CAJ | 학술논문

目的观察聚乙二醇(PEG)/骨形态发生蛋白(BMP)-2纳米基因复合物及载基因仿生骨与骨髓间充质干细胞(BMSCs)对骨缺损修复的共同作用.方法通过离子交联法制备PEG/BMP-2纳米基因复合物,通过溶液共混法制备聚乳酸(PLA)/聚已内酯(PCL)载基因仿生骨并接种BMSCs于仿生骨之上,应用免疫组化及免疫印迹检测BMSCs转染后BMP-2蛋白表达;酶联免疫方法检测转染后细胞培养上清中BMP-2分泌情况;实时聚合酶链反应检测转染后细胞BMP-2及骨钙素mRNA表达水平;截除新西兰大耳白兔双侧桡骨中段,植入载基因仿生骨材料,对骨缺损修复部位摄X线片、行HE染色和BMP-2免疫组织化学染色.结果制备出PEG/BMP-2纳米颗粒及PEG-BMP-2-PLA/PCL载基因仿生骨.PEG/BMP-2纳米颗粒转染BMSCs和载基因仿生骨BMSCs内BMP-2表达量明显上调,骨钙素mRNA表达和碱性磷酸酶活性有所增加;体内实验中,PEG-BMP-2-PLA/PCL载基因仿生骨组与对照组比较BMP-2表达升高,新生骨在骨缺损区域所占面积比也明显增加.结论 PEG-BMP-2-PLA/PCL对骨缺损修复具有良好的效果.
목적 관찰취을이순(PEG)/골형태발생단백(BMP)-2납미기인복합물급재기인방생골여골수간충질간세포(BMSCs)대골결손수복적공동작용.방법 통과리자교련법제비PEG/BMP-2납미기인복합물,통과용액공혼법제비취유산(PLA)/취이내지(PCL)재기인방생골병접충BMSCs우방생골지상,응용면역조화급면역인적검측BMSCs전염후BMP-2단백표체;매련면역방법검측전염후세포배양상청중BMP-2분비정황;실시취합매련반응검측전염후세포BMP-2급골개소mRNA표체수평;절제신서란대이백토쌍측뇨골중단,식입재기인방생골재료,대골결손수복부위섭X선편、행HE염색화BMP-2면역조직화학염색.결과 제비출PEG/BMP-2납미과립급PEG-BMP-2-PLA/PCL재기인방생골.PEG/BMP-2납미과립전염BMSCs화재기인방생골BMSCs내BMP-2표체량명현상조,골개소mRNA표체화감성린산매활성유소증가;체내실험중,PEG-BMP-2-PLA/PCL재기인방생골조여대조조비교BMP-2표체승고,신생골재골결손구역소점면적비야명현증가.결론 PEG-BMP-2-PLA/PCL대골결손수복구유량호적효과.
Objective To explore the efficacy of a novel tissue engineered bone in repairing bone defects using poly-lactic acid-polycaprolactone (PLA-PCL) scaffolding seeded with PEG-bone morphogenetic protein-2 (BMP-2) transfected rBMSCs (rabbit bone marrow stromal cells).Methods rBMSCs were harvested,transfected with PEG/BMP-2 or liposome/BMP-2 and then implanted into PLA-PCL tissue engineered bone.The protein level of BMP-2 was assessed by Western blot and immunohistochemistry.Enzyme-linked immunosorbent assay (ELISA) was employed to measure the amount of BMP-2 in culture media.The mRNA levels of BMP-2 and osteocalcin were assayed quantitatively by real-time polymerase chain reaction (PCR).The middle portion of bilateral radius in New Zealand rabbits was excised and implanted with tissue engineered bone.And the modified areas were monitored by radiology,hematoxylin-eosin staining and immunohistochemical staining of BMP-2.Results PEG-BMP-2 nanoparticles (NPs) and BMP-2 loaded PEG-PLA-PCL tissue engineered bones were successfully constructed.The novel PEG-PLA-PCL NPs/DNA complex was superior for transfecting BMP-2 in rBMSCs compared to traditional liposomes.Moreover,the mRNA level of osteocalcin and alkaline phosphatase activity also increased after a transfection of BMP-2 encapsulated NPs.Conclusion PEG-PLA-PCL NPs/BMP-2 complex facilitates bone repair so that it provides theoretic rationales for potential clinical treatments.