| The therapy of segmental bone defects remains a troublesome problem in orthopedic clinic. At present, all available options for bone reconstruction, such as autogenous bone, allograft bone, and alloplastic materials, each have some disadvantages. Bone morphgenetic proteins (BMPs) are capable of inducing bone formation during fracture healing, because of inducing pluripotential mesenchymal stem cells to differentiate into osteoblasts. Researchers believe that a part of bone nonunions are due to lack of BMP in fracture zone and osteogenic precursor cells that locate there, can't be induced into osteoblast. Now recombinant BMP has been used in the study of bone defects. However, the clinical usefulness of recombinant BMP as regenerative agent is limited by its defects, including short half-life, low bioactivity, high price, lack of ideal carriers, and so on. During recent years, with development of molecular biology rapidly, the study of BMP gene therapy has become more popular. Gene therapy to deliver BMP may offer several theoretical advantages over implantation of a recombinant BMP protein, shows an inspiring prospect. The one of key steps are choosing a kind of target cells properly in it. Fibroblasts distribute widely and culture easily, otherwise the experiments in vivo and in vitro have demonstrated that BMP gene can induce fibroblasts to differentiate into osteoblasts and has osteoinductive ability, too. Therefore, it will be promising that using humanfibroblasts as target cells for BMP gene study. In order to investigate the probability of human fibroblasts as target cells used in the study of BMP-2 gene therapy and guess the osteogeneic mechanism of fibroblasts being BMP-2 gene transfected in vivo, we assayed the effects of human BMP-2 gene on proliferation and differentiation of human fibroblast strain KMB-17, expect to provide some basis for its further study.In this study, an eukaryotic expression plasmid encoding for human BMP-2 (pBK-BMP2) was transfected into KMB-17 cells by liposome-mediated method, then in different periods after being transfected, the expression of BMP-2 gene was detected by Reserve transcriptase polymerase chain reaction (RT-PCR) and observed by immunocytochemistry (ICC) staining; the effect of BMP-2 gene transfection on KMB-17 cells proliferation was evaluated by 3(4,5 dimethythiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay and flow cytometry; Further more, the effect of BMP-2 gene transfection on differtiation of KMB-17 cells was investigated by morphologic and ultrastructural observation, alkaline phosphatase (ALP) activity assay, osteocalcin(OC) production assay and typeâ… collagen synthesis assay. In addition, the results of MTT, ALP activity , OC production and typeâ… collagen synthesis were compared with control groups. The results showed that mRNA of BMP-2 was detected in experimental group KMB-17 cells by RT-PCR, but control groups not; furthermore by ICC staining we found BMP-2 gene could expresse for at least 3 weeks in KMB-17 cell after being transfected; at the same time, BMP-2 gene expression triggered KMB-17 cells proliferation, there was statistically significant difference KMB-17 cells of experimental group as compared with control groups (P<0.05), proliferation index (PI) increased,too. The morphologic changes of KMB-17 cells were also very obvious, the shape of cells changed into scale-like or multiangle, and a lot of black granules were secreted; the ultrastructural features of KMB-17 cells showed that rough endoplasmic reticulum dilated and mitochondria hyperplasia, the dilating rough endoplasmic reticulums were full of abundant proteinoid substances. Moreover, BMP-2 gene transfecion could stimulate KMB-17 cells to increase ALP activity, OC pruduction, and collagen synthesis. Compared with control groups, there were statistically significant differences on ALP activity, OC production and collagen synthesis in BMP-2 gene transfection group (P<0.05).The study reveals that the eukaryotic expression vector encoding fo...
|
PDF Full Text Request