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This paper introduced multiple flexible teaching methods in medical immunology based on its characteristics including paying attention to introductory class,activating class atmosphere,integrating multiple teaching form.Results showed that these methods stimulating interests of the students,improving their comprehensive quality and ability of innovation,so teaching effect can be improved accordingly.
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BACKGROUND:Several studies have demonstrated that bone marrow mesenchymal stem cells (BMSCs) inhibit the proliferation and activation of T lymphocytes, exerting a role of negative immunological regulation. OBJECTIVE: To induce rat BMSCs to differentiate into neuron-like cells in vitro using all-trans retinoic acid and to investigate the effects of differentiated cells on T lymphocyte proliferation.DESIGN, TIME AND SETTING: A cytological in vitro observation was performed in Guangdong Medical College in March 2008.MATERIALS: Two male 4-week-old Sprague Dawley rats were provided by the Laboratory Animal Center of Guangdong Medical College. Fresh healthy human blood was provided by the Department of Clinical Laboratory, Guangdong Medical College. METHODS: Rat BMSCs were isolated by adherent method and were passaged in vitro. Cells of passage 5 were pre-induced with Low glucose-dulbecco's modified eagle's medium (LG-DMEM) containing 0.3 mg/L all-trans retinoic acid and 10% fetal bovine serum. Twenty-four hours later, aforementioned LG-DMEM was discarded and LG-DMEM containing 0.6 mg/L all-trans retinoic acid was added to induce cells differentiation into neuron-like cells. Fresh healthy human blood was taken to prepare responding cells. Rat BMSCs, as stimulator cells, were included for one-way mixed lymphocyte reaction. Four groups were designated. Control group: 100 μL of responding cells at a concentration of 1×109/L; Experimental group 1:1×104 neuron-like cells + 100 μL of responding cells; Experimental group 2:1×105 neuron-like cells + 100 μL of responding cells; Experimental group 3:1×106 neuron-like cells + 100 μL of responding cells.MAIN OUTCOME MEASURES: Morphological observation of induce-differentiated cells, identification of nerve cells, and one-way mixed lymphocyte reaction results.RESULTS: Fifty minutes after addition of inducing medium, under the optical microscope, BMSCs exhibited a typical morphology of perikaryon. Three hours later, most of cells became into bipolar or multipolar neuron-like cell appearance with cell bodies and processes. Immunocytochemical staining results showed that majority of cells exhibited neuron specific enolase- and nestin-positive expression and glial fibrillary acidic protein-negative expression. Compared with the control group, radionuclide counts per minute were significantly reduced in each experimental group (P<0.05). There was significant difference in radionuclide counts per minute between experimental groups (P<0.05). With increasing BMSCs amount, the inhibitory effects on T lymphocyte proliferation were more evident.CONCLUSION: AII-trans retinoic acid can induce neuron-like cell differentiation of rat BMSCs in vitro. The neuron-like cells can inhibit human T lymphocyte proliferation in a dose-dependent manner.
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Objective:Marrow mesenchymal stem cells differentiates into neuron-like cells when induced by Gastrodia,which verifies the multipotentiality of the stem cells.Here to detect the immunogenic properties of the neuron-like cells after differentiation.Methods:Human mesenchymal stem cells were isolated from bone marrow by wall sticking method,amplified by in vitro culture,and differentiated into neuron-like cells by oriented induction with Gastrodia.The morphology of cells was observed under light microscopy.Neuron-specific enolase (NSE),nestin and glial fibrillary acidic protein (GFAP) were detected by imnunocytochemistry.The expression of HLA-DR protein after induction was tested by flow cytometry(FCM).The immunogenic properties of the neuron-like cells induced from hMSCs were detected by one-way mixed lymphocyte reaction(MLR).Results:Human mesenchymal stem cells could be separated and amplified in vitro.After being induced by Gastrodia for 3 hours,most of the cells would be differentiated into neuron-like cells,revealing cytodendrite.By immunochemical staining,the cells showed positive of NSE,nestin,and negative of GFAP.HLA-DR was not detectable on differentiated hMSCs by FCM.MLR assays demonstrate that differentiated hMSCs fail to stimulate proliferative response of hPBLs.Conclusion:Human mesenchymal stem cells could be induced to differentiate into neuron-like cells by Gastrodia.MLR assays demonstrate that differentiated hMSCs fail to stimulate proliferative response of hPBLs.They are low immunogenic.
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BACKGROUND: How dose sodium nitroprusside, as a vasodilatator, affect the potential of bone marrow-derived mesenchymal stem cells (MSCs) in hematopoietic differentiation?OBJECTIVE: To observe the changes during the differentiation of MSCs into hematopoietic cells after adding sodium nitroprusside, and compare the results with those of simple MSCs transplantation.DESIGN: A randomized grouping, controlled observation.SETTINGS: Department of Microbiology and Immunology; Department of Pathophysiology, Guangdong Medical College.MATERIALS: The experiments were carried out in Guangzhou Medical College in August 2006. Twenty-seven clean-degree balb/c mice of 7-8 weeks, were used as recipients, and were randomly divided into MSCs transplantation group (n =9), sodium nitroprusside+MSCs transplantation group (n =9) and blank control group (n =9). Another 4-week-old SD rat was selected as the MSCs donor. Sodium nitroprusside for injection (50 mg/piece) was provided by Beijing Double-Crane Modern Pharmaceutical Technology Co., Ltd (National drug approval: No. H11020907).METHODS: ① Under aseptic condition, the femur of SD rat was collected. MSCs in it were isolated for culture and amplifying in vitro. MSCs of passages 6-7 were digested and centrifugated, and the density was adjusted to 1×109 L-1.Monoclonal antibody with fluorescence labeled was added into cell suspension, and the phenotype was detected with flow cytometry. ② Sodium nitroprusside (50 mg/piece) was adjusted to the terminal concentration of 200 mg/L by adding with saline. It should be used within 4 hours. ③ Before transplantation, all the mice were exposed to 5.0 Gy. X-ray for 4 hours, and the absorbed dose was 1.45 Gy per minute. After irradiation, mice in the MSCs transplantation group were directly infused via caudal vein with 0.3 mL MSCs suspension (containing 1.5×106 cells); The mice in the sodium nitroprusside+MSCstransplantation group were firstly injected with the dispensed 200 mg/L sodium nitroprusside (0.15 mL), and immediately infused with 0.3 mL MSCs suspension (containing 1.5×106 cells) after 1 minute; The mice in the blank control group were infused with isovolume serum-free culture medium. ④ At 60 days after transplantation,peripheral blood was drawn from orbits of the survived mice in each group, single cell suspensions of bone marrow and spleen were prepared after the mice were killed, the levels of rat-derived hematopoietic cells CD11a and CD45 were detected with flow cytometry.MAIN OUTCOME MEASURES: ① MSCs culture and amplification; ② Levels of rat-derived hematopoietic cells in different tissues.RESULTS: All the 27 balb/c mice as recipients survived to the end of the experiment. ① MSCs isolation and amplification: The MSCs attached with uniform shapes of spindle and proliferated rapidly after culture for 3 days, and 90% of the cells were fused without overlapping at 6 days. The cells attached completely within 24 hours after passage,extended and became spindle again, rapidly proliferated and grew, and became fused completely at 3 days. ② Levels of rat-derived hematopoietic cells in different tissues: In the MSCs transplantation group and sodium nitroprusside+MSCs transplantation group, Iow expressions of rat-derived CD11a and CD45 hematopoietic cells could be detected in peripheral blood, bone marrow and spleen, and they were obviously higher in the sodium nitroprusside+MSCs transplantation group than in the MSCs transplantation group (t=2.619, P ＜ 0.05), while negative ones in the blank control group.CONCLUSION: MSCs have the ability to differentiate into hematopoietic stem cells, which can be promoted by sodium nitroprusside.