Objective: To study the effects of recombinant human Chondromodulin-I protein in treatment of osteosarcoma. Methods: Human Chondromodulin-I gene was amplified from cartilage tissue by RT-PCH and was cloned into pro-caryotic expression vector Pet28a( + ). The recombinant plasmid Pet( CHM-I) was transformed into E. coll BL21 ( DE3 ) ; the product was fused with 6 ?His at N-terminal and was purified by Ni2 +2NTA ion exchange resin. The formation of tube-like cellular networks was assayed by co-incubation of HUVECs and recombinant human (Chondromodulin-I. The proliferation of the cells incubated with the recombinant human Chondromodulin-I protein was examined by MTT assay and cell number counter Null mice received local injection of recombinant human Chondromodulin-I protein into the tumors. Results: Rcombinant human CHM-I protein was successfully obtained. The purified CHM-I did not inhibit the proliferation of tumour cells,but inhibited the formation of tubelike cellar network in vitro and tumor growth. Conclusion:Recom-bitant human Chondromodulin-I protein can inhibit angiogenesis in vitro and growth of tumor in vivo.

In order to evaluate the efficacy of low intensity ultrasound and tissue engineering technique to repair segmental bone defects, the rabbit models of 1.5-cm long rabbit radial segmental osteoperiosteum defects were established and randomly divided into 2 groups. All defects were implanted with the composite of calcium phosphate cement and bone mesenchymal stem cells, and additionally those in experimental group were subjected to low intensity ultrasound exposure, while those in control group to sham exposure. The animals were killed on the postoperative week 4, 8 and 12 respectively, and specimens were harvested. By using radiography and the methods of biomechanics, histomorphology and bone density detection, new bone formation and material degradation were observed. The results showed that with the prolongation of time after operation, serum alkaline phosphatase (AKP) levels in both groups were gradually increased, especially in experimental group, reached the peak at 6th week (experimental group: 1.26 mmol/L; control group: 0.58 mmol/L), suggesting the new bone formation in both two group, but the amount of new bone formation was greater and bone repairing capacity stronger in experimental group than in control group. On the 4th week in experimental group, chondrocytes differentiated into woven bone, and on the 12th week, remodeling of new lamellar bone and absorption of the composite material were observed. The mechanical strength of composite material and new born density in experimental group were significantly higher than in control group, indicating that low intensity ultrasound could not only effectively increase the formation of new bone, but also accelerate the calcification of new bone. It was concluded that low intensity ultrasound could evidently accelerate the healing of bone defects repaired by bone tissue engineering.

Objective: To explore the inhibitiory effect of antisense oligodeoxynucleotide(ASODN) of survivin gene on the cultured osteosarcoma cell line MG63 and its sensitization effect to chemotherapy.Methods: survivin phosphorothioate ASODN was synthesized and transfected into MG63 cells by lipofectamine 2000.MTT assay was used to detect cell inhibition ratio.Apoptosis was observed by flow cytometry.Survivin mRNA and protein expression were determined by RT-PCR and Western blot respectively.Results: The proliferation of the cells transfected by lipofectamine 2000 was inhibited by survivin ASODN in a dose and time dependent manner.A higher total apoptosis rate(81.12?3.2)% could be induced in MG 63 cells in group Lip-ASODN than in group Lip-SODN(27.09?2.1)%(P

Objective To evaluate the hemorrheological changes after tourniquet deflation.Methods Twenty rabbits were subjected to lower extremity tourniquet inflation for 3 h. Venous blood samples were taken before tourniquet inflation and 2min after tourniquet deflation for measurement of hemorrheological parameters: low-shear viscosity and high-shear viscosity of whole blood; plasma high-shear viscosity , hematocrit; fibrinogen; aggreability,deformability and stiffness of erythrocyte; and blood sedimentation K value. The levels of parameters before tourniquet inflation served as baseline values. The gastrocnemius muscle samples were taken before tourniquet inflation and 5 min after torrniquet deflaion for observation of ultrastructure of small blood vessel.Results Compared with the baseline values, the levels of low- and high- shear viscosities of whole blood, plasma viscosity, fibrinogen, blood sedimentation K value, and aggreability and stiffness of erythrocyte increased significantly (P

Objective To observe the treatment and prophylaxis of deep venous thrombosis(DVT) following fracture of lower limb and the hemorheological changes before and after treatment.Methods 26 patients underwent fracture of lower limb for study received thromholytic therapy,decreasing blood viscosity,promoting blood circulation by eliminating stasis.Before treatment and on the tenth day after treatment,the hemorheological parameters were tested.There existed a statistical analysis for the data processing.Results Among 26 patients,therapeutic evaluation:12 patients was superior,13 patients were favorable,only one patient was infective,and the total effective rate was 96.2%.Curative effect was satisfied.Conclusions The most important was prevention first for the patient with DVT following fracture of lower limb.The critical measure was early diagnosis and early treatment.

Immortalized human precartilaginous stem cells (IPSCs) were established to provide stable cell resource for the study of the molecular mechanism of gene targeting on the differentiation of PSCs. Plasmid pCMVSV40T/PUR containing simian virus 40 large T antigen gene (SV40Tag) was transfected into human PSCs by using lipofectin transfection. Colonies were isolated by puromycin selection and expanded by multiple passages. Immunohistochemistry, RT-PCR and Southern blotting were used to identify the transfected cells and to detect the expression and integration of SV40Tag in expanded cell lines. The positive colonies were isolated and subcultured, designated immortalized precartilaginous stem cells (IPSCs), which were confirmed as fibroblast growth factor receptor-3 (FGFR-3) positive cells by immunohistochemistry and RT-PCR. SV40Tag cDNA was found in cultured IPSCs of passage 8 by Southern blotting, and the expressions of SV40Tag mRNA and protein were confirmed by RT-PCR. These findings suggested that IPSCs strain with SV40Tag was constructed successfully.
Cartilage/*cytology ; Cell Proliferation ; Cell Transformation, Viral ; Cells, Cultured ; Fetus ; Simian virus 40/*genetics ; Stem Cells/*cytology ; Transfection

Background and Purpose:Cyclooxygenase-2(COX-2) plays an important part in tumor genesis,growth,and angiogenesis.Many inhibitors of COX-2 could inhibit proliferation and induce apoptosis of cancer cells. This study investigated the impact of NS398 on anti-proliferation and the induction of apoptosis in human osteosarcoma cell line MG-63.Methods:Cell proliferation is measured by MTT method.Characteristic changes of apoptosis in morphology are observed by fluorescence microscopy、transmission electron microscopy(TEM) and quantitatively by TDT-mediated dUTP-biotin nick end-labeling(TUNEL) assay.The apoptotic rates are calculated by flow cytometry(FCM).Results:The growth inhibition rates of MG-63 cells treated with 1,10,50,100 and 200 ?mol/L NS398 are 14.7%,23.5%,33.6%,52.5% and 81.4%,respectively(P

In order to study the chondrogenic phenotype differentiation of adult sheep bone marrow-derived mesenchymal stem cells (MSCs) in a defined medium as potential seed cells for cartilage tissue engineering. MSCs were isolated by density centrifugation with Percoll solution from bone marrow aspirated from sheep iliac crest. The third passage of MSCs were induced with H-DMEM containing TGF-beta3, IGF-I, Dexamethasone and VitC. The shape and ultrastructure of cells were observed, toluidine blue stain for GAG and immunohistochemistry for type II collagen were applied for chondrogenic phenotype identification. After 14 days of induction, MSCs changed from a spindle-like appearance to a polynal shape, a large amount of endoplasmic reticulum, Golgi complex and mitochondria were observed, and the differentiation of MSCs chondrogenic phenotype was verified by positive staining of toluidine blue and immunohistochemistry. MSCs derived from bone marrow can differentiate to chondrogenic phenotype when induced in vitro and can be used as optimal seed cells for cartilage tissue engineering.
Bone Marrow Cells/*cytology ; Cell Differentiation ; Cell Separation ; Cells, Cultured ; Chondrocytes/*cytology ; *Chondrogenesis ; Mesenchymal Stem Cells/*cytology ; Phenotype ; Sheep ; Tissue Engineering

Objective To study the biological effects of pulsed electromagnetic fields (PEMFs) on the pro-liferation of immunomagnetically separated human precartilaginous stem cells (PSCs) in vitro. Methods The cells from an aborted fetus's metaphysis were digested using collagenase. The PSCs were isolated by magnetic cell sorting (MACS), then subcultured and amplified. Flow cytometry, immunohistochemistry, immunofluorescence and RT-PCR analysis were performed to identify the purified PSCs. The PSCs were stimulated by PEMFs at 50 Hz frequency and 1 mT intensity. Cell proliferation was measured at different time points using methyl thiazolyl tetrazolium ( MTT), and the cell growth curve was plotted. Flow cytometry was applied to measure the cell cycle and apoptosis. Results The PSCs were successfully cultured. There was fibroblast growth factor receptor-3 (FGFR-3) on their sur-face. Cell proliferation was promoted after 4 and 6 days of PEMF stimulation. The percentage of cells at the S phase was higher than in a control group. Early, late and total rates of apoptosis in the experimental group decreased signifi-cantly. Conclusion PEMFs can enhance the proliferation and inhibit the apoptosis of human PSCs, and it is possi-ble to cultivate the high density human PSCs in vitro.

Objective To establish the strain of immortalized human precartilaginous stem cells (PSCs) which can be a stable cell resource for study of the molecular mechanism of gene targeting on differ-entiation of PSCs. Methods Plasmid pCMVSV40T/PUR containing simian virus 40 large T antigen gene (SV40Tag) was transfected into human PSCs using lipofeetin transfection method. Colonies were isolated by puromycin selection and expanded by many passages. Immunohistochemistry, RT-PCR and Southern blot were used to identify the transfected cells and to detect the expression and integration of SV40Tag in expanded cell lines. Results The positive colonies were isolated and subcultured, named as immortalized precartilaginous stem cells (IPSCs), which were confirmed as positive to fibrnblast growth factor receptor-3 (FGFR-3). The existence of SV40Tag cDNA was detected by Southern blot and the expression of SV40Tag mRNA and protein by RT-PCR and immunohistochemistry. Conclusion IPSCs strain with SV40Tag can be constructed successfully.