BACKGROUND: In the healing process of fracture, if the callus tissue around the location of fracture is cleared, the end of fracture is difficult to be integrated completely, and more or less there would be some bone de fect. We deduce that these callus tissues are the necessary product during the process of bone reparation, which ought to be useful. Concerning this,we observed histological content and the volume of transforming growth factor-β1 (TGF-β1) with histological analysis and hybridization in situ of the frozen section of these calluses to investigate the effects of callus graft in the healing process of fracture.OBJECTIVE: To understand the salvaging value of bony callus organization around fracture in delayed operation by means of determining histological substances and contents of TGF-β1.DESIGN: Hybridization in situ of the callus tissue and randomized-grouping and controlled experiment.SETTING: Department of Orthopaedics, the Second Hospital of Xi'an Jiaotong University, and Department of Orthopaedics, Central Hospital of Taizhou City PARTICHANTS: Totally 51 inpatients who received delayed fracture operations in the Department of Orthopaedics of the Second Hospital of Xi 'an Jiaotong University and Central Hospital of Taizhou City from July 1994 and October 2003 were recruited. In some cases of delayed fracture operation, callus was obtained with the consent of patients, Dig labeled kit was bought from Boehringer Mannheim Corp. CTT GCT GTA CTGTGT GTC CAA, TGF-β1 oligonucleotide probe sequence, was synthe sized by DNA synthesizer. Computer X-ray system was bought from Japan Fuji Corp. METHODS: 2,4,6,8 weeks after fracture, a small amounts of callus was obtained. Frozen section was made from undecalcified callus tissue, and noneisotope Dig labeled hybridization in situ, observe gene expression of TGF-β1 in callus. In the operation, after clearing the bone stump and fas tening the fracture, original bony callus was grafted to the bone defect and around the fracture. Simple callus graft, flank bone graft or both were adopted respectively on patients in delayed operation with follow-up obser vations were given for 1-4 years, with the average of 1 year and 9 months,at outpatient clinic for recheck.MAIN OUTCOME MEASURES: ① Histological substance and TGF-β1 content of bony callus at different phases of the fracture. ② Effect of different mode of bone graft on the time of fracture healing. RESULTS: Totally 51 cases were treated, and 7 cases were not followed up for death or moving to other areas. Data of the followed-up 44 patients were collected into the stage of result analysis. ① Gene expression of TGF-β1 in bone callus: In the second week after fracture, there were a small amount of fibrous bone callus and comparatively much cartilage bone callus,and TGF-β1 was hypso-expressed in cartilage cells inside the cartilage islet; about four weeks later, TGF-β1 was notably expressed in os teoblasts; about eight weeks later, a host of calluses grew, mainly carti laginea, and the numbers of formed fibroblast, callus, osteoblasts and tra becular bone were increasing. TGF-β1 expression in all kinds of cells dis appeared after six weeks but had a tendency to rise again in bone matrix in the eighth week. ② There were no significant difference of healing time in different modes of bone graft .CONCLUSION: TGF-β1 plays an important role in balancing bone healing, and necessary connections exist between tissue change in bone callus and change in expression of TGF-β1. As callus is a product of recovery and reconstitution of organism, we regard callus graft salvage as a feasible method in reducing pain and damage to patients who need graft.

BACKGROUND:Previous studies designed and made titanium metal rubber cervical disc prosthesis, and performed feasible studies on its effect on movement and stress distribution by replicating intervertebral discs. OBJECTIVE:To further observe the changes in the stability of goat cervical vertebra after metal rubber cervical disc replacement. METHODS:Nine goats were randomly divided into experimental group (n=6) and normal control group (n=3). Goats in the experimental group received metal rubber cervical disc replacement at C4/5segment. Goats in the normal control group did not receive any treatment. Radiographic data at anteroposterior and lateral position, hyperextension and excessive flexion were taken to measure intervertebral height, range of motion and intervertebral angle at C4/5 segment before operation, immediately, 4, 8, 12 weeks after operation. Subsequently, slicing and embedding of hard tissue at surgical segment, picric acid-acid fuchsin staining and scanning electron microscopy were conducted.RESULTS AND CONCLUSION:No significant difference in the intervertebral height and spinal range of motion at C4/5 segment at different time points was detected between postoperative results in the experimental group and preoperative results in the experimental group, normal control group. The intervertebral height at C4/5 segment was higher immediately, 4 and 8 weeks after surgery than preoperative result in the experimental group (P < 0.05). No significant difference in intervertebral angle at C4/5 segment was detectable between 4, 8 and 12 weeks postoperatively in the experimental group and normal control group (P < 0.05). At 4 weeks after surgery, bone did not contact with the edge of the prosthesis in the experimental group. At 8 weeks, the gap between bone and the prosthesis became smal, and some new bone attached to the edge of the prosthesis. At 12 weeks, a few osteoblasts were observed on the surface of the prosthesis. New osteogenic tissue grew into the prosthesis. Results suggested that metal rubber cervical disc replacement in the intervertebral space could maintain intervertebral height and range of motion in a short period, and tightly bind to the vertebral body.

ts the shear stress, thus, facilitates the union of fracture and the restoration of function.

OBJECTIVE: To construct an effective lentiviral vector for RNA interference (RNAi) with human glucose transporter 3 (GLUT3)gene.
 METHODS: Four pairs of shRNA sequences against different parts of GLUT3-mRNA were separately cloned into the RNAi plasmid vector pLV-shRNA by recombinant DNA technology to construct shRNA expression vectors pLV-shRNA-GLUT3-1, pLV-shRNA-GLUT3-2, pLV-shRNA-GLUT3-3, and pLV-shRNA-GLUT3-4. The vectors were transfected into HeLa cells to detect the effectiveness of GLUT3 gene silencing. One of effective vectors was selected and co-transfected into 293T cells with lentivirus packaging plasmids to obtain packaged lentivirus particles LV-GLUT3. After viral titer determination, U251 glioblastoma cells were infected with LV-GLUT3 at a multiplicity of infection (MOI) of 10. Finally, the expression of GLUT3 protein was detected by Western blot. 
 RESULTS: DNA sequencing demonstrated that the shRNA sequences were successfully inserted into the pLV-shRNA vectors. In HeLa cells, the expression of GLUT3-mRNA was significantly down-regulated by the recombinant vectors compared with negative control. The recombinant lentivirus LV-GLUT3 harvested from 293T cells had a titer of 1.5×10(9) TU/mL. After infection with LV-GLUT3, the expression of GLUT3 protein in U251 glioblastoma cells was down-regulated. 
 CONCLUSION An effective lentiviral shRNA expression vector targeting the GLUT3 gene is successfully constructed and can be used for further study on the functions of GLUT3 gene.
Genetic Vectors ; Glucose Transporter Type 3 ; genetics ; HEK293 Cells ; HeLa Cells ; Humans ; Lentivirus ; Plasmids ; RNA Interference ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Transfection