腺病毒介导的骨形态发生蛋白-2基因治疗骨缺损的研究

Experiment of treatment of bone defect with adenovirus-mediated expression of bone morphogenetic protein-2

目的探讨以胶原海绵为支架,用含腺病毒介导的骨形态发生蛋白(BMP)-2的鼠肌母细胞修复小鼠胫骨缺损的可行性。方法制备鼠BMP-2腺病毒质粒并经PCR鉴定;将体外培养肌源性干细胞的贴壁细胞传代,取第3代细胞,在鼠肌母细胞中表达重组Ad-BMP-2质粒并孵育。采用间接免疫荧光法测定BMP-2阳性细胞的数量,碱性磷酸酶(ALP)法测定各组细胞ALP活性。建立胫骨近端骨缺损小鼠模型(n=80),分为4组:Ad-BMP-2转染组、Ad-LacZ转染组、空白组和Ad-BMP-2植入组。术后第3周观察各组小鼠植入区组织学变化和模型处放射学改变。结果间接免疫荧光法测定显示,Ad-BMP-2转染组中BMP-2阳性细胞多见,而Ad-LacZ转染组和空白组中未见阳性细胞,仅见微弱的背景染色。Ad-BMP-2转染组中ALP活性含量较高,而Ad-LacZ转染组和空白组ALP活性含量极低。Ad-BMP-2转染组模型处有明显的骨缺损修复、愈合成像,Ad-BMP-2植入组次之,Ad-LacZ转染组和空白组表现不明显。结论构建的Ad-BMP-2质粒能高效转染小鼠肌母细胞,且转染后的细胞能在机体持续分泌有效量的BMP-2。腺病毒介导的转染BMP-2的鼠肌母细胞具有明确的骨缺损修复能力,为临床促进骨折愈合提供了一种有效的方法和材料。

Objective To explore the feasibility of mouse muscle-derived cells with adenovirus-mediated expression of BMP-2 in the treatment of mouse tibia bone defect via collagen sponge as scaffold. Methods Mouse adenovirus BMP-2 plasmids were prepared and identified by PCR. The adherent cells of muscle-derived stem cells cultured in vitro were passaged, cells of the third generation were selected, and recombinant Ad-BMP-2 plasmids were expressed and incubated in mouse myoblasts. The number of BMP-2 positive cells was measured using indirect immunofluorescence method, and alkaline phosphatase （ALP） method was adopted to determine the cell activity in each group. The mouse models of proximal tibial bone defect were established （n = 80） and were divided into Ad-BMP-2 transfection group, Ad-LacZ transfection group, blank group and Ad-BMP-2 implantation group. The histological and radiologieal changes were observed 3 weeks after operation. Results It was revealed by indirect immunofluorescence that BMP-2 positive cells were frequently found in Ad-BMP-2 transfection group, while none were observed in Ad-LacZ transfection group and blank group. The ALP activity was higher in Ad-BMP-2 transfection group, while that was extremely low in Ad-LacZ transfection group and blank group. There was most typical imaging of repair and healing of bone defect in Ad-BMP-2 transfection group, followed by Ad-BMP-2 implantation group, while there was no typical imaging in Ad-LacZ transfection group and blank group. Conclusion The constructed Ad-BMP-2 plasmids can effectively transfect mouse muscle-derived cells, and the transfected cells can continuously express effective amount of BMP-2. The mouse muscle-derived cells with adenovirus-mediated expression of BMP-2 play a definite role in the treatment of bone defect, which provides an effective method and material for clinical application.