支架载体在BMP-9转导干细胞成骨基因治疗中特性的研究

Characterization of scaffold carriers for bmp-9 transduced cell-based gene therapy in bone regeneration

目的：比较3种可能的支架载体在以骨形态发生蛋白9（Bone morphogenetic protein 9,BMP-9）介导细胞为基础的裸鼠成骨基因治疗模型中的不同促成骨特性。方法：4~6周龄雌性裸鼠40只,体重15~20 g,随机分为4组。3种支架载体分别为I：型胶原纤维,羟基磷灰石—磷酸三钙,脱钙基质骨（Demineralized bone matrix,DBM）。分别将表达BMP-9重组腺病毒Ad-BMP-9感染后的小鼠成肌细胞系C2C12细胞注入埋植于皮下一侧的3种载体。将表达绿色荧光蛋白（Green fluorescent protein,GFP）重组腺病毒Ad-GFP感染后的C2C12细胞分别注入埋置于其对侧皮下的3种载体作为对照。第4组仅在双侧皮下分别注入Ad-BMP-9或Ad-GFP感染后的C2C12细胞。4周后处死实验裸鼠,行X线检查和组织学分析。结果：X线检测显示：Ⅰ型胶原纤维组90%样本出现新骨形成,HA-TCP组、DMB组和皮下注射组的新骨形成率分别为89%、50%和44%。组织学分析显示：Ⅰ型胶原纤维组和HA-TCP组新骨质量和数量更佳;Ⅰ型胶原纤维组样本中干细胞及新骨分布较其它组更趋均匀;DBM组样本中存活干细胞数量少于其它组。结论：在以BMP-9基因转导细胞为基础的基因治疗中,使用Ⅰ型胶原纤维或HA-TCP载体较脱钙基质骨载体可更有效成骨。同时,Ⅰ型胶原纤维和HA-TCP载体可为基因治疗中使用的细胞提供更好的组织工程环境。

Objective：To compare the ability of three carriers to promote osteogenesis in a mouse model of cell-based gene therapy using BMP-9. Methods：Forty athymic nude mice were randomized into four groups for subcutaneous implantation of carrier and C 2 C 12 cells transfected with adenovirus expressing BMP-9. Each mouse also had a control consisted of carrier with cells infected with adenovirus expressing green fluorescence protein （GFP）. One of the four groups had only transfected cells injected subcutaneously with no carrier. The following groups were defined： ① collagen type Ⅰ sponge,② Tri-calcium-phosphate/hydroxy-apatite（HA-TCP）,③Demineralized bone matrix （DBM）,and ④ the subcutaneous injection group.The mice were sacrificed at 4 weeks and plain radiographs obtained. Histologic evaluation of the implantation sites were performed. Results：In the collagen sponge group,9 of 10 （90%）had radiographic evidence of new bone formation,8 of 9（89%）in the HA-TCP group,3 of 6（50%）in the DBM group,and 4 of 9（44%）in the cell-only group. Histologic findings from selected samples included the following：better quality and larger amounts of bone were found in the collagen sponge and HA-TCP samples;a more uniform distribution of cells and bone were noted throughout the collagen sponge samples when compared to the other carriers;a decreased number of cells were noted in DBM samples when compared to the other carriers. Conclusion：Collagen type Ⅰ sponge and HA-TCP carriers consistently produce more bone than DBM carrier in cell-based gene therapy using BMP-9. Addtionally,collagen sponge and HA-TCP provide a better environment for cell based gene therapy as evidenced in the histologic data.