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Objective:To investigate the effects of Metarhiziun taii polysaccharide on the immunity activity of the diabetic mellitus model mouse.Methods:To build stables type Ⅰ diabetic model mouse by intraperitoneal injecting small dose streptozotocin ( STZ) continuously.Then observeing weight changes,detecting empty stomach serum glucose,immune organ index( include spleen and thymus),peritoneal macrophage phagocyte ability,spleen lymphocyte multiplication and immunoglobulin IgG′s and complement C3,C4 content in blood serum of the diabetic rats.Results:The weight of model mouse had been improved by Metarhiziun taii polysaccharide, serum glucose had been improved degrade of the ability of model mouse.T,B lymphocyte proliferation had been increased obviously by middle and high dose polysaccharid,spleen index,IgG′s and C3,C4 content in blood serum of the diabetic rats could be improved by high dose distinctively.Thymus index and the capacity of peritoneal macrophage phagocyte would enhanced by middle and high dose polysaccharide inject.Conclusion:The immune function and serum glucose of typeⅠdiabetic mode rats could be improved by intra-gastriced Metarhiziun taii polysaccharide.
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Objective To explore the proliferation and differentiation of osteoblasts from 2 sources co-cultured with SD rat Schwann cells(SCs) . Methods Bone marrow stromal cells (BMSCs) were obtained by washing the femoral and tibial bone marrow cavities in SD rats. Osteoblast differentiation of the third passage of BMSCs was induced by incubation in osteogenic medium. Primary rat calvarial osteoblasts were obtained by digestion of the calvarial bone in one day old SD rats. The cells were cultured in DMEM supplemented with 10% fetal bovine serum(FBS) . SCs of passage 2 were obtained by digestion of sciatic nerve. The SCs were identified by S-100. The proliferation of 2 kinds of osteoblasts co-cultured with SCs was tested using 96 co-culture plate by methyl thiazdyl tetrazolium(MTF). Real-time PCR was used to test the osteoblast differentiation through co-culturing with SCs in 3 d and 7 d. The osteoblasts were implanted in the subtus chamber. The SCs were implanted in the superior chamber. Results SCs enhanced significantly the proliferation of calvarial osteoblasts at 7 time points. The expression levels of OPN mRNA, OCN mRNA, ALP mRNA, and BMP-2 mRNA of the osteoblasts were significantly lower in the experiment group than in the control group in 3 d and 7 d. SCs also enhanced significantly the proliferation of the induced osteoblasts in 5 d, 7 d and 9 d. The expression levels of OPN mRNA, OCN mRNA, ALP mRNA, and BMP-2 mRNA of the induced osteoblasts were significantly higher in the experiment group than in the control group in 3 d and 7 d, except the level of ALP mRNA in 7 d.Conclusions The BMSCs-induced osteoblasts cocultured with SCs may be used as seed cells to construct neurotized tissue engineered bone.
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BACKGROUND: Nuclear factor kappa B (NF-κB) is possibly related to regulation of various cell signals that are derived from aqueous uveoscleral outflow pathway.OBJECTIVE: To explore effect of travoprost on the expression of NF-κB and inhibitor-κB (I-κB) in human ciliary muscle cells cultured in vitro. DESIGN, TIME AND SETTING: A contrast study, which was performed in the Laboratory of Zhongshan Ophthalmology Center from March 2005 to November 2006.MATERIALS: Eyeballs were obtained from the youth who died due to other diseases except eye disease no more than one hour. The relatives voluntarily provided the informed consent.METHODS: Travoprost (1 μmol/L) was added in human ciliary muscle cell culture medium, and then the samples were divided into four groups according to culture time, including 0-hour (control group), 6-hour, 12-hour, and 24-hour experimental groups. MAIN OUTCOME MEASURES: Expression of mRNA and protein of NF-κB p65 and I-κBα in the four groups by using real-time RT-PCR, immunofluorescence relative quantitative analysis and enzyme linked immunosorbent assay (ELISA) techniques. RESULTS: As compared to control group, mRNA expression of NF-κB p65 in the 6-hour, 12-hour, and 24-hour experimental groups was decreased (F=17.068, P=0.001); while mRNA expression of I-κBα was not changed remarkably in the 6-hour and 12-hour experimental groups (P > 0.05), but the expression was significantly higher than that in the 24-hour experimental group (F=32.742, P=0.000). Immunofluorescence relative quantitative analysis showed that the fluorescence intensity of NF-κB p65 in the 6-hour, 12-hour, and 24-hour experimental groups were weaker than that in the 0-hour control group (F=17.216, P=0.000); additionally, as compared to 0-hour control group, fluorescence intensity of I-κBα in the 6-hour experimental group was not changed remarkably (P=0.134), that in the 12-hour experimental group was weakened (P=0.032), and that in the 24-hour experimental group was strengthened (F=17.346, P=0.001). ELISA revealed that expression of phosphorylated NF-κB p65 was decreased gradually by the time of being induced by travoprost (F=15.4, P=0.001). CONCLUSION: Travoprost can down-regulate mRNA expression of NF-κB p65, inhibit nuclear translocation, and up-regulate mRNA expression of I-κBα in human ciliary muscle cells.
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BACKGROUND: As is well known that hematopoietic stem and progenitor cells (HSPCs) contain in bone marrow, peripheral blood, and cord blood. Recent studies found that human placenta tissue (PT) also exists in HSPCs. But so far the property and differentiation capacity of human PT-HSPCs is not yet known. Furthermore the composition of lymphocyte subpopulations and immunogenicity regarded to human PT-HSPCs are also unclear.OBJECTIVE: To verify whether there are more HSPCs in human PT than those in human umbilical cord blood (UCB), to investigate their capacities of proliferation and differentiation, and to analyze the phenotypes of lymphocyte subpopulations in human PT.DESIGN, TIME AND SETTING: Open eXperiments were performed at the Key Laboratory of Cell Engineering of Guizhou Provinee from January 2004 to December 2006.SETTING: Key Laboratory of Cell Engineering of Guizhou Province, the Affiliated Hospital of Zunyi Medical College.MATERIALS: Twelve human placenta and UCB samples through cesarean delivery were collected aseptically with the informed consents of parturients derived from Maternity Department of the Affiliated Hospital of Zunyi Medical College. The main reagents were detailed as follows: lymphocyte subpopulations analysis reagents Simultest IMK-lymphocyte Kit, CD34 absolute counting reagents Kit (Becton Dickinson); CD34 Multisort Kit, FITC conjugated CD38 monoclonal antibody, anti-FITC microbeads and MS/LS mini MACS segregating, columns (Miltenyi Biotec).METHODS: UCB samples were 1:1 diluted with RPMI-1640 containing 0.1 volume fraction of fetal bovine serum and the mononuclear cells (MNCs) were isolated on Ficoll-Histopaque by centrifugation for 30 minutes. The MNCs at the interface were collected and washed with PBS. Single cells suspension liquid of human PT was prepared by mechanical method combined with 0.25g/L collagenase digestion. After that, the placenta samples underwent the same protocol as used in UCB to isolate MNCs. The percentage of CD34+CD38-, CD34+CD38+ HSPCs and the phenotype of lymphocyte subpopulations derived from human PT-MNCs were analyzed by flow cytometry (FCM). CD34+CD38-, CD34+CD38- cell subsets isolated by magnetic-activated cell sorting (MACS) from human PT were used to carry out colony-forming culture including granulocyte/macrophage colony-forming unit (CFU-GM), burst forming unit-erythroid (BFU-E) and mixed colony-forming unit (CFU-Mix) in order to assess their capacities of hematopoietic progenitor cells' proliferation and differentiation. In parallel, UCB samples underwent the same protocols for comparison.MAIN 0UTCOME MEASURES: Percent compositions of CD34+ HSPCs, hematopoietic progenitors' lineage colony-forming capacities of CD34+ HSPCs, phenotypes and compositions of lymphocyte subpopulations both in PT and UCB.RESULTS: The percentage of CD34+ cells contained in human PT was 8.8 times higher than that of in UCB (P<0.01). The total number of lymphocytes, T cells (CD3+CD2+), B cells (CD19+), Th (CD3+CD4+) and Th/Ts ratio were apparently lower in human placenta, while the number of CD8+CD28- T suppressor cells were higher compared to UCB samples (P<0.01). Among PT, CFU-GM, BFU-E and CFU-Mix frequencies of CD34+CD38- cells subset were much higher than that of CD34+CD38- (P<0.01). Within the same phenotype of cell subsets, however, the number of each colony-forming unit was similar between PT and UCB (P 0.05).CONCLUSION: Human PT is richer in CD34+CD38-, CD34+CD38+ HSPCs and both of them have the abilities of proliferating and differentiating into CFU-GM, BFU-E and CFU-Mix. Considering that human PT have a lower lymphocyte subpopulations and higher Ts cells, human PT might be a alternative and suitable source of HSPCs for clinical transplantation.
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BACKGROUND:Travoprost can increase human ciliary muscle cell interspace, decrease uveoscleral outflow resistance and then decrease intraocular pressure. But whether this action pathway is conducted by enhancing the expression of matrix metalloproteinase (MMP) in the ciliary muscle cells remains unclear.OBJECTIVE:To observe the effect of travoprost on the expression of MMP-2 in the human ciliary muscle cells.DESIGN:Controlled observation analysis.SETTING:Zhongshan Ophthalmic Center,Sun Yat-sen University.MATERIALS:This study was Carried out in the Zhongshan Ophthalmic Center,Sun Yat-sen University between August 2005 and April 2006.Donor was from the unilateral eyeball of a youth patient,who was dead within one hour,had no any disease (informed consent was obtained from the relatives) in the Zhongshan Ophthalmic Center, Sun Yat-sen University.Rabbit anti.human MMP-2 polyclonal antibody (Boster Bioengineering Co.,Ltd,Wuhan),and travoprost (86610F,0.004% solution,ALCON company.USA) were used in this study.METHODS: Experimental intervention: 1μmol/L travoprost was added into bovine serum-free medium of human ciliary muscle cell, serving as experimental group,and meanwhile,the cells which were not interfered by drugs were taken as control group.In the experimental group,cells were harvested 6, 12,and 24 hours after travoprost being added.Experimental evaluation:MMP-2 gene and protein expressions in the human ciliary muscle cells in each group were detected by RT-PCR and ELISA methods.The activity of MMP-2 in each group was detected by Zymography technique.MAIN OUTCOME MEASURES:MMP-2 mRNA expression in the human ciliary muscle cell,MMP-2 protein expression and MMP-2 activity in the extracellular fluid.RESULTS:①In the experimental group, at 6,12 and 24 hours after travoprost being added,the relative expression of MMP-2 mRNA was gradually increased (F=236.959,P＜0.01).②In the experimental group,at 6,12 and 24 hours after travoprost being added,MMP-2 protein was also gradually increased with time (F=38.110,P＜0.01).③Zymography technique detection showed that in the experimental group,at 6,12 and 24 hours after travoprost adding,MMP-2 activity was gradually enhanced with time (F=74.348,P＜0.01).CONCLUSION:After human ciliary muscle cell cultured in vitro being subjected to the intervention of travoprost.MMP-2 expression is gradually increased with action time of travoprost, and meanwhile MMP-2 activity is also gradually enhanced.