0.05).The size of PCR fragments ranged from 144 to 188bp containing 19～41 CA repeats and the frequency of (CA)n ranged from 0.34%～14.1%.The heterozygosity(H) and polymorphism information content(PIC) were 0.85 and 0.83 respectively.Significant differences were found in frequencies of eleven alleles including 22-repeats,23-repeats,26-repeats,27-repeats,28-repeats,30-repeats,31-repeats,32-repeats,33-repeats,34-repeats and 35-repeats between Chinese and Caucasians.Conclusion:This site is referred to as a genetic marker with high polymorphism information content.And the distribution of this genetic marker is different in different ethnic groups.

[Objective]To investigate the relationship between degeneration of cartilage and the expressions of Matrix Metalloproteinase 7(MMP-7),Matrix Metalloproteinase 9(MMP-9),Matrix Metalloproteinase 13(MMP-13)and Tissue Inhibitor of Matrix Metalloproteinase 1(TIMP-1)in osteoarthritis.[Method]The histological changes of cartilages by hematoxyllin-eosin staining and immunohistochemical expression of Matrix Metalloproteinase 7(MMP-7),Matrix Metalloproteinase 9(MMP-9),Matrix Metalloproteinase 13(MMP-13)and Tissue Inhibitor of Matrix Metalloproteinase 1(TIMP-1)in osteoarthritis.were studied in 20 osteoarthritis cases and 2 normal controls.All data were statistically analyzed by Mann-Whitney U test and correlation analysis.[Result]The osteoarthritis cartilage underwent fibroplasias and tearing.The quantity of chondrocyte increased and the clustered and hypertrophic cells came into being.Little immunostaining of MMP-7 and MMP-13 was observed in normal cartilage,while their expressions increased in degenerated cartilage(P0.05),superficial layer of the moderate and end-stage osteoarthritis.However,it significantly increased in deep layer(P

[Objective] To study the bone metabolic biochemical index of the aged patients with hip fracture,for better predicting the future risk of the old people' s hip fracture.[Method]50 cases of sufferers(over 60 years old) with hip fracture(28 males,and 22 females) and 30 cases of healthy aged people(15 males,and 15 females) were selected to analyze Ⅰ Collagen crosslinked c-telopeptide(ICTP),deoxypyridino line(Dpd) in urine,and serum bone glaprotein(BGP).[Result](1)The mean level of ICTP and Dpd in urine in aged hip fracture group was higherthan that of the control group(P0.05).[Conclusion]Bone absorbability in the aged hip fracture patients is higher than in the aged healthy people.The analysis of ICTP and Dpd in urine may /might give some reference value in preventing and treatlng aged hip fracture patients.

PBL,problem based learning,is a kind of teaching model for resolving problems. Based on the present teaching situation of our university,my experience in NewYork University and my teaching experience for overseas students,this article elucidated the importance and necessity of PBL teaching model in overseas student education and put forward a suitable PBL teaching method.

Objective To screen promoter DNA-binding protein of inducible nitric oxide synthase gene by using phage display technique from human liver cDNA library, and to study the expression and regulation mechanism of iNOS gene. Methods The sequence of iNOS promoter was identified in GenBank by bioinformatics based on the open reading frame(ORF) of iNOS gene and amplified from HepG2 genome by polymerase chain reaction (PCR). The amplified product was subcloned into pCAT3-Basic reporter vector, named as pCAT3-iNOSp. The HepG2 cell line was then transfected with pCAT3-Basic to serve as negative control, and pCAT3-promoter which contains the promoter region of CMV served as the positive control subject, and pCAT3-iNOSp served as the test subject, respectively. The choloraphenical acetyltransferase(CAT)activity was determined by enzyme linked immunosorbent assay(ELISA) kit. The T7 Select human liver cDNA library was biopanned and positive clones were selected. After screening, positive plaque was performed to amplify and PCR products were sequenced. Results The expression of CAT in transfection of pCAT3-PS1TP1p was 4.2 times as higher as pCAT3-Basic plasmid. Sequence analysis was performed in 12 positive plaque, which were the iNOSp binding protein. Conclusion The iNOS gene promoter identified in this study has shown to have transcription activity,and iNOS promoter DNA-binding proteins havs been screened. The results will be useful for further study of the expression and regulation mechanism of iNOS in liver cell.

[Objective]To prove the feasibility of using the distance between sagittal plane of the spinal process and the enter point to individualize the placing of pedicle screw.[Method]Thirty spine specimen were collected and divided into two groups,data were measured,such as the width of the pedicle,distance between the enter point and anterior border of the vertebra,distance between sagittal plane of the spinal process and the enter point,angle from the longitudinal axis of the pedicle to sagittal axis of the vertebra,angle from the longitudinal axis of the pedicle to vertical line of the operating table.In group one the pedicle screws were placed with the help of the distance between sagittal plane of the spinal process and the enter point,the other by the method advised by Ebraheim.CT scan was applied to evaluate the place of the screws,according to the perforation extent,they were classified into 4 grades:A=totally in the pedicle;B=perforation extent4mm.[Result]The individualized group showed much lower perforation rate than the traditional method group in T3~10,and similar in T1,T2,T11,12.[Conclusion]It can obviously improve the accuracy of the pedicle screw placement to use the distance between sagittal plane of the spinal process and the enter point to localize the enter point,especially when anatomic landmark such as articulationes zygapophysiales and transverse process change.

Background and purpose:The screening of the genes being related closely with the mechanism of osteosarcoma metastasis was a difficult point in the realm of orthopaedics.We screened differential expression gene of human osteosarcoma MG-63 cell sublines with different metastatic capabilities with cDNA microarray,and studied the molecular mechanism of osteosarcoma metastasis.Methods:Total RNA of human osteosarcoma MG-63 cell sublines A1 and A2 was extracted,purified to mRNA and then reversely transcripted to cDNA probe respectively.The cDNA probe of A1 was labelled with Cy3 and the cDNA probe of A2 was labelled with Cy5.The two samples were hybridized with the cDNA microarray.The hybridization signals were scanned by Agilent Scanner and obtained data were analyzed using Ima Gene 3.0 software and Genespring software.Results:222 differential expression genes were found between cell sublines A1 and A2 by analyzing gene expression profile.There were 119 upregulated genes and 103 downregulated genes in cell sublines A1.All differential expression genes belonged to six main function groups and 49 genes of these had very obvious differentce in expression.Conclusions:There were many differently expressed genes between A1 and A2 cell sublines and only part of them were closely associated with mechanism of osteosarcoma metastasis.The technology of cDNA microarray could analyze effectively gene expression profile of human osteosarcoma MG-63 cell sublines, and supply a new approach to study the mechanism of osteosarcoma metastasis

The present paper is aimedto investigate the effect of basic fibroblast growth factor (bFGF) on proliferation, migration and differentiation of endogenous neural stem cell in rat cerebral cortex with global brain ischemia-reperfusion. A global brain ischemia-reperfusion model was established. Immunohistochemistry was used to observe the pathological changes and the expression of BrdU and Nestin in cerebral cortex. RT-PCR was used to measure the NSE mRNA in brain tissue. The results of measurements indicated that in sham operation group, there was no positive cell in cerebral cortex, and the content of NSE mRNA did not change. In the operation group, the expression of BrdU and Nestin increased significantly at the end of the 3rd day, and peaked on the 7th day. NSE mRNA expression did not significantly increase. In bFGF group, compared with sham operation group and model group, the number of BrdU-positive and Nestin-positive cells increased significantly at each time point (P<0. 05), and peaked at the end of the 11th day, and the content of NSE mRNA increased significantly (P<0. 05). This research demonstrated that the proliferation of endogenous neural stem cells in situ could be induced by global cerebral ischemia and reperfu- sion, and could be promoted and extended by bFGF. In additiion, bFGF might promote endogenous neural stem cells differentiated into neurons.
Animals ; Brain Ischemia ; pathology ; Cell Differentiation ; Cell Movement ; Cell Proliferation ; Cerebral Cortex ; cytology ; metabolism ; pathology ; Fibroblast Growth Factor 2 ; pharmacology ; Nestin ; metabolism ; Neural Stem Cells ; drug effects ; Rats ; Reperfusion Injury

BACKGROUND: The precartilaginous stern cells are limited regarding in vitro proliferative capacity, but the immortalized cell lines can provide a large number of stable immortalized cells, and simian virus 40 large T antigen gene (SV40Tag) is one of gene fragments which are commonly used and effective in vitro immortalized ceils. OBJECTIVE: To construct human immortalized precartilaginous stem cells (IPSCs) using human precartilaginous stem calls induced by SV40LTAg gane. METHODS: The human immortalized precartilaginous stem calls were isolated from aborted fetus and purified with enzyme digestion and immunomagnetic beads screening method. By using liposome-mediated gene transfection technology, plasmid pCMVSV40T/PUR containing SV40Tag was transfected in primary embryonic precartilaginous stem cells, while non-transfected cells sewed as negative controls. Positive clones were cultured to observe the cell morphology and the passage recovery, to calculate cell survival rata and population doubling time, to drew call growth curve. Immunofluorescence cytochemistry was used to detect the expression of IPSCs fibroblast growth factor receptor 3, the expressions of SV40Tag and fibroblast growth factor receptor 3 in the human precartilaginous stem cells were determined by RT-PCR. RESULTS AND CONCLUSION: Morphology of human IPSCs seemed coincidence with primary human precartilaginous stem cells. The survival rate of human IPSCs was not influenced by subculture, freezing and recovery, but the survival rate was descended in the human precartilaginous stem cells at the 6~(th) and 10~(th) passages (P < 0.01). Compared with cells at the 6~(th) and 10~(th) passages, the proliferation of human IPSCs was greater, with short population doubling time and high growth rate (P < 0.01). The immunofluorescence showed that fibroblast growth factor receptor 3 was positive in human IPSCs at the second passage, and the RT-PCR results of fibroblast growth factor receptor 3 revealed a specific amplification band at 400 bp,.while that of SV40Tag revealed at 560 bp. No band was seen in the primary cells. It is indicated that SV40Tag human IPSCs can be constructed successfully using immunomagnatic bead screening technology and liposome transfection technique.

BACKGROUND: Precartilaginous stem cells (PSCs) have strong proliferation ability and differentiation potential,but they are instable and prone to differentiate.Importing exogenous gene could immortalize them and leave phenotype character unchanged.OBJECTIVE: To establish immortalized precartilaginous stem cells (PSCs) from neonatal SD rats in vitro for the further related research about the differentiation mechanism and clinical application of precartilaginous stem cells.DESIGN,TIME AND SETTING: Single sample observation.The study was carried out in the Department of Orthopedics.Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology from October 2005 to September 2006.MATERIALS: Neonatal SD rats,irrespective of gender,24-hour old,were used for prepare PSCs.METHODS: By using LipofectamineTM 2000,a gene transfection reagent,plasmid pCMVSV40T/PUR containing the simian virus 40 large T antigene gene (SV40Tag) was transfected into the primary cultured PSCs isolated by immuniomagnetic beads coasted with the second antibody.Colonies were isolated by puromycin selection and expanded by many passages.MAIN OUTCOME MEASURES: Biological character of PSCs; plasmid identification; biological character of transfected cells and identification; RT-PCR; growth curve.RESULTS: Immunomagnetic beads separation system obtains PSCs,which was confirmed as fibroblast growth factor receptor-3 (FGFR-3) positive PSCs.Double restriction enzyme was cut,electrophoresis confirmed pCMV was 3 kb,SV40T was 2.3 kb.A particular anti-puromycin cell clone was acquired,which was confirmed as FGFR-3 positive PSCs.The total RNA was isolated from the positive cell clones,and a 588 bp fragment,which was specific for the SV40T antigene gene,was amplified.The transfected cells were expanded to immortalized cell strain,named as immortalized precartilaginous stem cells (IPSCs).Thepopulation doubling time of IPSCs was (22.98±2.77) hours,no significant effect of subculture,freezing and recovering had been found.CONCLUSION: Precartilaginous stem cells could be isolated from neonatal SD rats,cultured in vitro,and immortalized through the transfection of pCMVSV40T/PUR.