BMP-2转染间充质干细胞上清对软骨的影响

Effect of supernatant of BMP-2 transferred mesenchymal stem cells on cartilage

[目的]观察从腺病毒-骨形态发生蛋白-2-绿色荧光蛋白(Ad-BMP-2-GFP)转染骨髓间充质干细胞(BMSCs)获取的含有BMP-2的上清对软骨细胞体外凋亡和体内再生的影响。[方法]通过Ad-BMP-2-GFP转染BMSCs使BMP-2高表达,采用冰冻干燥法浓缩BMSCs细胞上清至10倍浓度,过滤后-20℃储存备用。体外实验:加入IL-1β(18 ng/ml)建立软骨细胞体外炎症环境,施加转染后和未转染浓缩上清,并以PBS处理为空白对照组,流式细胞术检测各处理组软骨细胞凋亡水平。体内实验:20只雄性新西兰大白兔随机分为4组,除假手术组外,其他3组建立兔膝关节软骨缺损模型,分别注射等量的纯生理盐水、转染组和未转染的培养上清。6周后取材,通过大体标本和番红O固绿染色观察关节软骨缺损修复效果。[结果]体外实验显示,转染上清组的软骨细胞凋亡水平较未转染上清组和空白对照组明显减弱(P<0.05);体内实验显示,转染上清组对软骨缺损修复效果优于未转染上清组和单纯生理盐水组。[结论]通过Ad-BMP-2-GFP转染BMSCs,获取含有BMP-2的上清可以抑制体外炎症环境下软骨细胞凋亡,促进体内软骨缺损修复。

[Objective]To explore the effect of the supernatant containing BMP-2 obtained from bone marrow mesenchymal stem cells(BMSCs)transfected with adenovirus-bone morphogenetic protein-2-green fluorescent protein(Ad-BMP-2-GFP)on chondrocyte apoptosis in vitro and cartilage regeneration in vivo.[Method]BMSCs of rabbits were transfected or not transfected with Ad-BMP-2-GFP to express BMP-2,and the supernatants were concentrated to 10-fold concentration by freeze-drying method,and stored at-20°C for later use.In vitro the IL-1β(18 ng/ml)was added the chondrocyte cultures to establish the inflammatory environment,after that,10%the transfected or untransfected supernatants were applied into the cultures while PBS used as blank control.The chondrocyte apoptosis was measured by flow cytometry.In vivo study 20 male New Zealand white rabbits were randomly divided into 4 groups,including the sham,normal saline,transfected and untransfected supernatant groups.The animals in the sham group had arthrotomy only without any intraarticular injection,while those in the latter 3 groups were made a cartilage defect 6 mm in diameter deep to subchondral bone on the central trochlea of femoral condyle,and were intraarticularly injected 500μl of normal saline,transfected and untransfected supernatants respectively.Six weeks later,repair of the cartilage defects was assessed by gross specimen and Safranin O green staining.[Results]The experiments in vitro showed that the level of chondrocyte apoptosis in the transfected supernatant group was significantly weaker than that in the untransfected supernatant group and the blank control group(P<0.05).In addition,the experiments in vivo revealed that the transfected supernatant group had significantly better repair than the non-transfected supernatant group and the normal saline group.[Conclusion]This supernatant containing BMP-2 obtained from BMSCs transfectedwith Ad-BMP-2-GFP does inhibit the apoptosis of chondrocytes in inflammatory environment in vitro and promote cartilage regeneration in vivo.