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Objective To evaluate the effect of mesenchymal stem cell (MSC) coated-islets on instant blood-mediated inflammatory reaction (IBMIR) after islet transplantation. Methods MSC labeled with tracer and human islets were placed into an ultra-low adsorption cell culture dish, shaken and mixed twice at an interval of 0.5 h, and then incubated at 37 ℃ and 5% CO2 for 24 h to obtain MSC-coated islets. The coating effect of MSC and in vitro function of the islets were assessed. A blood circulation tube-shaped model was established in vitro. In the blank control group, 0.2 mL of islet culture solution was added. In the islet group, 800 islet equivalent quantity (IEQ) of uncoated islets were supplemented. In the MSC-coated islets group, 800 IEQ of MSC-coated islets were added, and circulated for 60 min at 37 ℃. A portion of 0.5 mL blood sample was taken for routine blood test at 0, 30 and 60 min, respectively. After 60 min circulation, the blood sample was filtered with a 70 μm filter to collect plasma, blood clots and islets. Blood clots and islets were subject to hematoxylin-eosin (HE) staining and immunohistochemical staining. Morphological changes and the aggregation of CD11b-positive cells surrounding the islets were observed. The contents of plasma thrombin-antithrombin complex (TAT), tissue factor (TF), C3a, C5b-9, interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1 and IL-8 were determined by enzyme-linked immune absorbent assay. Results After 24 h co-incubation, the islets were coated by MSC, with a coating degree of approximately 80%. In the islet and MSC-coated islet group, a large quantity of neutrophils and monocytes were observed surrounding the blood clots and islets, and the quantity of CD11b-positive cells in the MSC-coated islet group was less compared with that in the islet group. After co-incubation with the whole blood for 0, 30 and 60 min, the quantity of platelets, neutrophils and monocytes was declined in the MSC-coated and islet groups, and gradually decreased over time. Compared with the blank control group, the quantity of platelets, monocytes and neutrophils was lower, whereas the TF content was higher in the MSC-coated islet group. Compared with the islet group, the quantity of platelets, monocytes and neutrophils was higher, whereas the TAT and TF contents were less in the MSC-coated islet group, the differences were statistically significant (all P < 0.05). Compared with the blank control group, the expression levels of C3a, C5b-9, IL-6, TNF-α and IL-8 were up-regulated in the MSC-coated islet group. Compared with the islet group, the expression levels of C3a, C5b-9, IL-1β, IL-6, TNF-α, IL-8 and MCP-1 were down-regulated in the MSC-coated islet group, and the differences were statistically significant (all P < 0.05). Conclusions MSC-coated islets may reduce the exposure of islet TF in the blood and prevent the incidence of IBMIR during the coagulation response stage, thereby mitigating the injury and loss of islet allograft in the early stage of islet transplantation.
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Objective To investigate the protective effect and mechanism of serum containing Euonymus fortunei on the rat pancreatic islet cells. Methods Forty male SD rats were randomly divided into 5 groups (n=8 in each group), including the control group (normal rat islet cells were cultured with normal rat serum), ischemic preconditioning group (abdominal aorta was blocked first and then re-opened before the pancreas was obtained, and the pancreatic islet cells were cultured with normal rat serum), Euonymus fortunei treatment group (normal rat islet cells were cultured with rat serum containing Euonymus fortunei), Euonymus fortunei group and blank group (normal rats were administered orally with Euonymus fortunei extract or distilled water for the preparation of rat serum). Diphenylthiocarbazone (DTZ) staining was utilized to observe and calculate the quantity of islets. Acridine orange (AO)/propidium iodide (PI) staining was adopted to calculate the survival rate of islet cells. The insulin release experiment was performed to calculate the stimulation index (SI) and evaluate islet cell function. The concentration of glutathione (GSH) and nitric oxide (NO) in islet cells was detected using GSH and NO kits. The expression level of inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) was quantitatively measured by reverse transcription polymerase chain reaction (RT-PCR). Results Islet cells were observed in specifically scarlet color after DTZ staining. The quantity of islet cells did not significantly differ among different groups (all P>0.05). Along with the prolongation of culture time, the activity of islet cells in each group was gradually decreased. At 72 h after isolation and culture, compared with the control group, the survival rate of the cells was significantly higher in the Euonymus fortunei treatment group (P<0.05). The insulin release test results demonstrated that compared with the control group, the SI of the ischemic preconditioning and Euonymus fortunei treatment groups was significantly increased (both P<0.05). Compared with the control group, the GSH contents of pancreatic islet cells in the ischemic preconditioning and Euonymus fortunei treatment groups were considerably enhanced, the NO content was significantly decreased, and the expression level of iNOS mRNA was significantly down-regulated (all P<0.05). Conclusions Euonymus fortunei can increase the survival rate of islet cells and enhance the function of pancreatic islets by increasing the level of GSH, down-regulating the expression of iNOS and decreasing the NO production.
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BACKGROUND: the development of magnetic separation technique,it is feasibility to in vitro sort and amplify CD4+CD25+Treg cells for transplantation; however,the application dosage and immune tolerance have been less reported yet.OBJECTIVE: To investigate dose-effect relationship of CD4+CD25+Treg cells during allograft transplantation.METHODS: SD rats which were considered as the donors and Wistar rats as receptors were used to establish allograff kidney transplantation models.CD4+CD25+Treg cells were separated from splenic cells of Wistar rats and induced phenotype of donor antigenic specificity in vitro.According to the quantities of CD4+CD25+Treg cells injecting through tail vein during the operation of allograft kidney transplantation,models were rolled into four experiment groups: group 1(2×105),group 2(5×105),group 3(1×106),and group 4(2×106).The models out injection were considered as controls.Survival status of kidney was detected at day 15 postoperatively; creatinine level and pathological changes were detected at days 4,9 and 15 according to Banff Schema diagnostic standard; semi-quantitative scores were measured Watanabe technique.RESULTS AND CONCLUSION: The death rate was the highest in control group(83.3%),and then group 1(66.7%),group 4(58.3%),and group 2(33.3%); but rats in the group 3 were all survival.Creatinine level in experimental groups was significantly less than control group at days 4,9,and 15 postoperatively(P<0.05,P<0.01); the creatinine levels in the group1 and group 2 were significantly greater than in the group 3 and group 4 at days 9 and 15 postoperatively(P<0.05),Semi-quantitative scores demonstrated that there was no significant difference between group 2 and group 1; but the scores in the group 3 and group 4 were significantly greater than control group(P < 0.05).The results indicated that CD4+CD25+Treg cells could improve kidney function following transplantation,and prolong survival time of transplanted kidney.The 1×106 was the best dosaae for application.
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Objective To study the influence of tumor necrosis factor-alpha (TNF-?) gene polymorphism on infections in old kidney allograft recipients. Methods The TNF-? genotype in -308 promoter position was determined in 87 old kidney transplant recipients from January 1998 to January 2001 by PCR using sequence -specific primer(PCR-SSP), and then the effects of genotypes on the frequency of rejection and infection, and the survival rate of the patient and graft were observed. Results 58 recipients were low TNF-? production genotype and 29 were high TNF-? production genotype. There were no significant differences in the frequency of rejection, median day of first infection and kinds of infections between the two genotypes. The frequency and times of infections were higher in low TNF-? genotype recipients than those in high TNF-? genotype recipients(67 2% vs 41 4%, P=0 021; 1 68 vs 0 86, P=0 017). The survival rates of the patients and grafts were equal in both the two groups. Conclusion The old recipients with low TNF-? production genotype may be susceptible to infections under routine immunosuppression. It is helpful for old recipients to perform more individual immunosuppression regimens according to TNF-? genotype.
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0.05 ). In Zenapax and OKT3 groups, 1 and 2 allografts were removed as a result of rupture respectively. 4 cases were suffered from delayed graft function(DGF) in Zenapax group and 9 in OKT3 group respectively with the difference being significant ( P