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Objective:To observe the effects of All-trans retinoic acid (ATRA) on the immune functions of AChR-specific lymphcytes via in vitro assays,and investigate the possibility of ATRA in the clinical treatment of myasthenia gravis (MG).Methods:CFA control group and EAMG experimental rats were established to obtain single lymphocytes suspension and cells were followed by AChR97-116 peptide with or without ATRA stimulation for 72 h,and then viable cell population,cell apoptosis,cell cycle and the distribution of Th cells were determined by flow cytometry.CCK-8 assay was selected to evaluate the effects of ATRA on proliferatory ability of lymphocytes.ELISA was used to detect the antibody secretion of B cells affected by ATRA.Results:Compared with CFA group,lymphocytes obtained from EAMG rats had higher ratios of living cells,and this ratio was obviously decreased after ATRA treatment,P＜0.001.Different concentrations of ATRA promoted the apoptosis of AChR-specific cells (P＜0.001),and the promoted effects were ATRA dose-dependent,however,cell cycles were not changed.ATRA markedly inhibited the proliferation of cells from both CFA and EAMG groups,moreover,AChR-specific cells were more sensitive to ATRA treatment (P＜0.01) than that of cells from CFA rats (P＜0.05).The ratio of AChR-specific CD4+T cells was reduced by ATRA (P＜0.01),and ATRA incubation significantly promoted the percentages of Th2,(PCD4+-4IL-4+＜0.001),Treg (PCD4+-Foxp3+＜0.001) cell types,but markedly inhibited the percentages ofThl7 (PCD4+-IL-17+＜0.05),Thl (PCD4+-IFN-γ+＜0.001) cells.ELISA data showed us that ATRA obviously down regulated the antibody secretion of AChR-specific B cells,P＜0.01.Conclusions:ATRA not only inhibited the functions of AChR-specific T cells,but also suppressed the roles of AChR-specific B cells,predicating a therapeutic effect of ATRA on myasthenia gravis therapy.
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Objective: To evaluate the mechanism of T follicular helper cells ( Tfh ) in experimental autoimmune encephalomyelitis (EAE) via in vivo experiments. Methods:C57BL/6 mice were randomly divided into four groups,CFA group,EAE group,anti-ICOSL group and control group. Lymphocytes of different time points isolated from draining lymph nodes and spleen were stained for T follicular helper cells surface marker and T cells activation surface marker and analyzed by FACS. Observed parameters include inflammatory infiltration,demyelination in spinal cord and germinal center in spleen. ELISA was used to measure the level of antigen specific antibodies. Results: Mice in anti-ICOSL treated group developed mild disease was with lower clinical scores when compared with the EAE group. HE staining results turned out with alleviated inflammation and Luxol Fast Blue staining( LFB) showed no demyelization in anti-ICOSL treated mice compared with non-treated EAE models. Flow cytometry results revealed that percentages of T follicular helper cells decreased though the whole activated degree T cells was not influenced in anti-ICOSL treated group. Fewer ger minal center was found in both anti-ICOSL group and CFA group with reduced secretion of MOG-specific Ab. Conclusion:T follicular helper cells supported the development of cognate B cells,promoted the formation of germinal center,facilitate pathogenic MOG-specific Ab secretion,thus enhance EAE.
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BACKGROUND: Bone marrow mesenchymal stem cell transplantation is superior to neural stem cell transplantation to repair spinal cord injury; however, the therapeutic effect is unstable and possibly related to microenvironment.OBJECTIVE: To study the differentiation of cultured in vitro bone marrow mesenchymal stem cells (BMSCs) into neurocytes by establishing a microenvironment and to observe differential expression of protein.DESIGN, TIME, AND SETTING: Observational contrast study was performed at the Laboratory of Neurobiology, Basic Medical College, Harbin Medical University from July 2005 to May 2007.MATERIALS: Adult Wistar rats and newborn fetal rats were used in this study.METHODS: Spinal cord was obtained from fetal rats to culture neurocytes. While, BMSCs were separated from bone marrow of adult rats, and they were then cultured in vitro, proliferated, and labeled with red fluorescin PKH26. BMSCs and neurocytes were individually cultured in the BMSCs group and the neurocyte group, respectively. In addition, BMSCs and neurocytes were co-cultured in vitro in double-layer culture dish in the co-culture group and the layered combination group, respectively.MAIN OUTCOME MEASURES: The obtained cells after 7-day culture were immunofluorescently detected by neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP). Surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) technique was used to analyze associated protein that was apparently changed during the differentiation from BMSCs into neurocytes.RESULTS: Seven days after co-culture, BMSCs were morphologically shared like neurocytes. Immunofluorescence indicated that NSE- and GFAP-positive ratios of BMSCs in the co-culture group were significantly higher than the layered combination group (P < 0.05); while, the ratios in the layered combination group were significantly higher than BMSCs alone group (P < 0.05). Five protein expressions were changed during the differentiation from BMSCs into neurocytes, for example, TIP39_RAT and CALC_RAT expressions increased in the layered combination group, which were 5.344 and 2.805 times as the primary expressions; INSL6_RAT, PNOC_RAT, and PCSKI_RAT expressions decreased, which were 0.380, 0.499, and 0.437 times as the primary expressions.CONCLUSION: By a microenvironment, both BMSCs and neurocytes in the co-culture and layered combination groups can differentiate into neuroblasts; while, contact differentiation ratio is higher than non-contract one. The differentiation is closely related to five proteins, including TIP39_RAT, CALC_RAT, INSL6_RAT, PNOC_RAT, and PCSK1_RAT.
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BACKGROUND: Tissue engineering method has been employed to recover cartilage defect and overcome many traditional shortages.OBJECTIVE: In vitro marrow mesenchymal stem cells (MSCs) of adult rat was induced to differentiate into chondrocyte phenotype so as to probe into the feasibility of MSCs to be cartilage seed cell in tissue engineering.DESIGN: Completely randomized design and controlled experimental study.SETTING: Department of Histology and Embryology, and Department of Neurobiology, Harbin Medical University MATERIALS: Six Wistar rats of either gender, cleanness grade, were provided by Experimental Animal Center, Affiliated Second Hospital, Harbin Medical University. Permission number of experimental animal production was SCXK(black) 20020002.METHODS: The MSCs of the second generation adult rat was taken and divided into test group and control group. The test group was induced with serum free and control group was induced with 10% fetal calf serum. Collagen type Ⅱ immunohistochemical staining, toluidine blue staining was performed to detect differentiation.MAIN OUTCOME MEASURES: Identification of chondrocyte, comparison of the positive rate of Collagen type Ⅱ immunohistochemical staining at different induction time point.RESULTS: Induced MSCs had identical characteristic to the chondrocyte.CONCLUSION: In vitro culture can induce MSCs to differentiate directly to chondrocyte-like cell. The results suggest that it is feasible to use MSCs as seed cells in the cartilage tissue engineering.
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AIM: To study the effect and mechanism of arsenous oxide (As_2O_3) on pancreatic cancer PC_2 strain. METHODS: The pancreatic cancer PC_2 strain was chosen in this experiment. Apoptosis was detected by TUNEL test. Expression of survivin gene was detected by reverse transcriptase PCR. RESULTS: ① After administration of As_2O_3, the survival number of pancreatic cancer cells decreased significantly in a time-dependent manner (P
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AIM: To observe the effect of phosphorylation protein kinase C delta (PKC?) on the procedure of PC12 cells apoptosis induced by 6-hydroxydopamine(6-OHDA) and to investigate the potential molecular pathogenesis of Parkinson disease.METHODS: TUNEL staining and transmission electron microscope were applied to measure apoptosis when dopaminergic PC12 cells exposed to the excitomotors and inhibitors of PKC before 6-OHDA for 18 hours. The expression of phosphorylation of PKC? was detected by Western blotting. RESULTS: PMA, an activating agent of PKC?, significantly increased PC12 cell apoptosis induced by 6-OHDA. Rottlerin, an inhibitor of PKC?, protected PC12 cells apparently. As contrast, bisindolylmaleimide I, an inhibitor of general PKC and G6976, the inhibitor of calcium-dependent PKC, did not show any protective role. CONCLUSION: The phosphorylation PKC? is one of the important links in the process of PC12 cell apoptosis induced by 6-OHDA. PKC? may directly participate in neurodegeneration process in parkinsonian.
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Objective:To investigate whether interferon-gamma inducible protein-10(IP-10) and interferon-gamma(IFN-?) participated in the human cerebral ischemia injury.Methods:Twenty-one cerebral ischemia specimens, collected from patients died of cerebral infarction, were divided into three groups: less than 7 days, 7-14 days and 15-21 days according to the lasting time of cerebral infarction. The infiltrating of inflammatory cells were observed using HE stain as non-ischemic hemisphere was for controls. Expression of IP-10 and IFN-? in sections both postmortem ischemic hemisphere and non-ischemic hemisphere were detected using immunohistochemistry.Results:In the groups of less than 7 days and 7-14 days large quantity of inflammatory cells were infiltrated in ischemia tissue. Expression of IP-10 in three groups was elevated in the ischemic hemisphere compared with non-ischemic hemisphere(1.74-folds, 1.41-folds and 1.52-folds increases respectively, P0.05).Conclusion:These results showed IP-10 and IFN-? are expressed in human cerebral ischemia. It was suggested that IP-10 and IFN-? involve in cerebral ischemia injury. Moreover, it was also suggested that IP-10 participate in the repair for the later stage of cerebral ischemia injury.