【摘要】 目的 探讨骨形成蛋白-2（bone morphogenetic protein-2，BMP-2）对室管膜前下区（anterior subventricular zone，SVZa）神经干细胞DLX5表达的影响。 方法 体外培养SVZa神经干细胞，用BMP-2及其拮抗剂Noggin诱导SVZa神经干细胞，分别用免疫荧光染色和逆转录-聚合酶链反应（RT-PCR）检测DLX5表达变化。 结果 BMP-2组SVZa神经干细胞DLX5蛋白表达和DLX5mRNA表达水平明显高于对照组（Plt;0.05），且该效应能被其拮抗剂Noggin特异性地抑制。 结论 BMP-2是DLX5上游调节基因，可促进SVZa神经干细胞DLX5的表达。【Abstract】 Objective To investigate the effect of bone morphogenetic protein-2 (BMP-2)on expression of DLX5 of neural stem cells in anterior subventricular zone (SVZa). Methods The neural stem cells of SVZa were separated and cultured in vitro, which were induced by BMP-2 and Noggin.Immunofluorescence staining and RT-PCR were employed to assay the expression of DLX5. Results The percentages of expression of DLX5 protein and DLX5 mRNA in BMP-2 group were much higher than those in the control group (Plt;0.05). And this induction could be specifically blocked by Noggin. Conclusion BMP-2 is an upstream gene of DLX5; BMP-2 can promote the expression of DLX5 of the neural stem cells of SVZa.
Objective To construct the recombined DNA pcDNA3.1-hBMP-2 and transfect into human marrow stromal stem cells (MSCs) in vitro, and to explore theeffects of transfection on cellular proliferation and expression of vascular endothelial growth factor (VEGF). Methods The expression of human bone morphogenetic protein 2(hBMP-2) in these cells after transfection was determined by in situ hybridization and immunohistochemical analysis and Western blot analysis. The changes of cell proliferation were observed by flow cytometry. The effects of BMP-2 gene transfection on expression of VEGF in the cells were analyzed by in situ hybridization of VEGF cDNA probe. Results Stable expressionof hBMP-2 in pcDNA3.1-hBMP-2 transfected MSCs was confirmed in the levels of mRNA and protein.Cellular proportion in S period increased, which indicated that the synthesis of cell DNA increased. The expression of VEGF in the cells increased obviously. Conclusion With the help of lipofectamine, the pcDNA3.1-hBMP-2 were transfected into human MSCs successfully. hBMP-2 plays an important role in promoting cellular proliferation and vascular generation during bone repair.
OBJECTIVE: To construct a co-expressing vector of human bone morphogenetic protein 2 (BMP-2) and osteoprotegerin (OPG) and to determine the expression of BMP-2 and OPG in myoblast C2C12. METHODS: Using the isolated total RNA from osteosacoma cell line MG63 as a template, the cDNA encoding region of human OPG was amplified by reverse transcription-polymerase chain reaction (RT-PCT) method and cloned into sites EcoR 1 and BamH I of mammalian expressing vector pIRES2-EGFP, and the cDNA encoding region of human BMP-2 was cloned into endonucleases site BstX I. Then the recombinant plasmid pIRES2-BMP-2-OPG was transformed into C2C12 cell line, the expression of OPG and BMP-2 were determined by Western blot assay. RESULTS: The sequence of OPG cDNA obtained was the same as that reported, recombinant plasmid pIRES2-BMP-2-OPG was constructed successfully. Human OPG and BMP-2 co-expression cell line C2C12 was selected and confirmed by Western blot analysis. CONCLUSION: The co-expressing vector of OPG and BMP-2 is constructed and can expressed stably in myoblast C2C12. The co-expression of human OPG and BMP-2 may be logical approach for treatment of osteoporosis and bone metastasis.
Objective To determine whether fibroblasts can be used to promote endochondral bone formation in vivo by transfer of human bone morphogenetic protein-2(hBMP-2) into fibroblasts. Methods pcDNA3-hBMP-2 was constructed by use of gene clone and recombined technique.NIH3T3 fibroblasts were transfected with pcDNA3hBMP-2. The positive cell clones were selected with G418. In NIH3T3 fibroblaststransferred with pcDNA3-hBMP-2, the expression of hBMP-2 was determined by in situ hybridization and immunohistochemical analysis; alkaline phosphatase activity was measured. hBMP-2producing fibroblasts were implanted into nude mouse muscle to observe endochondral bone formation in vivo. Results pcDNA3-hBMP-2 was successfully constructed. In NIH3T3 fibroblasts transfected with -pcDNA3-hBMP-2,the BMP-2 expression was stable; alkaline phophatase activity was much higher than that in nontransfectedNIH3T3 cells. Endochondral bone formation invivo was observed at the site of implantation 4 weeks later.Conclusion Fibroblasts transfected by hBMP-2 gene can be used to promote endochondral bone formation in vivo.
Objective To investigate and compare the osteogenic potential of three kinds of calcium phosphate ceramic as carriers for recombinant human bone morphogenetic protein-2(rhBMP-2) in vivo.Methods BCPceramics (HA,TCP,HA/TCP) impregnated with rhBMP-2 (experimental groups) and without rhBMP-2(control groups) were implanted into 6 muscles pockets on the dorsum of 3month-old Wistar rabbits. The rabbits were sacrificed 2, 4 and 8 weeks after implantation and bone induction was estimated by alkaline phosphatase(ALP) activity measurement. The implants were also examined histologically and histomorphometrically by HE staining and computerized graphical analysis. Results The ALPactivity of implants withrhBMP-2 was higher than that of control groups(P＜0.05), but there was no difference between 2 and 4 weeks in experimental groups. In all experimental groups,theimplants exhibited that new bone formation increased with the lapse of time. The amount of new bone formation is more in -HA/rhBMP-2 group than in the other two group in the 2nd and 4th weeks, but there was no difference between them (P＞0.05).In the 8th week, the amount of bone formation was most in HA/TCP with -rhBMP-2, and was more than that in the 2nd and 4th weeks. Whereas in control groups, there was only fibrous connective tissue. Conclusion HA/TCP- is a good carriers of rhBMP-2 and can be used as bone substitutes clinically.
Objective To evaluate the osteogenic potential of human bone marrow mesenchymal stem cells (MSCs) transferred with human bone morphogenetic protein 2(BMP 2) gene by adenovirus. Methods The MSCs were isolated from human bone marrow and cultured in vitro. They were divided into 3 groups: Adv hBMP 2 transduced group; Adv βgal transduced group; untransduced group. Western immunoblot analysis, alkaline phosphatase(ALP) staining, Von Kossa staining, and a quantitative ALP activity assay were performed. Nine unde mice received injection into a thigh muscle to test the osteoinductivity of the three types of cells. Results In the Adv-hBMP-2 transprotein; most MSCs were stained positively for ALP activity 9 day after transduction; the MSCs reached the peak of ALP activity 12 day after transduction; the calcified nodes formed 21 days after transduction. The ectopic bones formed in the thigh muscles of the nude mice. Little bone formation was observed in the other groups 4 weeks after cell injection. Conclusion Adenovirus mediated hBMP-2 gene transfection can induce osteogenesis of human bone marrow mesenchymal stem cells.
Objective To investigate a new grafting material of bone xenograft with b bone inductive and conductive capacity. Methods Based on successful clinical application of the reconstituted bone xenograft (RBX), a new xenograft was made by combining recombinant human bone morphogenetic protein-2 (rhBMP-2) with antigen-free bovine cancellous bone (BCB). Sixty male BALB/C mice aged 4 weeks were divided into study group of 30 and control group of 30 randomly. rhBMP-2 / BCB was implanted in the left thigh muscle pouch in the study group andBCB in the control group. The mice were sacrificed at 7 d, 14d and 21d after implantation. Inductivity of rhBMP-2/BCB was detected by histological observation and biochemical determination of the samples. Results Histological examinationshowed that rhBMP-2/BCB induced chondrogenesis on the 7th day, with woven boneformed on the 14th day, and lamellar bone and marrow on the 21st day, while BCBfailed to induce chondrogenesis or osteogenesis on the 7th, 14th and 21st days. The alkaline phosphatase activities and calcium content in study group were higher than those in control group with significant difference (P＜0.01). Conclusion rhBMP-2/BCB is an ideal grafting material with b bone inductive and conductive capacity without evoking immune reaction.
Objective To evaluate the bone regenerative potential of reconbinant human bone morphogenetic protein 2(rhBMP-2) / collagen on adult rat calvarial bone. Methods A tight subperiosteal pocket was produced under both sides ofthe temporal muscle in rats. rhBMP-2 / collagen was implanted in one side and collagen alone was implanted in the other side as control. The rats were sacrificed 2, 4 and 8 weeks after operation. The specimen was harvested and examined histologically. For morphometric analysis, the thickness of the temporal bone of both sides was measured and compared. Results The rhBMP-2 / collagen onlay implant resulted in active bone formation and the augmented bone was connected directly with the original bone, whereas the collagen alone resulted in neither bone nor cartilage production. The ossification process in the rhBMP-2 / collagen occurred directly through bone formation, similar to intramembranous ossification. Conclusion rhBMP-2 / collagen is an effective material as a biological onlay implant.
OBJECTIVE To improve the osteoinduction of coral and provide a perfect bone graft substitute for clinical bone defects. METHODS By combining coral with collagen and recombinant human bone morphogenetic protein-2(rhBMP-2), coral/collagen/rhBMP-2 composite was obtained. The composite was implanted into the back muscle pouches of mice, and coral/collagen or coral/rhBMP-2 were implanted as control. The osteoinduction of the composite was assessed by histology and image analysis system. RESULTS The chondrocyte differentiation and matrix formation were observed in local sites after one week, lamellar bone with bone marrow were formed after 4 weeks, and coral were absorbed partially. The quantity of osteoinduction was time-related and rhBMP-2 dose-related(P lt; 0.01). Coral/collagen and coral/rhBMP-2 implants did not show any bone or cartilage formation. CONCLUSION The coral/collagen/rhBMP-2 composite possesses a superior osteoinduction and will be a new type of bone substitute to be used in orthopedic and maxillofacial surgery.