rhBMP-2和rhVEGF转染ADSCs后体外表达及诱导骨缺损成骨修复的实验研究  

Expression of rhBMP-2 & rhVEGF gene in ADSCs and application those to bone defect repair

作　　者：杨在亮[1] 张波[1] 周继红[1] 黄宏[1] 张良[1] 刘大维[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所第四研究室创伤,烧伤复合伤国家重点实验室,重庆400042

出　　处：《创伤外科杂志》2007年第4期296-300,共5页Journal of Traumatic Surgery

基　　金：国家"973"项目资助(2005CB522603)

摘　　要：目的观察重组人骨形成蛋白2(rhBMP-2)和血管内皮生长因子(rhVEGF)转染脂肪源性干细胞(ADSCs)后的体外表达情况,并以含该转染体系的组织工程骨进行大动物负重骨缺损的修复研究。方法将外源性基因rhBMP-2和rhVEGF通过脂质体LipofectamineTM2000介导的方式转染第四代ADSCs,以G418筛选出阳性克隆并扩大培养后,用RT-PCR和SABC免疫组化法在转录和蛋白质水平观察其转染后第2天和第4周表达情况。建立小香猪自体前肢尺骨中段1.5cm骨缺损模型,分3组进行对比试验:(A组)转染了生物活性因子的ADSCs与脱细胞骨基质材料(ACBM)复合而成新型组织工程骨植入修复组;(B组)未转染生物活性因子的ADSCs复合ACBM植入修复组;(C组)未处理组。以X线和组织切片评价其修复效果。结果转基因ADSCs在转染后瞬时和4周时都可表达rhBMP-2和rhVEGF。大动物骨缺损修复实验中,前2周内3组动物均未出现明显的急性炎症反应;连续X线观察和骨切片显示:A组在术后第3个月已完全修复,且修复效果和进程明显优于B组,C组缺损则主要由纤维结缔充填。结论rhBMP2及rh-VEGF转染ADSCs后可获得4周内稳定的表达。携带该缓释体系的新型组织工程骨是一种具有显著成骨能力的优良骨缺损修复材料。Objective To observe the expression of rhBMP-2 ＆ VEGF gene that were transfected into adipose -derived stem cells （ADSCs） in vitro,and application of the tissue-engineered bone with the transfected compound to repair the bone defect of ulna of Guizhou minipigs. Methods The ADSCs were transfected with rhBMP-2＆ VEGF by LipofectamineTM2000 and selected by G418. The expression of rhBMP-2 ＆ VEGF gene in ADSCs was tested with im-munohistochemistry and RT-PCR in the 48h and 4th week after transfected. The minipigs were divided into 3 groups ： the 1.5cm bone defect was made on the midpiece of ulna. In A group,the defect was repaired by ACBM with the AD-S, Cs that transfected by rhBMP-2 ＆ VEGF;in B group,the defect was repaired by ACBM with the ADSCs only;in C group, no treatment for bone defect. The effects of repair were evaluated by X-ray and histological section. Results The rhBMP-2 ＆ VEGF gene could be expressed in 48h and 4th week after transfected. In all 3 groups there were no acute inflammatory reaction and focal necrosis in two weeks after operation. The X-ray and histological evidence showed completely repair in A group,and their capacity of repairing bone defect were better than those of B group. In C group, bone defect were filled by fibrous tissue. Conclusion The rhBMP-2 ＆ VEGF gene in ADSCs have stable expression in four weeks after transfected. The tissue-engineered bone that are constructed by ACBM and ADSCs with rhBMP-2 ＆ VEGF gene is a satisfactory and effective osteogenetic repair substitute for bone defect.

关 键 词：重组人骨形成蛋白2 血管内皮生长因子 转染 转染脂肪源性干细胞 表达 组织工程骨 骨缺损 修复 

分 类 号：R683[医药卫生—骨科学]