Résumé
The effects of targeted silencing of heparanase gene by small interfering RNA (siRNA) on invasiveness and metastasis of osteosarcoma cells (MG63 cells) were investigated in the present study.Two complementary oligonucleotide strands were synthesized and inserted into pGenesil-1 vector based on the mRNA sequence of heparanase gene.The expression vector containing short hairpin RNA (pGenesil-shRNA) was constructed successfully.MG63 cells were randomly allocated into 3 groups:blank group,empty vector (pGenesil) transfected group and expression vector (pGenesil-shRNA) transfected group.Under the induction of Lipofectamine 2000,the recombinants were transfected into MG63cells.Heparanase gene expression level was detected by RT-PCR and Western blotting.Cell proliferation was measured by MTT assay.Cell invasiveness and metastasis were examined by cell adhesion and Transwell-ECM assays.HUVECs migration assay was applied for the detection of angiogenesis.As compared with negative controls,the mRNA and protein expression levels ofheparanase were down-regulated by 76.1％ (P＜0.01) and 75.3％ (P＜0.01) respectively in the pGenesil-shRNA transfected group.Meanwhile,the proliferation,adhesiveness,invasiveness and angiogenesis properties of MG63 cells were all significantly inhibited.It was suggested that targeted silencing of heparanase gene by siRNA could dramatically inhibit the invasiveness and metastasis of osteosarcoma cells.
Résumé
The mobilization efficiency of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) to bone marrow mononuclear cells (MNCs) in mice was observed,and the changes of CXCL12/CXCR4 signal were detected in order to find out the mobilization mechanism of stem cells.Kunming mice were randomly divided into two groups.The mice in treatment group were subjected to subcutaneous injection of G-CSF at a dose of 100 μg/kg and SCF at a dose of 25 μg/kg every day for 5 days,and those in control group were given isodose physiological saline.The MNCs were separated,counted and cultured,and the colony-forming unit-fibroblast (CFU-F) was evaluated.CD34+CXCR4+ MNCs were sorted by flow cytometry.The expression of CXCL12 protein in bone marrow extracellular fluid was detected by ELISA,and that of CXCL12 mRNA in bone marrow was measured by RT-PCR.The results showed that the counts of MNCs in peripheral blood and bone marrow were increased after administration of G-CSF/SCF (P＜0.01).The factors had a dramatic effect on the expansion capability of CFU-F (P＜0.05).Flow cytometric of bone marrow MNCs surface markers revealed that CD34+CXCR4+ cells accounted for 44.6%±8.7% of the total CD34+ MNCs.Moreover,G-CSF/SCF treatment induced a decrease in bone marrowCXCL12 mRNA that closely mirrored the fall in CXCL12 protein.In this study,it is evidenced that G-CSF/SCF can effectively induce MNCs mobilization by disrupting the balance of CXCL12/CXCR4 signaling pathway in the bone marrow and down-regulating the interaction of CXCL12/CXCR4.
Résumé
To explore the expression of Beclin1 in osteosarcoma and investigate the effects of down-regulation of autophagy on the chemotherapeutic sensitivity to cisplatin (DDP),the expression of Beclin1 in 28 specimens of osteosarcoma (group A) and 19 specimens of normal bone tissues (group B) were immunohistochemically detected. The expression of Beclin1 mRNA in MG63 cells treated with different concentrations of DDP was examined with RT-PCR. After down-regulation of autophagy in MG63 cells by an autophagy inhibitor,3-methyladenine (3-MA),the cell proliferation inhibition rate of MG63 cells treated with DDP was evaluated by using the MTT assay. The positive rates of Beclinl were 67.85% in group A and 94.73% in group B. Its expression was lower in osteosarcoma than in normal bone tissues,with a significant difference found between them (P＜0.05).RT-PCR showed that the expression of Bcclin1 mRNA in the cells treated with high-dose DDP were higher than that in the non-treated cells,and no significant difference in the expression of Beclin1 mRNA was found between the cells treated with low-dose DDP and the non-treated cells. There was a positive correlation between the level of Beclin1 mRNA expression and the concentration of DDP.MTT assay showed that the proliferation inhibition rates of the cell treated with 3-MA and DDP combined were substantially increased when compared with those treated with DDP alone (P＜0.01).This study demonstrated that autophagy may be implicated in the carcinogenesis of osteosarcoma,and DDP may induce autophagy in the MG63 cells. It also suggests that the down-regulated autophagy could increase chemotherapeutic sensitivity of DDP to osteosarcoma.
Résumé
In order to investigate the effect ofArg-Gly-Asp (RGD) peptide-modified silk biomaterial on the adhesion and proliferation of bone marrow-derived mesenchymal stem cells (MSCs),MSCs of third generation were seeded onto the surface of RGD-decorated silk (silk-RGD group),silk alone (silk group) or tissue culture plate (TCP group).After incubation for 4 or 12 h,MSCs were examined quantitatively by using precipitation method for cell attachment.The cell proliferation,which was de-fined as cell density,was compared among the three groups after culture for 1,2,3,and 4 days.Cell skeleton,which was labeled fluorescently,was observed under laser confocal microscope after 24 h of culture.The results showed that cell adhesion rate in silk-RGD group was higher than in silk group (P<0.05),but similar to that in TCP group after incubation for 4 or 12 h (P>0.05).There were no sig-nificant differences in the cell proliferation among the three groups at different time points (P>0.05 for all).Laser confocal microscopy revealed that in silk-RGD group,MSCs,strongly fluorescently stained,spread fully,with stress fibers clearly seen,while in silk group,actin filaments were sparsely aligned and less stress fibers were found.It was concluded that RGD peptide could improve the ad-hesion of MSCs to the silk scaffold,but had no impact on the proliferation of the cells.
Résumé
To investigate the efficacy of a combination therapy on gluteal muscle contracture, 286 definitely diagnosed patients were subjected to surgical treatment, and then functional exercises and physical therapy. The patients with severe symptoms were asked to have a set of specially-designed functional exercises. All the patients were followed up for 3 to 24 months by hospital visit, correspondenee or telephone interview. The effective rate was 100%, and the curative rate was up to 94.6%. Few patients developed complications and relapse was rare. It is concluded that the combination therapy, including surgical removal of diseased tissues, functional exercises and physical therapy, is an effective approach for the treatment of severe juvenile gluteal muscle contracture.
Résumé
Summary: The inhibitory effect of niacinamide on tumor necrosis factor-α (TNF-α) induced annulus fibrous (AF) degradation was assessed, and the mechanism of the inhibition was investigated. Chiba's intervertebral disc (IVD) culture model was established. Forty-eight IVDs from 12 adult Japanese white rabbits were randomly divided into 4 groups (12 IVDs in each group), and various concentrations of niacinamide and TNF-α were added to the medium for intervention: negative control group, niacinamide control group (0.5 mg/mL niacinamide), degeneration group (10 ng/mL TNF-α), and treatment group (0.5 mg/mL niacinamide and 10 ng/mL TNF-α). After one week's culture, AFs were collected for glycosaminoglycan (GS) content measurement, safranin O-fast green staining, and immunohistochemical staining for typeⅠ,Ⅱ collagen and cysteine containing aspartate specific protease-3 (Caspase-3). It was found that the GS content in treatment group was increased by about 48% as compared with degeneration group (t=16.93, P<0.001), and close to that in niacinamide control group (r=0.71, P=0.667). Safranine O-fast green staining exhibited higher staining density and better histological structure of AF in the treatment group as compared with the degeneration group. Immunohistochemical staining for both Type Ⅰ and Ⅱ collagen demonstrated that lameilar structure and continuity of collagen in treatment group were better reserved than in degeneration group. Positive staining rate of Caspase-3 in AFs of negative control group, niacinamide control group, degeneration group and treatment group was 3.4%, 4.3%, 17.9% and 10.3% respectively. The positive rate in treatment group was significantly lower than in degeneration group (P<0.01). It was concluded that niacinamide could effectively alleviate TNF-α induced destruction and synthesis inhibition of matrix ingredients in AFs. The inhibition may be related with reduction of expression of Caspase-3. Thus, niacinamide is of potential for IVD degeneration clinical treatment.