By using decoy-oligodeoxynucleotides (decoy-ODNS) technique, the effects of Stathmin gene on the proliferation and differentiation of in vitro cultured precartilainous stem cells (PSCs) were investigated. The Stathmin decoy-ODNs were transfected into PSCs in rats by using gene transfection technique. Under the induction of cortisol (1 micromol/L), electrophoretic mobility shift assay was used the inhibitory effects of decoy-ODNS on Stathmin gene. MTT and cytometry were used to test the cell proliferation. The expression of collagen II and V and Stathmin protein was detected by using Western blot. The results showed that Stathmin decoy-ODNs inhibited the Stathmin activity in a dose-dependent manner. When the concentration of decoy-ODNs was 10 times of standard concentration, the proliferation of PSCs was obviously suppressed and the differentiation happened. Compared to the control group, the difference was significant (P<0.05). It was concluded that decoy-ODNs could inhibit the proliferation and promote the differentiation of PSCs by antagonizing Stathmin activity.
Cartilage/*cytology ; Cell Differentiation/*drug effects ; Cell Proliferation/drug effects ; Cells, Cultured ; Oligodeoxyribonucleotides/genetics ; Oligodeoxyribonucleotides/*pharmacology ; Rats, Sprague-Dawley ; Stathmin/*genetics ; Stathmin/pharmacology ; Stem Cells/*cytology

BACKGROUND: Compared with artificial intervertebral disc replacement,prothetic nucleus replacement is easier to perform with fewer risks and requiting smaller incision.OBJECTIVE: To evaluate the effect of prosthetic disc nucleus replacement for treatment of lumbar disc herniation.DESIGN: A self-controlled trial.SETTING: Department of Orthopaedics, Zhujiang Hospital, Southern Medical University.PARTICIPANTS: Thirty-three patients including 21 male and 12 female patients with lumbar disc herniation received prosthetic disc nucleus replacement in Zhujiang Hospital of Southern Medical University from January 2002 to October 2003.METHODS: All the patients received prosthetic lumbar nucleus replacement and 3 weeks after the operation, X-ray examination was performed for observing the height and morphology of the intervertebral space. All the patients were followed up for 8 months and the therapeutic effects were assessed in a four-grade system(excellent, good, improved, poor). MAIN OUTCOME MEASURES: The change in the symptoms of low-back pain and the range of movement (ROM) of the lumbar spine.RESULTS: According to the intention-to-treat analysis, none of the 33 patients was lost to the analysis. During the follow-up, 29 patients had excellent, 3 good, and 1 poor outcome in terms of the symptoms of low-back pain and ROM of the lumbar spine, with a total rate of good outcome of 96. 97%(32/33) . X-ray examination in the 3rd month revealed normal size of the intervertebral space in 29 cases, and increased but still narrowed intervertebral space in 3 cases. No aggravation of retrograde changes was found in the adjacent segments.CONCLUSION: Prosthetic disc nucleus replacement can effectively relieve low-back pain in lumbar disc herniation, restore the spinal movement and decrease retrograde changes of adjacent intervertebral space and articular process.

[Objective]To evaluate the feasibility of the minic microgravity as a method and the layered cylindric collagen-chitosan-?-tricalcium phosphate composite as a scaffold for the cartilage tissue engineering after an observation of how it absorbs the chondrocytes and affects the cell behavior.[Method]The chondrocytes were isolated and multiplied in vitro,and then the chondrocytes were seeded onto the porous collagen-chitosan-?-tricalcium phosphate composite scaffold and were cultured in both minic microgravity and ordinary environment for 3 weeks.The effects of the composite scaffold on the cell adhesivity,proliferation,morphological changes and synthesis of the extracellularmatrix were observed by the growth curve,phase-contrast microscopy,histology,scanning electron microscopy and immunohistochemistry.[Result]The chondrocytes that adhered to the scaffold increased significantly and secreted extracellular matrix in the center of the porous scaffold around the chondrocytes under minic microgravity compared with ordinary environment.Immunohistochemistry of type Ⅱ collagen was positive.[Conclusion]The minic microgravity environment will be a good method for the cartilage tissue engineering.And the layered cylindric collagen-chitosan-?-tricalcium phosphate composite scaffold has a good cellular compatibility.It will be an ideal scaffold for the cartilage tissue engineering.

[Objective]To evaluate the effectiveness of recombinant human bone morphogenetic protein-2 in the repair of rabbit rotator cuff injury.[Method]Forty-eight male New Zealand rabbits,aged 8 months,received an rotator cuff acute injury and reconstruction of the insertion of supraspinatus tendon on greater tuberosity of humerus.The rabbits were randomly divided into 3 groups postoperatively:(1) rhBMP-2 group:fibrin sealant(FS) containing rhBMP-2 was applied to the interface between the bone-tendon interface; (2)FS control group:only FS was applied;(3)blank control group:untreated after the surgery.Harvested 36 specimens underwent biomechanical analysis at the 2nd,4th,8th postoperative weeks respectively.Harvested 12 specimens underwent histological analysis at the 8th postoperative week.[Result]Histological examination showed that Sharpey's fibers were found in the interface with the formation of four-layer indirect insertion in the rhBMP-2 group at the 8th postoperative week.In the FS control group and blank group,the tendon-bone interface was filled with granulation tissue and part of Sharpey's fibers and the newly generated bone tissue.Biomechanical analysis displayed that the tensile strength and stiffness of bone-tendon interface increased time-dependently in all groups,and it in the rhBMP-2 group was significantly higher than those in the other two groups at any time-points (both P

Objectives To evaluate the effectiveness of transcarotid artery chemotherapy for gliomas after surgery, and selection of drug, avenue of administration, and optional time for the therapy. Methods Beginning from 4 to 30 days after operation, Nimustine (ACNU, Japan) 2.5mg/kg was injected per carotid artery, once every week for three times as one course. A second course of treatment was given after an interval of 4 to 6 weeks. Results With the above regime, the effect was marked in 39 cases (18.8%), fairly effective in 44 cases (20.8%), only slightly effective in 59 cases (27.8%), no effect in 61 cases (28.8%), and failure in 5 cases (2.4%), the mean survival time was nearly 100 weeks. Conclusion Transcarotid artery chemotherapy for gliomas is helpful in prolonging survival period, with little side effects, easy to carry out, less expensive, and better accepted by the patients.

0.1), while in Yunnan Baiyao group, there were significant reductions in all scores (P

Objective To investigate the status and difference of learning burnout between Korean-Chinese nursing students and Chinese nursing students;and to compare the influencing factors of learning burnout between them.Methods A total of 307 nursing students in Yanbian University were recruited by convenience samphng method.Data were analyzed by SPSS 20.0,using descriptive statistics,t-test,ANOVA,correlation analysis,and linear regression analysis.Results The emotion and learning burnout of Korean-Chinese nursing students scored (3.18±0.52)points and (2.84±0.33)points,which were higher than (3.04±0.53)points and (2.69±0.36) points of Chinese nursing students (t=5.72,4.19,all P ＜ 0.05);The significant predictors of learning burnout of Korean-Chinese nursing students were effective commitment,continuance commitment and ideality commitment;and the significant predictors of Chinese nursing students were effective commitment,continuance commitment and behavior self-efficacy.Conclusions Because of the special background of Korean-Chinese nursing students,it is suggested payed attention to improve their major commitment and learning self-efficacy,then to decrease their learning burnout.

This study examined the effect of small interfering RNA-mediated β-catenin knockdown on the survival, invasion and chemosensitivity of human osteosarcoma cells (U2-OS cells). The siRNA against β-catenin was constructed and transfected into U2-OS cells. The expression of β-catenin was detected by qRT-PCR and Western blotting. Cell growth and apoptosis was detected in the presence or absence of doxorubicin by MTT and flow cytometry, respectively. Cell invasion ability was measured by transwell assay. The results showed that the transfection of β-catenin siRNA resulted in decreased expression of β-catenin, suppression of invasion and motility of U2-OS cells, reduced chemosensitivity to doxorubicin in vitro, and little change in cell growth and apoptosis. Additionally, down-regulated MT1-MMP expression was found after transfection. It was concluded that knockdown of β-catenin gene may decrease the invasive ability of human osteosarcoma cells through down-regulated MT1-MMP expression, and the chemosensitivity of osteosarcoma cells against doxorubicin.

This study examined the construction of eukaryotic expression plasmid of human transforming growth factor-β3 (hTGF-β3) and its inducing effect on the differentiation of precartilaginous stem cells (PSCs) into chondroblasts. hTGF-β3 gene was amplified by using polymerase chain reaction (PCR) and then inserted into the eukaryotic expression plasmid pcDNA3.1 to construct the eukaryotic expression plasmid pcDNA3.1(+)-hTGF-β3. Rat PSCs were isolated and purified by employing an immunomagnetic cell sorting system. pcDNA3.1(+)-hTGF-β3 was transfected into purified PSCs with the use of linear polyamines. The expression of TGF-β3 and cartilage-specific extracellular matrix (ECM) components was detected after transfection by real-time quantitative PCR, ELISA, immunochemistry and Western blotting, respectively. The results showed that the eukaryotic expression plasmid pcDNA3.1(+)-hTGF-β3 was successfully established as identified by enzyme digestion and DNA sequencing. Real-time quantitative PCR and ELISA revealed that hTGF-β3 was strongly expressed in pcDNA3.1(+)-hTGF-β3-transfected PSCs. Real-time quantitative PCR, immunochemistry and Western blotting showed that the cartilage-specific ECM markers, i.e., cartilage oligomeric matrix protein (COMP), Aggrecan, collagen type X and II were intensely expressed in the pcDNA3.1(+)-hTGF-β3-transfected cells. It was concluded that hTGF-β3 could be stably expressed in pcDNA3.1(+)-hTGF-β3-transfected PSCs and induce the differentiation of PSCs into chondroblasts.

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.