Objective To explore the feasibility and efficacy of lentivirus-mediated co-transfection of rat bone marrow mesenchymal stem cells （MSCs） with human vascular endothelial growth factor 165 （hVEGFI65） gene and human...Objective To explore the feasibility and efficacy of lentivirus-mediated co-transfection of rat bone marrow mesenchymal stem cells （MSCs） with human vascular endothelial growth factor 165 （hVEGFI65） gene and human bone morphogenetic protein 2 （hBMP2） gene. Methods The hVEGF165 and hBMP2 cDNAs were obtained from human osteosarcoma cell line MG63 and cloned into lentiviral expression vectors designed to co-express the copepod green fluorescent protein （copGFP）. The expression lentivector and packaging Plasmid Mix were co-transferred to 293TN cells, which produced the lentivirus carrying hVEGF165 （Lv-VEGF） or hBMP2 （ Lv-BMP） , respectively. MSCs of Wistar rats were co-transfected with Lv-BMP and Lv-VEGF （BMP ＋ VEGF group）, or each alone （BMP group and VEGF group）, or with no virus （ Control group）. The mRNA and protein expressions of hVEGF165 and hBMP2 genes in each group were detected by real-time PCR and enzyme linked immunosorbent assay （ELISA）. Results Lentiviral expression vectors carrying hVEGF165 or hBMP2 were correctly constructed and confirmed by restriction endonucleses analysis and DNA sequencing analysis. A transfer efficiency up to 90% was archieved in all the transfected groups detected by the fraction of fluorescent cells using fluorescent microscopy. From the results generated by real-time PCR and ELISA, VEGF165 and BMP2 genes were co-expressed in BMP ＋ VEGF group. No significant difference of BMP2 expression was detected between BMP ＋ VEGF and BMP groups （ P 〉 0. 05）. Similarly, there was no significant difference of VEGF165 expression between BMP ＋ VEGF and VEGF groups （ P 〉 0. 05）. Conclusion VEGF165 and BMP2 genes were successfully co-expressed in MSCs by lentivirus-mediated co-transfection, which provided a further foundation for the combined gene therapy of bone regeneration.展开更多
Objective: To evaluate the osteogenic potential of bone morphogenetic protein (BMP)-2 gene transfected goat bone marrow-derived mesenchymal stem cells (MSCs). Methods: Goat bone marrow- derived MSCs were transfected b...Objective: To evaluate the osteogenic potential of bone morphogenetic protein (BMP)-2 gene transfected goat bone marrow-derived mesenchymal stem cells (MSCs). Methods: Goat bone marrow- derived MSCs were transfected by Adv-human bone morphogenetic protein (hBMP)-2 gene(Group 1), Adv-beta gal transfected MSCs (Group 2)and uninfected MSCs(Group 3). Western blot analysis, alkaline phosphatase staining, Von Kossa staining and transmission electron microscopy were adopted to determine the phenotype of MSCs. Then the cells were injected into thigh muscles of the nude mice. Radiographical and histological evaluations were performed at different intervals. Results: Only Adv-hBMP-2 transfected MSCs produced hBMP-2. These cells were positive for alkaline phosphatase staining at the 12th day and were positive for Von Kossa staining at the 16th day after gene transfer. Electron microscopic observation showed that there were more rough endoplasmic reticulum, mitochondria and lysosomes in Adv-hBMP-2 transfected MSCs compared to MSCs of other two groups. At the 3rd and 6th weeks after cell injection, ectopic bones were observed in muscles of nude mice of Group 1. Only fibrous tissue or a little bone was found in other two groups. Conclusions: BMP-2 gene transfected MSCs can differentiate into osteoblasts in vitro and induce bone formation in vivo.展开更多
Objective: To explore reciprocal action between BMP 2 (bone morphogenetic protein 2) and BMP 3 for better understanding of the mechanism of BMP during bone fracture union. Methods: rhBMP 2 was added into the cultured ...Objective: To explore reciprocal action between BMP 2 (bone morphogenetic protein 2) and BMP 3 for better understanding of the mechanism of BMP during bone fracture union. Methods: rhBMP 2 was added into the cultured fibroblasts with the concentration of 1 200 ng/ml. The expression of BMP 3 in fibroblasts was detected by immunohistochemistry. Eukaryotic expression vector pcDNA3 BMP 3 was transfected into the fibroblasts. After the effective expression of BMP 3 was identified, BMP 2 was also detected by immunohistochemistry in BMP 3 expression cells. The fibroblasts transfected with empty vector pcDNA3 were used as the control. Results: Exogenous rhBMP 2 could promote the expression of BMP 3 in fibroblasts. BMP 3 also could be detected in these cells.Conclusions: BMP 2 and BMP 3 could reciprocally adjust the expression in fibroblasts.展开更多
Objective： To investigate the effect of a new biomaterial combining calcium citrate and recombinant human bone morphogenetic protein-2 （rhBMP-2） on bone regeneration in a bone defect rabbit model. Methods： Totall...Objective： To investigate the effect of a new biomaterial combining calcium citrate and recombinant human bone morphogenetic protein-2 （rhBMP-2） on bone regeneration in a bone defect rabbit model. Methods： Totally 30 male New Zealand white rabbits were randomly and equally divided into calcium citraterhBMP-2 （CC-rhBMP-2） group and rhBMP-2 only group. Two 10 ram-long and 5 ram-deep bone defects were respectively created in the left and right femoral condyles of the rabbits. Subsequently 5 pellets of calcium citrate （10 mg） combined with rhBMP-2 （2 rag） or rhBMP-2 alone were implanted into the bone defects and compressed with cotton swab. Bone granules were obtained at 2, 4 and 6 weeks after procedure and received histological analysis. LSD t-test and a subsequent t-test were adopted for statistical analysis. Results： Histomorphometric analysis revealed newlyformed bones, and calcium citrate has been absorbed in the treatment group. The percent of newly formed bone area in femoral condyle in control group and CC-rhBMP-2 group was respectively 31.73%±1.26% vs 48.21%±2.37% at 2 weeks; 43.40%±1.65% vs 57.32%±1.47% at 4 weeks, and 51.32%±7.80% vs 66.74%±4.05% at 6 weeks （P〈0.05 for all）. At 2 weeks, mature cancellous bone was observed to be already formed in the treatment group. Conclusion： From this study, it can be concluded that calcium citrate combined with rhBMP-2 signifcantly enhances bone regeneration in bone defects. This synthetic gelatin matrix stimulates formation of new bone and bone marrow in the defect areas by releasing calcium ions.展开更多
基金Supported by Key Program of Shanghai Science and Technology Committee (054119520)
文摘Objective To explore the feasibility and efficacy of lentivirus-mediated co-transfection of rat bone marrow mesenchymal stem cells （MSCs） with human vascular endothelial growth factor 165 （hVEGFI65） gene and human bone morphogenetic protein 2 （hBMP2） gene. Methods The hVEGF165 and hBMP2 cDNAs were obtained from human osteosarcoma cell line MG63 and cloned into lentiviral expression vectors designed to co-express the copepod green fluorescent protein （copGFP）. The expression lentivector and packaging Plasmid Mix were co-transferred to 293TN cells, which produced the lentivirus carrying hVEGF165 （Lv-VEGF） or hBMP2 （ Lv-BMP） , respectively. MSCs of Wistar rats were co-transfected with Lv-BMP and Lv-VEGF （BMP ＋ VEGF group）, or each alone （BMP group and VEGF group）, or with no virus （ Control group）. The mRNA and protein expressions of hVEGF165 and hBMP2 genes in each group were detected by real-time PCR and enzyme linked immunosorbent assay （ELISA）. Results Lentiviral expression vectors carrying hVEGF165 or hBMP2 were correctly constructed and confirmed by restriction endonucleses analysis and DNA sequencing analysis. A transfer efficiency up to 90% was archieved in all the transfected groups detected by the fraction of fluorescent cells using fluorescent microscopy. From the results generated by real-time PCR and ELISA, VEGF165 and BMP2 genes were co-expressed in BMP ＋ VEGF group. No significant difference of BMP2 expression was detected between BMP ＋ VEGF and BMP groups （ P 〉 0. 05）. Similarly, there was no significant difference of VEGF165 expression between BMP ＋ VEGF and VEGF groups （ P 〉 0. 05）. Conclusion VEGF165 and BMP2 genes were successfully co-expressed in MSCs by lentivirus-mediated co-transfection, which provided a further foundation for the combined gene therapy of bone regeneration.
文摘Objective: To evaluate the osteogenic potential of bone morphogenetic protein (BMP)-2 gene transfected goat bone marrow-derived mesenchymal stem cells (MSCs). Methods: Goat bone marrow- derived MSCs were transfected by Adv-human bone morphogenetic protein (hBMP)-2 gene(Group 1), Adv-beta gal transfected MSCs (Group 2)and uninfected MSCs(Group 3). Western blot analysis, alkaline phosphatase staining, Von Kossa staining and transmission electron microscopy were adopted to determine the phenotype of MSCs. Then the cells were injected into thigh muscles of the nude mice. Radiographical and histological evaluations were performed at different intervals. Results: Only Adv-hBMP-2 transfected MSCs produced hBMP-2. These cells were positive for alkaline phosphatase staining at the 12th day and were positive for Von Kossa staining at the 16th day after gene transfer. Electron microscopic observation showed that there were more rough endoplasmic reticulum, mitochondria and lysosomes in Adv-hBMP-2 transfected MSCs compared to MSCs of other two groups. At the 3rd and 6th weeks after cell injection, ectopic bones were observed in muscles of nude mice of Group 1. Only fibrous tissue or a little bone was found in other two groups. Conclusions: BMP-2 gene transfected MSCs can differentiate into osteoblasts in vitro and induce bone formation in vivo.
基金ThisprojectwassupportedbytheNationalNaturalScienceFoundation (No .3 9870 792 )
文摘Objective: To explore reciprocal action between BMP 2 (bone morphogenetic protein 2) and BMP 3 for better understanding of the mechanism of BMP during bone fracture union. Methods: rhBMP 2 was added into the cultured fibroblasts with the concentration of 1 200 ng/ml. The expression of BMP 3 in fibroblasts was detected by immunohistochemistry. Eukaryotic expression vector pcDNA3 BMP 3 was transfected into the fibroblasts. After the effective expression of BMP 3 was identified, BMP 2 was also detected by immunohistochemistry in BMP 3 expression cells. The fibroblasts transfected with empty vector pcDNA3 were used as the control. Results: Exogenous rhBMP 2 could promote the expression of BMP 3 in fibroblasts. BMP 3 also could be detected in these cells.Conclusions: BMP 2 and BMP 3 could reciprocally adjust the expression in fibroblasts.
文摘Objective： To investigate the effect of a new biomaterial combining calcium citrate and recombinant human bone morphogenetic protein-2 （rhBMP-2） on bone regeneration in a bone defect rabbit model. Methods： Totally 30 male New Zealand white rabbits were randomly and equally divided into calcium citraterhBMP-2 （CC-rhBMP-2） group and rhBMP-2 only group. Two 10 ram-long and 5 ram-deep bone defects were respectively created in the left and right femoral condyles of the rabbits. Subsequently 5 pellets of calcium citrate （10 mg） combined with rhBMP-2 （2 rag） or rhBMP-2 alone were implanted into the bone defects and compressed with cotton swab. Bone granules were obtained at 2, 4 and 6 weeks after procedure and received histological analysis. LSD t-test and a subsequent t-test were adopted for statistical analysis. Results： Histomorphometric analysis revealed newlyformed bones, and calcium citrate has been absorbed in the treatment group. The percent of newly formed bone area in femoral condyle in control group and CC-rhBMP-2 group was respectively 31.73%±1.26% vs 48.21%±2.37% at 2 weeks; 43.40%±1.65% vs 57.32%±1.47% at 4 weeks, and 51.32%±7.80% vs 66.74%±4.05% at 6 weeks （P〈0.05 for all）. At 2 weeks, mature cancellous bone was observed to be already formed in the treatment group. Conclusion： From this study, it can be concluded that calcium citrate combined with rhBMP-2 signifcantly enhances bone regeneration in bone defects. This synthetic gelatin matrix stimulates formation of new bone and bone marrow in the defect areas by releasing calcium ions.
