【作者】 郭瑞；

【导师】 高长有； 马列；

【作者基本信息】 浙江大学 ， 材料学， 2010， 博士

【摘要】 本文将基因治疗与组织工程支架相结合,构建了基因活性支架。首先,合成了阳离子基因载体N-三甲基壳聚糖(TMC),TMC可以通过静电作用压缩DNA形成纳米复合粒子。将TMC/DNA复合粒子通过物理吸附的方法负载到胶原-壳聚糖支架上,发现TMC/DNA微粒在支架中有一定的缓释能力。释放出来的TMC/DNA微粒中的DNA有超螺旋结构的存在,且具有较高的细胞转染能力。将TMC/pDNA-VEGF微粒负载到支架上制备了基因活性支架,体外实验表明：基因活性支架具有较高的细胞活性,HUVECs可以在支架上保持细胞的表型,支架上的TMC/pDNA-VEGF微粒具有较高的转染效率,可以转染细胞,分泌更多的VEGF。在本课题组前期的研究工作中,制备了胶原-壳聚糖／硅胶膜双层人工真皮替代物(BDEs)。为了增强人工真皮替代物的血管化能力,将TMC/pDNA-VEGF微粒负载到空白人工真皮替代物上,制备了基因活性人工真皮替代物(gene-activated BDEs)。猪创伤全层皮肤缺损的修复结果表明：术后7天、10天和14天,基因活性真皮替代物处理的创面具有最高的新生血管数和成熟血管数。PCR结果证实TMC/pDNA-VEGF微粒可以有效转染细胞,表达VEGF。基因活性真皮替代物处理10天的创面,即可进行二次移植。超薄皮片移植112天后,创面破裂强度可以达到正常皮肤的80%。愈合的全层皮肤表皮层和真皮层连接紧密,乳头层分化较好,胶原束和正常皮肤的结构相似。同时探讨了基因活性人工真皮替代物的使用在真皮修复过程中对相关因子的影响及其可能的修复机理。TMC/pDNA-VGF微粒在体内可以实现长达70天的细胞转染、表达。基因活性人工真皮替代物处理的创面具有最高的VEGF和TGF-β3 mRNA的表达水平,而TGF-β1 mRNA的表达水平较低,可以减少创面愈合后疤痕组织的产生。愈合后的创面真皮、表皮连接紧密,乳头层分化较好。空白真皮替代物处理的创面愈合结果优于凡士林纱布处理的创面。烧伤创面是个动态创面,会随着时间的增加而加深,其修复比创伤创面要困难很多。将基因活性人工真皮替代物用于烧伤全层皮肤缺损的修复,结果表明：术后7天、14天和21天,基因活性真皮替代物处理的创面具有最高的新生血管数和成熟血管数,说明真皮替代物的血管化明显增强。基因活性真皮替代物处理的烧伤创面,14天即可进行二次移植。超薄皮片移植105天后,创面的破裂强度可以达到正常皮肤的70%。愈合的全层皮肤表皮层和真皮层连接紧密,乳头层分化较好。将空白真皮替代物和基因活性真皮替代物与临床产品桀亚J-1 ADM用于创伤、烧伤修复的效果做了对比研究,结果表明：对于创伤全层皮肤缺损的修复,基因活性真皮替代物优于J-1 ADM和空白真皮替代物,空白真皮替代物和J-1ADM具有相近的修复效果。对于烧伤全层皮肤缺损的修复,基因活性真皮替代物优于J-1 ADM和空白真皮替代物,J-1 ADM的修复效果略好于空白真皮替代物。更多还原

【Abstract】 Gene therapy and tissue-engineered scaffolds were combined to fabricate bioactive scaffolds. A cationic gene delivery vector, N,N,N-trimethyl chitosan chloride (TMC), was synthesized and used to condense DNA. Then, the TMC/DNA complexes were incorporated into collagen-chitosan scaffold. The in vitro releasing test showed that TMC/DNA complexes had a faster releasing rate in the initial stage, and then slowly release until 28 days. The released plasmid DNA with supercoiled structure was detected; the released TMC/DNA complexes were still capable of transfecting a high percentage of cells. TMC/pDNA-VEGF complexes were incorporated into the collagen-chitosan scaffold to build a gene-activated scaffold. The in vitro results demonstrated that cells kept their phenotype and had a higher viability on the scaffold. The loaded TMC/pDNA-VEGF complexes possessed higher transfection efficiency too.In the previous studies, collage-chitosan/silicone membrane bilayer dermal equivalents (BDEs) were prepared. To develop the next generation BDE with higher performance, a faster rate of angiogenesis is highly demanded. Therefore, the TMC/pDNA-VEGF complexes were incorporated into BDEs to obtain the gene-activated BDEs. To evaluate the angiogenesis property in vivo, the gene-activated BDEs were transplanted into the full thickness incisional wounds. At day 7,10 and 14 after surgery, immunohistochemistry and immunofluorescence results showed that the gene-activated BDE group had the highest number of newly-formed and mature blood vessels. RT-qPCR results showed that the TMC/pDNA-VEGF complexes could effectively transfect cells in vivo and express VEGF. The ultra-thin skin graft was further transplanted onto the dermis regenerated by the gene-activated BDEs at day 10 and well survived. At 112 days after grafting, the healing skin had a similar structure and～80% tensile strength of the normal skin. The epidermis had formed papillary structure, conveying its well differentiation.Meanwhile, the effects and the possible dermal repair mechanism of the gene-activated BDEs were evulated in the full-thickness incisional wound repair process. The in vivo transfection of the TMC/pDNA-VEGF complexes was sustained as long as 70 days. The wound tissue treated by gene-activated BDEs had higher VEGF and TGF-β3 mRNA expression, but the TGF-β1 mRNA expression was lower. The healed skin had a tighter connection between epidermis and dermis, and the papillary structure was also observed. The use of the gene-activated BDE could reduce the formation of scar tissue. Moreover, the healing effects of the blank BDE were better than petrolatum gauze.Burn wounds are dynamic wounds, which can deepen with time, thereby the repair of burn wounds are more difficult than the incisional wounds. The gene-activated BDEs were also used to repair the full-thickness burn wounds. The in vivo results showed that, at day 7,14 and 21 after surgery, the gene-activated BDE group had the highest number of newly-formed and mature blood vessels, and the enhanced angiogenesis was realized. The ultra-thin skin graft was further transplanted onto the dermis regenerated by the gene-activated BDEs at day 14 and well survived. At 105 days after grafting, the healing skin had a similar structure and～70% tensile strength of the normal skin.Finally, the blank BDE and the gene-activated BDE were compared with a clinical dermal equivalent J-1 ADM in the treatment of incisional and burn wounds. The results showed that as for incisional wounds treatment, the gene-activated BDE was superior to blank BDE and J-1 ADM, and the blank BDE was nearly equal to J-1 ADM. As far as burn wounds treatment was concerned, the gene-activated BDE was superior to blank BDE and J-1 ADM, and J-1 ADM was slightly better than the blank BDE.更多还原