养的结果证明，aFGF在多层膜中仍然保持一定的生物活性，可促进成纤维细胞增殖并促进Ⅰ型胶原和白介素6的分泌。体外PBS浸泡实验显示(aFGF／heparin)／PEI多层膜具有一定的稳定性，但浸泡48h后，多层膜的生物活性显著下降，而保存在-20℃可以有效地保持多层膜的生物活性。将层层自组装技术成功地应用于胶原多孔支架的改性，构建了具有生物活性的胶原支架。成纤维细胞在该支架中的MTT活性有所提高，但不显著。针对生长因子活性不易保持、难以控释、价格昂贵等缺点，我们进行了将表达生长因子的基因及其载体与组织工程支架相结合，从而构建活性支架的探索。我们制备了N-三甲基壳聚糖(TMC)和N-三甲基壳寡糖(TMO)，可通过反应时间控制其季铵化程度。TMC／TMO可与DNA通过静电作用形成几百纳米大小的微粒，其尺寸受N／P值调控。较高的季铵化程度可以获得较小的复合物微粒。载体与DNA的结合能力随着季铵化程度的增加而增大，形成复合物的表面电势亦随之增大。载体的毒性随季铵化程度和分子量的增加而增大。细胞对复合物微粒的吞噬量随着TMC的季铵化程度增加而增加，但载体的基因转染能力随季铵化程度增加而先增加后降低，其转染能力优于PEI，但仍低于Lipofectamine2000。制备了聚N-异丙基丙烯酰胺和TMC的接枝共聚物(TMC-g-PNIPAAm)，其LCST为32℃。TMC-g-PNIAAm共聚物的LCST随盐溶液浓度的增大而减小，但不受pH值影响。TMC-g-PNIPAAm／DNA微粒的大小在200～900nm之间，温度对微粒粒径的影响不显著。微粒的表面电势和TMC-g-PNIPAAm与DNA的结合力受温度调控。接枝PNIPAAm对37℃下的细胞吞噬行为没有明显影响。TMC-g-PNIPAAm／DNA微粒的基因转染效率受温度调控，降低温度可以显著提高TMC-g-PNIPAAm的基因转染效率。其最佳转染效率与Lipofectamine2000相当，而细胞毒性则显著低于Lipofectamine2000。以二氧化硅微粒为模型，在其表面接枝Tat多肽。Tat接枝量可由投料量控制，并且接枝Tat对微粒的物理性质无显著影响。发现Tat多肽可介导细胞对微粒的吞噬，同时还会改变胞吞后微粒在细胞内的分布，可将微粒导入细胞核区。使用戊二醛可以有效地将Tat多肽接枝到TMO／DNA微粒上，通过反应条件可控制Tat接枝量。接枝Tat多肽对TMO／DNA微粒的物理性质和细胞毒性无显著影响，但可显著提高细胞吞噬量和基因转染效率。以表达VEGF的质粒DNA为模型，将载体／DNA微粒通过物理吸附的方法复合到胶原支架中，结合量受微粒浓度调控。载体／DNA微粒在支架中有一定的缓释能力。将负载载体／DNA微粒的胶原支架埋植到大鼠体内，发现可显著促进血管长入支架并提高支架内血管密度。并且随着DNA浓度提高和载体的转染效率提高，其促血管生成能力也相应提高。证明负载基因的活性支架在诱导组织／血管再生方面有良好的应用前景。更多还原

【Abstract】 One strategy for regenerative medicine is to induce tissue regeneration at damaged tissues or organs with either transplanted cells or host cells. This cell induced tissue regeneration is achieved by providing a local environment which enables cells to promote proliferation and differentiation. It has been recognized that the environment is naturally composed of biological signal molecules, extracellular matrix （ECM） molecules, mechanical stress, and cell-cell interactions. Thus, "bioactive" scaffold with an appropriate combination of the biological cues may modify cell activities for tissue regeneration.Initially, we tried to realize bioactive scaffold by incorporating growth factors. A class of thin film with bioactivity was constructed by using the layer-by-layer self-assembly technique. Acid fibroblast growth factor （aFGF） in the presence of heparin was used as negatively charged polyelectrolytes, while poly（ethyleneimine） （PEI） was chosen as a positively charged counterpart. The self deposition process and surface morphology of the resultant multilayers were monitored and detected by UV-Vis absorbance spectroscopy, advanced contact angle measurements and scanning force microscopy （SFM） observations, respectively. Cell culture was performed to assess the efficiency of the growth factors. The fibroblasts proliferated faster on surface assembled with 5 bilayers of （aFGF/heparin）/PEI with apparent higher cytoviability than on those surfaces modified by 1 bilayer of （aFGF/heparin）/PEI, 5 bilayers of aFGF/PEI or 5 bilayers of heparin/PEI, and tissue culture polystyrene. Enhanced secretion of collagen type I and interleukin 6 （IL-6） by the fibroblasts seeded on the 5 bilayers of （aFGF/heparin）/PEI was also verified by immunohistochemical examination. The bioactivity of the （aFGF/heparin）/PEI multilayers could be largely preserved when stored at -20℃. Using the same method, a bioactive collagen scaffold was constructed by （aFGF/heparin）/PEI assembly. By monitoring the linear increase of FITC-labeled aFGF fluorescence intensity with the assembled layer, the self-assembled process was proved. Cell viability test showedthat the assembled aFGF has positive effect on the cell viability.As growth factors typically have half-lives only on the order of minutes and easy to denature during preparation, we intended to promote cell proliferation and differentiation or the secretion of ECM components for tissue regeneration by the transfection of genes encoding growth factors.N,N,N-trimethyl chitosan chloride （TMC） with different quaternization degree and molecular weight was synthesized. Their molecular structure was characterized by ¹HNMR. The size and the morphology of TMC/DNA particles were observed by dynamic light scattering （DLS） and transmission electron microscopy （TEM）, respectively. The particle size ranged from 150nm to 600nm depending on the N/P ratio and was less influenced by the quaternization degree. The zeta potential of these particles was increased along with the N/P ratio, while higher quaternization degree TMC resulted in particles with higher zeta potential. The affinity between DNA and TMC was examined by ethidium bromide competitive binding assay. Those vectors with higher positive charge strength have stronger ability to combine with DNA. HEK293 cell line was chosen as a model to study the cytotoxicity, cellular uptake of particles, and gene transfection. The short term contact experiments showed good biocompatibility of TMC, but long term contact experiments revealed the high toxicity of TMC. The higher quaternization degree and molecular weight lead to higher toxicity of TMC. The higher quaternization degree leaded to higher cellular uptake of TMC/DNA particles, yet exhibited lower transfection efficiency of delivered gene. The optimized gene transfection efficiency achieved is comparable to PEI, but still lower than Lipofectamine 2000.N,N,N-trimethyl chitosan-g-poly（N-isopropylacrylamide） （TMC-g-PNIPAAm）, a thermoresponsive copolymer, was synthesized by coupling PNIPAAm-COOH to TMC. Their molecular structures were characterized by ¹HNMR. The lower critical solution temperature （LCST） of TMC-g-PNIPAAm in PBS was measured as 32℃ by DLS, regardless of the grafting ratios. Upon mixing with DNA, TMC/DNA particles were formed, whose size and morphology were investigated by DLS and TEM, respectively. The particle size ranged from 200nm to 900nm depending on the N/Pratio and was less influenced by the temperature variation. The zeta potentials of these particles were increased along with the N/P ratio. At a given N/P ratio, the zeta potentials were almost constant at 25℃ regardless of the existence of serum proteins. However, the values were significantly decreased at 37℃ in a solution containing serum protein. TMC-g-PNIPAAm has stronger ability to combine with DNA at 40℃ when the PNIPAAm chain is collapsed. The grafting of PNIPAAm won’t affect cellular uptake of the particles at 37℃. The level of gene transfection could be thermally controlled. By using a temperature variation protocol, i.e. incubation of the cultured cells at 25℃ for a while, the gene transfection efficiency was significantly improved. Finally, the optimized gene transfection efficiency achieved by TMC-g-PNIPAAm is comparable to Lipofectamine 2000. No obvious cytotoxicity was detected for the TMC-g-PNIPAAm/DNA particles.In order to increase cellular uptake and nuclear targeting, Tat peptide was used to modify SiO₂ particles as a model. The grafting amount of Tat peptide could be controlled by feeding amount. The morphology and zeta potential of the modified particles were similar to the original ones. Grafting of Tat peptide would largely increase the cellular uptake of the particles at 4℃ and 37℃, and affect the subcellular distribution of particles, leading to enter of the particles in the nucleus. According to these results, Tat peptide was grafted to TMC/DNA particles via glutaraldehyde crosslinking. The size and zeta potential of modified particles were similar to the original particles. The grafting of Tat peptide would largely increase the cellular uptake of the particles and gene transfection efficiency at 37℃. No obvious cytotoxicity was detected for the Tat modified TMC/DNA particles. Finally, a bioactive collagen scaffold was realized by combination of vector/DNA particles. The in vitro releasing test showed that the DNA had a faster releasing rate in the initial stage, with subsequent slower release lasted for 96h. Plasmid DNA encoding human vascular epithelial growth factor （VEGF） was used to induce angiogenesis of scaffold in vivo. After embedded in SD mice, the pDNA loaded scaffolds showed good tissue compatibility, and had the ability to enhance the angiogenesis of scaffold in vivo.更多还原