Résumé
BACKGROUND:The narrowing of intervertebral space induced by the intervertebral disc degeneration is mainly characterized by the expression of proteoglycan in nucleus pulposus cells and the reduction of col agen type II. OBJECTIVE:To quantitatively observe col agen type II protein in adult normal and degenerative intervertebral disc nucleus pulposus cells by immunofluorescence staining and safranin O staining. METHODS:The nucleus pulposus specimens were col ected from adult scoliosis patients and patients with intervertebral disc protrusion, who were al volunteers. After culture, 26 cells in each patient were measured. There were 78 cells in both normal group and degeneration group. The normal and degenerative intervertebral disc nucleus pulposus cells were subjected to safranin“O”staining, and gray values were determined;intracellular col agen type II was detected by immunofluorescence staining. RESULTS AND CONCLUSION:Immunofluorescence staining revealed that, degenerative intervertebral disc nucleus pulposus cells were only mildly stained, with the fuzzy staining, the shape was round, spindle, fusiform and irregular. There were a very smal amount of fluorescent particles within cells. The expression of col agen type II was decreased significantly compared with normal cells (P0.05). The degenerative intervertebral disc nucleus pulposus cells have a smal quantity and partial y become apoptotic, the content of col egen type II protein is decreased significantly compared with normal nucleus pulposus cells.
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Objective To observe the effect of Sox9 gene overexpressian on chondrogenesis of esenchymal stem ceils (MSCs) in vitro. Methods Rabbit MSCs were obtained and purified by gradient centrifuge and adhesion culture in vitro. MSCs were transfected by recombinant Sox9 plasmid. RT-PCR and Western blot analysis were used for transfection confirmation. Chondrogenic molecules such as type I1 collagen,aggrecan and Sox9 were detected by immunohistology, RT-PCR and western blot. Cell proliferation was tested by MTT assay. Results MSCs were successfully transfected with Sox9 gene and verified by RT-PCR and Western blot analysis. The overexpression of Sox9 had no effect on the proliferation of MSCs. Transfeeted cells expressed more chondrogenic markers than the control groups. Conclusion Sox9 gene overexpression can enhance chondrogenic differentiation of MSCs.
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BACKGROUND: Different isolation and culture methods for bone marrow mesenchymal stem cell (BMSCs) will result in varying cell activity and purity, which will influence repair effect of tissue engineering. OBJECTIVE: To investigate the optimized methods of isolation and culture of BMSCs in vitro and validate the efficacy of cell acquisition. DESIGN, TIME AND SETTING: In vitro cytology trial was performed at the laboratory of Department of Orthopedics, Second Hospital of Shanxi Medical University from October to December 2007. MATERIALS: Two 3-month-old New Zealand rabbits were provided by Shanxi Institute of Livestock. METHODS: Lymphocyte isolation solution was added to DMEM culture solution containing fresh bone marrow. BMSCs were obtained and purified by gradient centrifuge and adhesion culture in vitro. Non-attached cells were moved to new culture flask every 8 hours. The solution was changed firstly after 5 days of culture. Trypsinization was conducted at cell confluence of 90%. The first, third and fifth passages of cells were harvested. MAIN OUTCOME MEASURES: The morphology of BMSCs was observed with phase contrast microscope; the growth curve was drawn to determine doubling time. The percentage of the wel1 growth P2 cells were identified by CD44 staining by flow cytometry. RESULTS: Primary cultured BMSCs were oval, spindle-shaped or polygonal, and adhered to plastic surface within 48 hours, exhibiting spindle-shaped or polygonal with clear nucleus, and reached 90% confluence within 14 days. The first, third and fifth passages of BMSCs showed S-type, and were in latent phase at 1-3 days, in log phase 3 days later, and cells came into platform phase at 7-8 days. The mean doubling time was 24.22 hours. CD44-positive cells were found in the second passage, with positive rate of 93.0%. CONCLUSION: BMSCs are easily isolated and cultured in vitro with good growth and high purity by gradient centrifuge and adhesion culture with lymphocyte isolation solution.
Résumé
Objective To observe the effects of Xinshuaiheji (XSHJ) on cardiac function, plasma angiotensin Ⅱ (AngⅡ) and histomorphology in rats with heart failure after acute myocardial infarction. Methods A model of heart failure (HF) induced by myocardial infarction (MI) in rats was made, 10 days after MI, rats were treated for 4 weeks with bidist Water, Captopril, high dosage of XSHJ, low dosage of XSHJ. The effects of Xinshuaiheji on cardiac function (stroke volume, SV, cardiac output, CO, cardiac index, CI ) and AngⅡ were observed. We also observed and compared the changes of heart weight/body weight ratio (HW/BW), ratio of ventricular wall thinning in MI. Results After treatment with XSHJ, the cardiac function (SV, CO, CI) of HF rats improved (P