携载BMP4基因的核壳结构纳米粒子修复兔桡骨临界骨缺损的研究
发布时间：2018-08-13 16:02
【摘要】：背景及目的:骨缺损常因创伤、感染、肿瘤等所致,传统的治疗方法如自体骨移植、异体骨移植或非骨性材料等并发症多且效果不理想。因此,研究者们将骨组织工程与基因治疗技术结合,探讨能够在体外安全有效缓释并表达目的基因的载体。最近研究表明,壳聚糖(CS)也可以作为基因载体,其具有独特的生物安全性和生物相容性,可与基因形成稳定的纳米粒子,在体内缓慢降解而缓释基因。骨形成蛋白(BMP)在骨修复及骨再生中起着非常重要的作用,BMP4蛋白是BMP家族中重要的蛋白之一,具有诱导骨细胞分化和促进新生骨的形成作用。本研究旨在探究修饰后的壳聚糖包裹BMP4基因形成的核结构及核壳结构在体外转染效率及体内对兔桡骨临界骨缺损的修复作用。方法:巯基烷基化壳聚糖(TACS)与BMP4质粒通过正负电作用形成核结构,外包裹聚乙二醇修饰羟丁基壳聚糖(EG-HBC)形成壳结构,对HEK293T细胞进行体外转染,Western blot,免疫荧光实验探索核及核壳结构的转染效率。构建兔子双前肢18mm完全性临界骨缺损模型,分别植入含有TACS@EG-HBC/pBMP4或TACS/pBMP4的明胶海绵,对照组仅植入单纯的明胶海绵。术后2、4、8、12周取标本做分别处理,检测缺损部位的大体标本、骨密度及骨矿物质含量、X线、HE染色、免疫组化法、生物力学。结果:Western blot显示TACS@EG-HBC/pBMP4蛋白表达最高,免疫荧光结果示TACS@EG-HBC/pBMP4转染效率高于TACS/pBMP4组和对照组。大体标本示:TACS@EG-HBC/pBMP4/G组2周可见新生骨痂,4周缺损处骨痂接近桥接,中间有纤维组织,8周骨痂骨化并桥接,12周骨缺损完全骨化修复;骨密度及骨矿物质含量显著高于TACS/pBMP4/G组和G组;X线示:术后12周骨缺损处已完全修复,骨髓腔已再通;HE染色:4周时可见大量软骨细胞,后形成骨小梁相互连接成片,到12周时新生骨小梁相互连接成板层骨;免疫组化:BMP4棕色蛋白染色明显;生物力学测定:所形成的新生骨与正常骨组织生物力学无统计学差异。结论:TACS@EG-HBC/pBMP4/G具有良好的缓释基因和成骨能力。
[Abstract]:Background and objective: bone defect is often caused by trauma, infection, tumor and so on. The traditional treatment methods such as autogenous bone transplantation, allograft bone graft or non-bone material have many complications and the effect is not satisfactory. Therefore, the researchers combine bone tissue engineering with gene therapy to explore vectors that can effectively and safely release and express the target gene in vitro. Recent studies have shown that chitosan (CS) can also be used as gene carrier, which has unique biological safety and biocompatibility, and can form stable nanoparticles with gene, and slow degradation and slow release of genes in vivo. Bone morphogenetic protein (BMP) plays a very important role in bone repair and bone regeneration. BMP4 protein is one of the most important proteins in the BMP family, which can induce bone cell differentiation and promote the formation of new bone. The purpose of this study was to investigate the transfection efficiency of the modified chitosan coated BMP4 gene and its core-shell structure in vitro and the repair effect of the modified chitosan on the critical bone defect of rabbit radius in vivo. Methods: mercaptoalkylated chitosan (TACS) and BMP4 plasmids were used to form nuclear structure by positive and negative electric interaction, and EG-HBC was coated with polyethylene glycol to form shell structure. HEK293T cells were transfected with Western blot in vitro. The transfection efficiency of nuclear and core-shell structures was investigated by immunofluorescence assay. A complete critical bone defect model of 18mm in rabbit forelimbs was established and gelfoam containing TACS@EG-HBC/pBMP4 or TACS/pBMP4 was implanted into the control group. The specimens were collected for 12 weeks after operation, and the gross specimens were detected, bone mineral density (BMD) and bone mineral content (BMD), bone mineral content (BMD) and bone mineral content (BMD) were stained with HE, immunohistochemistry and biomechanics. Results the highest expression of TACS@EG-HBC/pBMP4 protein was detected by Western blot, and the transfection efficiency of TACS@EG-HBC/pBMP4 was higher than that of TACS/pBMP4 group and control group. Gross specimens showed that the callus at the defect of 4 weeks was close to the bridge in the group of EG-HBC / pBMP4 / G with fibrous tissue at 8 weeks and complete ossification of bone defect at 12 weeks. Bone mineral density (BMD) and bone mineral content (BMD) in TACS/pBMP4/G group and G group were significantly higher than those in TACS/pBMP4/G group and G group. At 12 weeks after operation, bone defects had been repaired completely, and a large number of chondrocytes could be seen in the medullary cavity at 4 weeks after HE staining, and trabeculae were connected to each other. At the 12th week, trabeculae were connected to each other to form lamellar bone; immunohistochemical staining of brown protein of BMP4 was obvious; biomechanical measurement: there was no significant difference in biomechanics between the formed new bone and normal bone tissue. Conclusion the EG-HBC / pBMP4 / G has good ability of sustained release gene and osteogenesis.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R687
本文编号：2181482
[Abstract]:Background and objective: bone defect is often caused by trauma, infection, tumor and so on. The traditional treatment methods such as autogenous bone transplantation, allograft bone graft or non-bone material have many complications and the effect is not satisfactory. Therefore, the researchers combine bone tissue engineering with gene therapy to explore vectors that can effectively and safely release and express the target gene in vitro. Recent studies have shown that chitosan (CS) can also be used as gene carrier, which has unique biological safety and biocompatibility, and can form stable nanoparticles with gene, and slow degradation and slow release of genes in vivo. Bone morphogenetic protein (BMP) plays a very important role in bone repair and bone regeneration. BMP4 protein is one of the most important proteins in the BMP family, which can induce bone cell differentiation and promote the formation of new bone. The purpose of this study was to investigate the transfection efficiency of the modified chitosan coated BMP4 gene and its core-shell structure in vitro and the repair effect of the modified chitosan on the critical bone defect of rabbit radius in vivo. Methods: mercaptoalkylated chitosan (TACS) and BMP4 plasmids were used to form nuclear structure by positive and negative electric interaction, and EG-HBC was coated with polyethylene glycol to form shell structure. HEK293T cells were transfected with Western blot in vitro. The transfection efficiency of nuclear and core-shell structures was investigated by immunofluorescence assay. A complete critical bone defect model of 18mm in rabbit forelimbs was established and gelfoam containing TACS@EG-HBC/pBMP4 or TACS/pBMP4 was implanted into the control group. The specimens were collected for 12 weeks after operation, and the gross specimens were detected, bone mineral density (BMD) and bone mineral content (BMD), bone mineral content (BMD) and bone mineral content (BMD) were stained with HE, immunohistochemistry and biomechanics. Results the highest expression of TACS@EG-HBC/pBMP4 protein was detected by Western blot, and the transfection efficiency of TACS@EG-HBC/pBMP4 was higher than that of TACS/pBMP4 group and control group. Gross specimens showed that the callus at the defect of 4 weeks was close to the bridge in the group of EG-HBC / pBMP4 / G with fibrous tissue at 8 weeks and complete ossification of bone defect at 12 weeks. Bone mineral density (BMD) and bone mineral content (BMD) in TACS/pBMP4/G group and G group were significantly higher than those in TACS/pBMP4/G group and G group. At 12 weeks after operation, bone defects had been repaired completely, and a large number of chondrocytes could be seen in the medullary cavity at 4 weeks after HE staining, and trabeculae were connected to each other. At the 12th week, trabeculae were connected to each other to form lamellar bone; immunohistochemical staining of brown protein of BMP4 was obvious; biomechanical measurement: there was no significant difference in biomechanics between the formed new bone and normal bone tissue. Conclusion the EG-HBC / pBMP4 / G has good ability of sustained release gene and osteogenesis.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R687
【参考文献】
相关期刊论文 前1条
1 Sébastien Gittens;The ectopic study of tissue-engineered bone with hBMP-4 gene modified bone marrow stromal cells in rabbits[J];Chinese Medical Journal;2005年04期
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