pCDNA3.1/BMP-4的构建与家兔骨干缺损的基因治疗
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摘要
目前的骨折治疗手段，包括外科和物理等疗法能使大部分骨折得到有效的愈合。然而，即使如此，许多研究人员仍在不懈努力以探寻其它新的方法来确保较复杂的骨折能得到有效的治疗。目前，从细胞水平控制和影响骨折愈合的生长因子及其转移系统的研究尤为引人注目，其中有关重组蛋白如骨形态发生蛋白的研究更备受关注。但直接应用这类生长因子往往因其半衰期过短等原因而受限制，因此有关骨折的基因治疗又成为新的研究热点。针对这一新的研究趋势，我们构建了pCDNA3.1/BMP-4质粒并对BMP-4基因进行了测序，结果表明BMP-4基因序列完整。将pCDNA3.1/BMP-4质粒转染成纤维细胞并通过RT-PCR、免疫蛋白印迹反应等技术证明BMP-4基因能够进行表达，且表达所分泌的BMP-4可以诱导骨髓基质细胞分化为成骨细胞。基于以上结果，我们设计了直接注射pCDNA3.1/BMP-4脂质体用于家兔尺骨缺损模型和火器性家兔股骨缺损模型治疗的两项实验研究，以探讨基因治疗在骨折愈合中的作用。
pCDNA3.1/BMP-4治疗家兔尺骨缺损：48只家兔被随机平均分为4组，两侧尺骨中段均被截除15mm。A组12只动物于术后1周注射40μg pCDNA3.1/BMP-4脂质体于尺骨缺损之间组织内；B组12只动物除术后1周注射40μg pCDNA3.1/BMP-4脂质体外，术后2、4周分别注射20ng TGF-β_1。对照组C组12只动物术后仅注射pCDNA3.1，而D组12只动物仅手术造成尺骨缺损。术后2、4、8、12周对骨缺损间局部组织进行碱性磷酸酶、钙含量、病理学和放射学检查。主要结果和结论如下：
1.BMP一基因可在骨缺损间局部组织内进行转录和翻译，并持续6周
时间。
2.A、B组局部组织术后4周时出现软骨细胞，软骨化骨过程开始。
12周时B组成骨过程接近完成，A组成骨过程则仍在继续，C组和D组
未检测到成骨过程。
3.X线检查发现，A、B组8周时已有骨痴生成，12周时缺损区完全
由新生骨填充，但B组新骨生成的质量明显优于A组。
4.B组在8周时ALP活性达到高峰，12周时降至稳定水平;而A组
动物8周和12周时的ALP活性则无显著差异。钙含量检测发现B组8周
和12周时显著高于A组。
5.以上结果表明，基因转移后所表达分泌的BMP一4能够诱导间充质
细胞向骨组织分化和促进骨折愈合，但与TGF一已l合用效果更佳。
pcDN冉3.1瓜Mp一4合用TGF一pl治疗家兔火器性股骨骨缺损:24只
家兔随机平均分为两组，以钢珠自股骨肌肉射出造成股骨中部企8~缺
损。经清创、延期缝合及固定处理，12只动物注射pCDNA3 .1忍MP一4脂
质体与TGF一日，作为治疗组(A组)，其余12只动物仅行手术处理以资对
照(B组)。骨折处理后2、4、8、12周检测同样指标，主要结果与结论如
下:
1.BMP一基因可表达为相应的mRNA和蛋白质达6周。
2.A组动物骨折处理后4周局部组织开始软骨化骨，8周时成骨积极
活跃，12周时成骨过程基本完成。B组动物虽可见同样的成骨过程，但成
骨质量和速度均不如A组。。
3.X线检查发现，A组动物8周时新骨生成，12周时缺损区完全由新
骨填充。B组12周时才有明显的新骨生成。
4.A组动物8周时ALP活性达到高峰，12周时下降;而B组动物8
Vll
//
周和12周时则保持在同一水平。A、B组动物局部组织的钙含量自第4周
开始均不断增加，但B组动物的钙含量却明显低于同时期的A组动物。这
些结果提示，BMP并表达基因合用TGF一旦;对成骨细胞的增殖、成熟和钙
分泌有促进作用。
5.上述结果证明，pCDNA3.1忍MP一4与TGF一p，合用，同样能够促
进损伤重、污染重的火器性骨缺损的愈合。
The current practice for fractures including surgical methods and physical methods makes most of fractures treated effectively. However, many researchers are still making efforts to find alternative therapeutic strategies with purpose of ensuring proper healing of complex fractures. Substantial efforts also have been devoted to development of biological factors and their delivery to influence or control the cellular events associated with bone formation and repair. Recombinant proteins like the bone morphogenetic proteins (BMP) have shown particular promise as therapeutic alternatives. Unfortunately, direct use of these factors has been limited by its short half-life etc. Fracture healing lends itself well to application of gene therapy because of transient and localized need for enhancing factors, thus many studies were focused on the gene therapy of fracture healing in this study. The pCDNA3.1/BMP-4 was constructed and the integrity of BMP-4 gene was verified by sequencing. The BMP-4 gene was transcribed a
nd translated when it was transferred into flbroblasts in vitro and its mRNA and protein were detected with RT-PCR, Western Blot, and the excretion of BMP-4 induced marrow stroma cells to differentiate into osteoblast. Based on those results, we designed two studies to explore the effect of BMP-4 gene therapy through direct injection of pCDNA3.l/BMP-4 liposome. The first experiment was the treatment of long segmental defects in rabbits with the model of ulna defect (Part One), and the second was the
treatment of firearm femur defect (Part Two).
Part One: 48 adult rabbits were averagely divided into four groups randomly. Both the two ulnas of each rabbit were operated and removed 15mm section from the central part of ulnas. 12 animals were directly injected 40 g pCDNA3.1/BMP-4 liposome into the tissues between the two ends of defects 1 week after operation (Group A ); pCDNA3.1/BMP-4 liposome was directly injected into another 12 animals as in Group A, also, 20ng TGF was injected at the 2" and the 4th week respectively after operation (Group B ); 12 animals with only pCDNA3.l liposome injection or only operation were used as control (Group C and Group D). The ALP activity, calcium content, pathological and radiological changes of the local tissues was examined at the 2nd, the 4th,the 8th and the 12th week after operation. The main results and conclusions are as the following:
1) The BMP-4 gene was transcribed and translated in local tissues between the two ends of ulna defects for six weeks.
2) The local tissues started the cartilaginous ossification marked with the appearance of cartilage at the 4th week after operation in Group A and Group B. The ossification was nearly completed at the 12th week in Group B, while the ossification was still lasting in Group A. No ossification was observed in Group C and Group D.
3) The X-ray examination showed that the new bone began to form at the 8th week and it stuffed the defects completely at the 12th week in Group A and Group B, but the quality and quantity of new formed bone were higher in Group B than that in Group A.
4) The ALP activity reached its peak at the 8th week and reduced to a
relatively stable level at the 12th week in GroupB, while it remained the same level in Group A. The calcium content at the 8th and 12th week was significantly higher in Group B than that in Group A.
5) The results above indicated that the expression and secretion of BMP-4 after transfection can induce the differentiation of mesenchymal cells into osseous tissue and promote the fracture healing, and BMP-4 gene therapy combined with TGF can more effectively promote the fracture repair.
Part Two: 24 adult rabbits were averagely divided into two groups randomly. The central part of one femur of each rabbit was made a 6-8mm defect with a fire of steel ball through the anterior muscular layer. After debridement, delayed suture and internal fixation, 12 animals were directly injected pCDNA3.l/BMP-4 liposome and TGF (Group A), and the other 12 animals w
pCDNA3.1/BMP-4治疗家兔尺骨缺损：48只家兔被随机平均分为4组，两侧尺骨中段均被截除15mm。A组12只动物于术后1周注射40μg pCDNA3.1/BMP-4脂质体于尺骨缺损之间组织内；B组12只动物除术后1周注射40μg pCDNA3.1/BMP-4脂质体外，术后2、4周分别注射20ng TGF-β_1。对照组C组12只动物术后仅注射pCDNA3.1，而D组12只动物仅手术造成尺骨缺损。术后2、4、8、12周对骨缺损间局部组织进行碱性磷酸酶、钙含量、病理学和放射学检查。主要结果和结论如下：
1.BMP一基因可在骨缺损间局部组织内进行转录和翻译，并持续6周
时间。
2.A、B组局部组织术后4周时出现软骨细胞，软骨化骨过程开始。
12周时B组成骨过程接近完成，A组成骨过程则仍在继续，C组和D组
未检测到成骨过程。
3.X线检查发现，A、B组8周时已有骨痴生成，12周时缺损区完全
由新生骨填充，但B组新骨生成的质量明显优于A组。
4.B组在8周时ALP活性达到高峰，12周时降至稳定水平;而A组
动物8周和12周时的ALP活性则无显著差异。钙含量检测发现B组8周
和12周时显著高于A组。
5.以上结果表明，基因转移后所表达分泌的BMP一4能够诱导间充质
细胞向骨组织分化和促进骨折愈合，但与TGF一已l合用效果更佳。
pcDN冉3.1瓜Mp一4合用TGF一pl治疗家兔火器性股骨骨缺损:24只
家兔随机平均分为两组，以钢珠自股骨肌肉射出造成股骨中部企8~缺
损。经清创、延期缝合及固定处理，12只动物注射pCDNA3 .1忍MP一4脂
质体与TGF一日，作为治疗组(A组)，其余12只动物仅行手术处理以资对
照(B组)。骨折处理后2、4、8、12周检测同样指标，主要结果与结论如
下:
1.BMP一基因可表达为相应的mRNA和蛋白质达6周。
2.A组动物骨折处理后4周局部组织开始软骨化骨，8周时成骨积极
活跃，12周时成骨过程基本完成。B组动物虽可见同样的成骨过程，但成
骨质量和速度均不如A组。。
3.X线检查发现，A组动物8周时新骨生成，12周时缺损区完全由新
骨填充。B组12周时才有明显的新骨生成。
4.A组动物8周时ALP活性达到高峰，12周时下降;而B组动物8
Vll
//
周和12周时则保持在同一水平。A、B组动物局部组织的钙含量自第4周
开始均不断增加，但B组动物的钙含量却明显低于同时期的A组动物。这
些结果提示，BMP并表达基因合用TGF一旦;对成骨细胞的增殖、成熟和钙
分泌有促进作用。
5.上述结果证明，pCDNA3.1忍MP一4与TGF一p，合用，同样能够促
进损伤重、污染重的火器性骨缺损的愈合。
The current practice for fractures including surgical methods and physical methods makes most of fractures treated effectively. However, many researchers are still making efforts to find alternative therapeutic strategies with purpose of ensuring proper healing of complex fractures. Substantial efforts also have been devoted to development of biological factors and their delivery to influence or control the cellular events associated with bone formation and repair. Recombinant proteins like the bone morphogenetic proteins (BMP) have shown particular promise as therapeutic alternatives. Unfortunately, direct use of these factors has been limited by its short half-life etc. Fracture healing lends itself well to application of gene therapy because of transient and localized need for enhancing factors, thus many studies were focused on the gene therapy of fracture healing in this study. The pCDNA3.1/BMP-4 was constructed and the integrity of BMP-4 gene was verified by sequencing. The BMP-4 gene was transcribed a
nd translated when it was transferred into flbroblasts in vitro and its mRNA and protein were detected with RT-PCR, Western Blot, and the excretion of BMP-4 induced marrow stroma cells to differentiate into osteoblast. Based on those results, we designed two studies to explore the effect of BMP-4 gene therapy through direct injection of pCDNA3.l/BMP-4 liposome. The first experiment was the treatment of long segmental defects in rabbits with the model of ulna defect (Part One), and the second was the
treatment of firearm femur defect (Part Two).
Part One: 48 adult rabbits were averagely divided into four groups randomly. Both the two ulnas of each rabbit were operated and removed 15mm section from the central part of ulnas. 12 animals were directly injected 40 g pCDNA3.1/BMP-4 liposome into the tissues between the two ends of defects 1 week after operation (Group A ); pCDNA3.1/BMP-4 liposome was directly injected into another 12 animals as in Group A, also, 20ng TGF was injected at the 2" and the 4th week respectively after operation (Group B ); 12 animals with only pCDNA3.l liposome injection or only operation were used as control (Group C and Group D). The ALP activity, calcium content, pathological and radiological changes of the local tissues was examined at the 2nd, the 4th,the 8th and the 12th week after operation. The main results and conclusions are as the following:
1) The BMP-4 gene was transcribed and translated in local tissues between the two ends of ulna defects for six weeks.
2) The local tissues started the cartilaginous ossification marked with the appearance of cartilage at the 4th week after operation in Group A and Group B. The ossification was nearly completed at the 12th week in Group B, while the ossification was still lasting in Group A. No ossification was observed in Group C and Group D.
3) The X-ray examination showed that the new bone began to form at the 8th week and it stuffed the defects completely at the 12th week in Group A and Group B, but the quality and quantity of new formed bone were higher in Group B than that in Group A.
4) The ALP activity reached its peak at the 8th week and reduced to a
relatively stable level at the 12th week in GroupB, while it remained the same level in Group A. The calcium content at the 8th and 12th week was significantly higher in Group B than that in Group A.
5) The results above indicated that the expression and secretion of BMP-4 after transfection can induce the differentiation of mesenchymal cells into osseous tissue and promote the fracture healing, and BMP-4 gene therapy combined with TGF can more effectively promote the fracture repair.
Part Two: 24 adult rabbits were averagely divided into two groups randomly. The central part of one femur of each rabbit was made a 6-8mm defect with a fire of steel ball through the anterior muscular layer. After debridement, delayed suture and internal fixation, 12 animals were directly injected pCDNA3.l/BMP-4 liposome and TGF (Group A), and the other 12 animals w
引文
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2． Coonolly JF, Guse R, Tideman J, et al: Autologous marrow injection for delayed unions of the tibia. J Orthop Trauma, 1989, 3: 276-282．
3． Flanagan AM, Chambers AJ: Stimulation of bone nodule formation in vitro by prostaglandins El and E2． Endocrinology, 1992, 130: 443-448．
4． Goldring MB, Goldring SR. Skeletal tissiue response to cytokines. Clin Orthop, 1990,258:245-249．
5． Yang LJ, Jin Y. Immunohistochemical observation on bone Morphogenetic protein in normal and abnormal conditions. Clin Othop, 1990,257:249-254．
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