Effect Of Reconstruction And Expression Of RhBMP -4/7 Fusion Protein On Cultured Bone Marrow Stem Cells

Bone morphogenetic protein has been demonstrated to be a potential growth factor in facilitate cell proliferation and differentiation into cells of the osteoblast lineage, and then accelerate bone formation both in vitro and in vivo. BMP combined with many kinds of seed cells and scaffolds has been widely used in bone tissue engineering procedure, aiming to achive bone repair and reconstruction via steoconduction and osteoinduction. At present , the family of BMPs were found more than ten and most strong ability was the BMP-2,BMP-4,BMP-7. According to the reported , the heterodimer of BMPs is more kinds in body but at the same time the homodiner of BMPs can be found.moreover, the ability of the homodiner of BMPs is stronger than the heterodimer of BMPs.The mechanism needs go deep into research.This study design to clone the cDNA of mature peptide of human bone morphgenetic protein 4 ,7 and construction and expression of rhBMP-4/7 fusion protein in E.coliafter transfromation and induction.The fusion protein was applied to induce the cultured bone marrow stem cell to observe the effect of rhBMP -4/7 fusion protein on cultured bone marrow stem cells proliferation and ability of ALP,OC change in cultured bone marrow stemcells. The research offer a new method and testimony for the bone repair and establish experimental basis for the bone tissue engineering and gene therapy for bone defect. Part I: Cloning and sequence of mature peptide of human bone morphognetic protein-4 cDNA To clone the cDNA of mature peptide of human bone morphgenetic protein-4, two primers were designed according to the hBMP-4 sequence reported in genebank. The mature peptide of hBMP-4 cDNA was gained by one step RT-PCR from the mRNA, which was extracted form human placenta. Then it was cloned into the vector of PGEM-T plasmid and the sequence of the plasmid PGEM-T –bmp-4 was detected by DNA sequence machine. DNA agarose gel electrophoresis showed that the product of RT-PCR was about 374bp, it was corresponed with the result of two enzyme digestion of the recombinanted plasmid. The result of sequence assay was in agreement with the reported hBMP-4 sequencein genebank. The mature peptideof hBMP-4 cDNA was cloned successfully and the sequence was testified correctly. Part II: Cloning and sequence of mature peptide of human bone morphognetic protein-7 cDNA To clone and sequence the cDNA gene of mature peptide of human bone morphgenetic protein-7 from fetus cartilage cells, Total RNA was extracted form culture human fetus cartilage cells, two primers were designed according to the hBMP-7 sequence reported in genebank. The mature peptide of hBMP-7 cDNA was gained by one step RT-PCR from the mRNA, which was extracted form fetus cartilage cells. Then it was cloned into the vector of PGEM-T plasmid and the sequence of the plasmid PGEM-T –bmp-7 was detected by DNA sequence machine. DNA agarose gel electrophoresis showed that the product of RT-PCR was about 454bp, it was corresponed with the result of two enzyme digestion of the recombinanted plasmid. The result of sequence assay was in agreement with the reported hBMP-7 sequencein genebank. The mature peptide of hBMP-7 cDNA was cloned from fetus cartilage cells successfully and the sequence was testified correctly. Part III: Construction and expression of rhBMP-4/7 fusion protienof the test group increased significantly than that of the control group.The result showed that the fusion protein of the hBMP-4/7 have the natural biological function and this protein can induce bone marrow stemcells to differentiation to osteoblast cells. From the above we could draw the conclusion that the cDNA of mature peptide of human bone morphgenetic protein-4 can be cloned from the human placenta and the mature peptide of hBMP-7cDNAas cloned from fetus cartilage cells successfully. The fusion gene was constructed in a plasmid in which the coding regions of human BMP-4 and BMP-7 mature peptied were connected by a synthetic linker sequence encoding a short peptieds through DNA recombinant techniques and expressed in E. coli after transfromation and induction. The fusion protein could induce the culture bone marrow stemcells. The result showed that the fusion protein of the hBMP-4/7 have the natural biological function. In a word, reconstruction method of the rhBMP -4/7 fusion protein and increase expression of the BMPs offer a new method and testimony for the inducement ossification and Establish experiment base for the bone tissue engineeringof the test group increased significantly than that of the control group.The result showed that the fusion protein of the hBMP-4/7 have the natural biological function and this protein can induce bone marrow stemcells to differentiation to osteoblast cells. From the above we could draw the conclusion that the cDNA of mature peptide of human bone morphgenetic protein-4 can be cloned from the human placenta and the mature peptide of hBMP-7cDNAas cloned from fetus cartilage cells successfully. The fusion gene was constructed in a plasmid in which the coding regions of human BMP-4 and BMP-7 mature peptied were connected by a synthetic linker sequence encoding a short peptieds through DNA recombinant techniques and expressed in E. coli after transfromation and induction. The fusion protein could induce the culture bone marrow stemcells. The result showed that the fusion protein of the hBMP-4/7 have the natural biological function. In a word, reconstruction method of the rhBMP -4/7 fusion protein and increase expression of the BMPs offer a new method and testimony for the inducement ossification and Establish experiment base for the bone tissue engineering...