Objective: To explore the effect of gamma irradiation on nuclear factor-kappa B in cultured bone marrow stromal cells. Methods: Immunocytochemistry, Western blot and electrophoretic mobility shift assay (EMSA) were us...Objective: To explore the effect of gamma irradiation on nuclear factor-kappa B in cultured bone marrow stromal cells. Methods: Immunocytochemistry, Western blot and electrophoretic mobility shift assay (EMSA) were used. Results: The expression of NF-kB in cultured mouse bone marrow stromal cells (BM-SCs) on the level of protein was elevated after exposure to 60Co in the dosage of 8. 0 Gy with the use of im-munocytochemistry and Western blot. The activity of nuclear factor-kappa B in cultured BMSCs was significantly increased after exposure to gamma irradiation by using EMSA. The activity peak was at the 4th h after irradiation. Conclusion: Our results suggest that the activation of nuclear factor-kappa B in the BMSCs after irradiation may be involved in the protection of BMSCs against apoptosis and in the recovery of hematopoiesis after radiation.展开更多
Objective:Large segmental bone defect repair remains a clinical and scientific challenge with increasing interest focusing on combining gene transfection with tissue engineering techniques.The aim of this study is to ...Objective:Large segmental bone defect repair remains a clinical and scientific challenge with increasing interest focusing on combining gene transfection with tissue engineering techniques.The aim of this study is to investigate the effect of connective tissue growth factor(CTGF) on the proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells(MSCs).Methods:A CTGF-expressing plasmid(pCTGF) was constructed and transfected into MSCs.Then expressions of bone morphogenesis-related genes,proliferation rate,alkaline phosphatase activity,and mineralization were examined to evaluate the osteogenic potential of the CTGF gene-modified MSCs.Results:Overexpression of CTGF was confirmed in pCTGF-MSCs.pCTGF transfection significantly enhanced the proliferation rates of pCTGF-MSCs(P<0.05).CTGF induced a 7.5-fold increase in cell migration over control(P<0.05).pCTGF transfection enhanced the expression of bone matrix proteins,such as bone sialo-protein,osteocalcin,and collagen type I in MSCs.The levels of alkaline phosphatase(ALP) activities of pCTGF-MSCs at the 1st and 2nd weeks were 4.0-and 3.0-fold higher than those of MSCs cultured in OS-medium,significantly higher than those of mock-MSCs and normal control MSCs(P<0.05).Overexpression of CTGF in MSCs enhanced the capability to form mineralized nodules.Conclusion:Overexpression of CTGF could improve the osteogenic differentiation ability of MSCs,and the CTGF gene-modified MSCs are potential as novel cell resources of bone tissue engineering.展开更多
To further investigate the osteogen ic potential of rabbit marrow stromal stem cells cultured in vitro. Methods: Rabbit marrow stromal stem cells were isolated by dens ity gradient centrifugation method and amplified ...To further investigate the osteogen ic potential of rabbit marrow stromal stem cells cultured in vitro. Methods: Rabbit marrow stromal stem cells were isolated by dens ity gradient centrifugation method and amplified in the flasks, using the osteog enic inducing conditions (OGC) as the culture media. The osteogenic potential of marrow stromal stem cells were investigated by means of bone seeking fluoresce nce (tetracycline) labelling, Alizarin red S (ARS) staining, Alcian blue Sirius red (AS) staining, and scanning electron microscope. Results: After being passaged, the marrow stromal stem cells in creased in number, became confluent and formed multi layer structure. The strom al stem cells excreted innumerable tiny granules, heaping up on the cell body an d merging gradually into foggy substances. These foggy substances kept on enlarg ing and formed round, oval, or flake like nodules. These nodules revealed brigh t golden yellow fluorescence under fluorescence microscope when labelled with te tracycline. Histochemical study with specific new bone staining with ARS reveale d positive calcium reaction, both denoting that they were newly formed bone tiss ues. After they were stained with AS, collagen and acid mucopolysaccharide were shown. Under scanning electron microscope, three types of cells with different c onfigurations were found. They were globular cells, spindle shaped cells and po lygonal or polygonal cells. Granules were excreted from the cells and heaped up on the cell body. Needle shaped and irregularly rectangular crystals also appea red and agglomerated with the granules to form nodules and trabecula like or fl ake like structures.Conclusions: Sequence of events of bone formation by rabbit mar row stromal stem cells cultured in vitro is fully depicted and confirmed, which provides the foundation for further investigating the mechanisms of osteoblast d ifferentiation from marrow stromal stem cells and the possible application in or thopaedics.展开更多
基金Supported by the "Ninth Five" Obligatory Budget of PLA. No. 96L045
文摘Objective: To explore the effect of gamma irradiation on nuclear factor-kappa B in cultured bone marrow stromal cells. Methods: Immunocytochemistry, Western blot and electrophoretic mobility shift assay (EMSA) were used. Results: The expression of NF-kB in cultured mouse bone marrow stromal cells (BM-SCs) on the level of protein was elevated after exposure to 60Co in the dosage of 8. 0 Gy with the use of im-munocytochemistry and Western blot. The activity of nuclear factor-kappa B in cultured BMSCs was significantly increased after exposure to gamma irradiation by using EMSA. The activity peak was at the 4th h after irradiation. Conclusion: Our results suggest that the activation of nuclear factor-kappa B in the BMSCs after irradiation may be involved in the protection of BMSCs against apoptosis and in the recovery of hematopoiesis after radiation.
基金supported by the National Basic Research Program (973) of China(No.2005CB623900)
文摘Objective:Large segmental bone defect repair remains a clinical and scientific challenge with increasing interest focusing on combining gene transfection with tissue engineering techniques.The aim of this study is to investigate the effect of connective tissue growth factor(CTGF) on the proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells(MSCs).Methods:A CTGF-expressing plasmid(pCTGF) was constructed and transfected into MSCs.Then expressions of bone morphogenesis-related genes,proliferation rate,alkaline phosphatase activity,and mineralization were examined to evaluate the osteogenic potential of the CTGF gene-modified MSCs.Results:Overexpression of CTGF was confirmed in pCTGF-MSCs.pCTGF transfection significantly enhanced the proliferation rates of pCTGF-MSCs(P<0.05).CTGF induced a 7.5-fold increase in cell migration over control(P<0.05).pCTGF transfection enhanced the expression of bone matrix proteins,such as bone sialo-protein,osteocalcin,and collagen type I in MSCs.The levels of alkaline phosphatase(ALP) activities of pCTGF-MSCs at the 1st and 2nd weeks were 4.0-and 3.0-fold higher than those of MSCs cultured in OS-medium,significantly higher than those of mock-MSCs and normal control MSCs(P<0.05).Overexpression of CTGF in MSCs enhanced the capability to form mineralized nodules.Conclusion:Overexpression of CTGF could improve the osteogenic differentiation ability of MSCs,and the CTGF gene-modified MSCs are potential as novel cell resources of bone tissue engineering.
文摘To further investigate the osteogen ic potential of rabbit marrow stromal stem cells cultured in vitro. Methods: Rabbit marrow stromal stem cells were isolated by dens ity gradient centrifugation method and amplified in the flasks, using the osteog enic inducing conditions (OGC) as the culture media. The osteogenic potential of marrow stromal stem cells were investigated by means of bone seeking fluoresce nce (tetracycline) labelling, Alizarin red S (ARS) staining, Alcian blue Sirius red (AS) staining, and scanning electron microscope. Results: After being passaged, the marrow stromal stem cells in creased in number, became confluent and formed multi layer structure. The strom al stem cells excreted innumerable tiny granules, heaping up on the cell body an d merging gradually into foggy substances. These foggy substances kept on enlarg ing and formed round, oval, or flake like nodules. These nodules revealed brigh t golden yellow fluorescence under fluorescence microscope when labelled with te tracycline. Histochemical study with specific new bone staining with ARS reveale d positive calcium reaction, both denoting that they were newly formed bone tiss ues. After they were stained with AS, collagen and acid mucopolysaccharide were shown. Under scanning electron microscope, three types of cells with different c onfigurations were found. They were globular cells, spindle shaped cells and po lygonal or polygonal cells. Granules were excreted from the cells and heaped up on the cell body. Needle shaped and irregularly rectangular crystals also appea red and agglomerated with the granules to form nodules and trabecula like or fl ake like structures.Conclusions: Sequence of events of bone formation by rabbit mar row stromal stem cells cultured in vitro is fully depicted and confirmed, which provides the foundation for further investigating the mechanisms of osteoblast d ifferentiation from marrow stromal stem cells and the possible application in or thopaedics.
