基因修饰的组织工程骨修复眶骨缺损的实验研究

目的 探索应用基因转染的组织工程技术修复眶骨缺损的可行性.方法 实验研究.体外分离、扩增兔骨髓基质干细胞(BMSC),将人骨形成蛋白2(BMP-2)基因通过腺病毒转染的方式导入BMSC,采用RT-PCR、免疫印迹法检测目的 基因的表达情况,并观察基因增强后的BMSC体外增殖特性和成骨能力.将BMP-2基因修饰后的BMSC复合天然珊瑚材料构建组织工程骨.24只成年新西兰白兔制造双侧眼眶骨下缘12 mm长的节段性骨缺损,共计48侧.随机数字表法分为4组,每组12侧眶骨缺损,行组织工程骨回植修复.A组为以转染BMP-2的自体BMSC构建的组织工程骨,B组为未转染基因的自体BMSC构建的组织工程骨、C组为单纯珊瑚材料,D组为旷置组.分别在手术后4、8、16周取材进行大体观察、micro-CT骨密度分析、组织学观察和组织形态学检测,比较骨缺损修复效果.采用单因素3水平设计定量资料方差分析处理新生骨和正常骨密度之间的差异,并采用LSD法对3个平均值进行两两比较;采用两因素析因设计定量资料方差分析处理新生骨占骨缺损面积的百分比.结果 BMP-2基因修饰后的BMSC能够高表达目的 基因,并且转染后的细胞体外成骨能力明显增强;构建的组织工程骨能够良好修复自体眶骨缺损,新生骨密度、成骨面积值均显著高于各对照组(F=11.46,F=7180.97;P<0.05).结论 BMP-2基因修饰的组织工程骨具有良好修复眶骨缺损的能力.(中华眼科杂志,2009,45:66-72)

Repairing rabbit orbital defects with hnman BMP-2 gene modified bone marrow stromal cells and coral

Objective To investigate the efficacy of tissue-engineered bone (human BMP-2 genetic modified BMSC combined with coral) in healing the segmental orbital defect in rabbits. Methods Rabbit BMSC were isolated and cultured in vitro, and cells of passage 2 were infected with adenovirus-mediated transfection of human BMP-2 gene (150 pfu/cell). After infection, the expression of BMP-2 was determined by RT-PCR and Western blot analysis, and cell proliferation and osteogenic differentiation were observed by flow cytometry, ALP and Alizarin red staining. A 12 mm bone defect in the infraorbital rim was induced by surgery in both orbits of 24 New Zealand white rabbits. The defects were repaired with modified tissue-engineered bone constructed with coral plus BMP-2 transfected BMSC (Group A, n=12), constructed by coral plus non-transfected BMSC (Group B, n=12) and grafts of coral alone (Group C, n=12), with untreated group (Group D, n=12) served as control. The osteogenesis of bone defect was monitored by gross observation, micro-CT measurement, histological and histomorphologic analysis at 4, 8, and 16 weeks after the implantation. Results After transfection, the BMP-2 expression was confirmed by RT-PCR and western blot, and the osteogenesis activity of BMSC could be obviously enhanced. The 12 mm segmental defect of rabbit orbit couldn't heal alone. Gross observation and micro-CT demonstrated well the bony-union in experimental group, with higher bone mineral density and more bone volume than other control groups (F=11.46,F=7180.97;P<0.05). Conclusion This study demonstrated that the rabbit orbital defect could be successfully repaired by tissue-engineered bone constructed with human BMP-2 gene modified BMSC and coral. (Chin J Ophthalmol, 2009,45:66-72)