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BACKGROUND: Under specially physical environment, for example weightlessness, metabolism of bone tissue may have remarkable changes; however, osteoblast is a core of bone metabolism and bone formation, so it is very sensitive to changes of gravity environment.OBJECTIVE: To observe the effects of simulated weightlessness on bone marrow stromal cell count and osteogentic capacity of weight bearing bone in rats so as to reveal the mechanism of bone loss.DESIGN : Randomized pairing and controlled study.SETTING: College of Aerospace Medicine and Department of Pathology of Stomatology College, the Fourth Military Medical University of Chinese PLA.MATERIALS: A total of 20 adult healthy male SD rats were selected in this study. At the beginning of experiment, rats based on their body mass were randomly divided into control group and suspension group with 10 in each group. Alkaline phosphatase kit was provided by Beijing Zhongsheng Bioengineering High-technological Company.METHODS: The experiment was carried out in the Department of Pathology, Collage of Stomatology, the Fourth Military Medical University of Chinese PLA from November 1999 to July 2000. Rats were randomly divided into tail suspension group and control group with 10 in each group. Rats in the tail suspension group were given tail suspension for 28 days. Their heads maintained 30° low position, and their hindlimbs freely suspended and were not given weight loading. While, rats in the control group were fed normally. At the end of experiment, bone marrow stromal cells were obtained from femur for primary and transferring cultures.MAIN OUTCOME MEASURES: Cell counting and methylthianolyldiphenyl-tetrazolium bromide (MTT) assay were used to draw growth curve of cells in primary and transferring cultures and to measure activity of alkaline phosphatase and forming quantity of mineralized nodules in vitro.RESULTS : ① Activity of alkaline phosphatase: Activity of alkaline phosphatase of cells in the primary and transferring cultures in the suspension group was lower than that in the control group, and there was significant difference between them (P＜0.05). ② Forming quantity of mineralized nodules: Forming quantity of mineralized nodules in the suspension group was less than that in the control group, and there was significant difference between them (P＜0.05). ③ Cell growth: Growth curve of femoral stromal cells in primary and transferring culture manifested as S. Doubling time of cells in the suspension group was similar to that in the control group. ④ Amount of bone marrow stromal cells in femur: Amount of bone marrow stromal cells in primary culture in the suspension group was decreased as 50% as that in the control group (P＜0.05).CONCLUSION: Under simulated weightlessness, amount of bone marrow stromal cells decreases obviously; in addition, amount of osteoblast also decreases in weight bearing bone of hindlimbs and osteogentic capacity in vitro decreases simultaneously.
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<p><b>OBJECTIVE</b>To observe the heterotopic osteogenesis of autogenous marrow stromal cells loading on porous calcium phosphate ceramic scaffolds with rhBMP2 gene transfection in a Sprague-Dawley rat model.</p><p><b>METHODS</b>Autogenous marrow stromal cells were obtained from left femurs and tibias of 20 male adult SD rats under general anesthesia and sterile condition and cultured in alpha-Minimal Essential Medium supplemented with 15% fetal bovine serum. RhBMP2 gene was transfected into stromal cells by means of LipofectAMINE 2000 reagent five days after primary culture. The stably gene expressive cells were selected with G-418 for 14 days and mixed with stromal cells without transfection. The mixture cells were seeded and subcultured for another 10 days in porous calcium phosphate bioceramic that had been subjected to surface-modification via soaking in human plasma fibronectin. The cell-ceramic compound was implanted subcutaneously and intramuscularly in the corresponding rat. Lab animals were sacrificed at two-week intervals till twenty weeks postoperatively and the involved samples were removed.</p><p><b>RESULTS</b>Morphologic and histological study demonstrated that cell-ceramic compound had an ability of heterotopic osteogenesis, which was similar to that of autogenous osteoblasts in previous study.</p><p><b>CONCLUSION</b>It seems that autogenous stromal cells with rhBMP2 transfection acts as a bioreactor promoting proliferation and differentiation of stem cells when they are replanted into the corresponding animals.</p>
Subject(s)
Animals , Male , Rats , Bone Marrow Cells , Cell Biology , Physiology , Bone Morphogenetic Protein 2 , Bone Morphogenetic Proteins , Genetics , Calcium Phosphates , Pharmacology , Ceramics , Pharmacology , Osteogenesis , Rats, Sprague-Dawley , Recombinant Proteins , Genetics , Stromal Cells , Cell Biology , Physiology , Transfection , Transforming Growth Factor betaABSTRACT
砄bjective: To investigate Human telomerase reverse transcriptase(hTRT) mRNA expression in oral squamous cell carcinoma(OSCC) and the relationship between hTRT activity and proliferation cell nuclear antigen(PCNA) expression.Methods: The expression of hTRT mRNA and PCNA was detected by in situ mRNA hybridization and immuno histochemical technique in 52 cases of OSCC and 22 cases of OSCC adjacent tissues including histologically normal mucosas and dysplastic lesions. Results: hTRT mRNA was observed in 82.7% cases of OSCC, 41.7% of dysplasia and 20% of normal mucosa( P
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Objective:To study the effects of parathyroid hormone (PTH) on BMP3 expression of cultured human dental papilla mesenchymal cells (DPMCs). Methods:DPMCs were obtained by cell culture, BMP3 mRNA expression was studied by in situ hybridization. BMP3 gene expression of human DPMCs after exposure to 33.3 nmol/L PTH for 5 days were measured. The cells cultured in the medium without PTH served as control.Results: Stronger BMP3 positive signals were observed in PTH treated cells than that in the control cells.Conclusion:PTH stimulats BMP3 systhesis of cultured DPMCs.
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Objective:To study the effects of doxycycline on the proliferation and invasion of adenoid cystic carcinoma cells.Methods:MTT assay was applied to study the effects of doxycycline on proliferation of SAcc83 cells. Activity of type IV collagenase was detected by the method of gelatin incorporated SDS PAGE electrophoresis.Monolayer cell organ culture was carried out to study the invasion of the cells.Results:Doxycycline at 5～50 ?g/ml inhibited the proliferation of adenoid cystic carcinoma cell in a dose and time dependant manner. 10 ?g/ml of the drug inhibited type Ⅳ collagenase and decreased the invasion of adenoid cystic carcinoma cells by 48%.Conclusion:Doxycycline can inhibit proliferation and invasion of SAcc83 cells.
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objective: To study the effects of BMP on the growth of osteosarcoma cells. Methods: Human antisense BMP2 retrovirus expression vector was constructed and transfected into human osteosarcoma OS 9901 cells by Lipofect AMINE. Positive cell clones were selected with G 418. The expression of BMP and PCNA were determined by immunohistochemical methods, image analysis and expressed as grey level. The morphology and cell cycle distribution of the cells were studied by electronmicroscope and flowcytometry respectively. Results:The grey levels of BMP and PCNA in the transfected cells were 198.4?8.51 and 197.3?3.22, those of the control were 135.1?12.32 and 142.9?8.47,respectively. G 1, G 2 and S phase cells were 51.9%,18.2% and 22.7% in the cell cycle of transfected cells, while those of the control were 52.8%,11.1% and 36.1%, respectively.Increased lysosome and small pieces of chromatin were observed in the transfected cells under electronmicroscope. Conclusion:Transfection of antisense BMP2 may inhibite the expression of BMP and proliferating activity of osteosarcoma cells.
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Objective: To study the significance of DNA content in oral squamous cell carcinoma (SCC).Methods: DNA content and cell cycle of the cells in 18 cases of oral SCC were analized by image cytometry (ICM). Results: The DNA index (DI) and proliferation index (PI) were remarkably heigher in SCC than in normal epithelium ( P
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Objective To probe into the mechanisms of bone morphogenetic protein-2 (BMP-2) in osteosarcoma and to provide basis for gene therapy. Methods A 1.0 kb cDNA fragment of human BMP-2 gene was inserted reversely into PDOR and Human antisense BMP-2 retrovirus expression vector was constructed. The recombinant retroviral vector was transfected into human osteosarcoma cells OS-9901 with liposome AMINE that expressed abundant BMP. Positive cell clones were selected with G 418. The expression of BMP and proliferating cell nuclear antigen (PCNA) was determined by immunohistochemical ABC methods. The osteosarcoma cell cycle was analysed by flowcytometry, and the changes were observed by electronmicroscope. Results The expression of cellular BMP and PCNA in the transfected osteosarcoma cells decreased obviously (t=24.01， 26.09, respectively, P
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Objective:To evaluate the immunogenicity of the recombinant plasmid pcDNA 3.1(+)/kgp_ cd.Methods:BALB/c mice were immunized with recombinant plasmid pcDNA 3.1(+)/kgp_ cd,KGP_ cd protein(control) or vector pcDNA 3.1(+)(control) by quadriceps injection or targeted submandibular gland(TSG) injection. Serum IgG and salivary sIgA levels were assayed by indirect ELISA after immunization. The expression of the protein KGP_ cd in quadriceps and submandibular gland was detected by immunohistochemistry techniques.Results: Serum specific anti-KGP_ cd IgG elicited by pcDNA 3.1(+)/kgp_ cd and KGP_ cd protein was significantly higher in both the quadriceps injection group and the TSG group than that in the pcDNA 3.1(+) group (P
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objective: To investigate the expression of Schwann cell marker S100 protein in salivary adenoid cystic carcinoma and try to find out the correlation of S100 expression and neural invasion of the tumor. Methods: Samples from 20 cases of adenoid cystic carcinoma(ACC) and 18 cases of mucoepidermoid carcinoma(MEC) were immunohistochemical stained with S100 antibody. Results: S100 was positive in all of the samples of the 20 cases of ACC and negative in all of MEC. Neural invasion was observed in 11 out of the 20 cases of ACC and in 2 of MEC. Conclusion: There may be Schwann cell differentiation in salivary adenoid cystic carcinoma, and it may be the histologic base of neural invasion of the tumor.
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Objective To study the effect of baicalin on the expression of receptor activator of nuclear factor-?B ligand (RANKL) and osteoprotegerin (OPG) in cultured human periodontal ligament cells (HPDL cells) as well as its action mechanism. Methods We first constructed small interferring RNA (siRNA) eukaryotic expression vector targeted transforming growth factor ?Ⅱ receptor (TGF-?RⅡ),and then transfected it into T cells. Then HPDL cells together with T cells transfected with siRNA or not were placed in medium that had been added with lipopolysaccharide (LPS) and baicalin. They were divided into six groups and cultured for 48 hours. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to observe the effect of baicalin on OPG-RANKL expression in HPDL cells. Results The clone sequence correctly identified by RT-PCR was consistent with the designed target sequence. The recombinant vector was constructed successfully and the expression of TGF-?ⅡR of T cells which had been transfected with siRNA1 was inhibited obviously. Ratio of RANKL/OPG in each group differed significantly (P