| Objective: To study the correlations between the EMPs levels, the EMPs phenotype and the acute allograft rejection after kidney transplantion in rats, and observe the affect of EMPs on monocyte function in vitro. To clarificate the partial mechanism of EMPs participating in acute allograft rejection.Methods: 1. Optimization and establishment a stabile orthotopic kidney transplantation model in rats: we improved the orthotopic kidney transplantation model in rats on the foundation of reference the classic methods of the kidney transplantation in rats. 2. Divided experimental animals into two groups: (1) Syngeneic controls (9 animals): the donors and the recipients were both Sprague-Dawely(SD) rats.(2) Allotransplantion groups (9 animals):the donors were Wistar rats, the recipients were Sprague-Dawely(SD) rats.Parallel groups of syn- and allo- transplanted rats were sacrificed on day 5 post-transplantation, the grafts were collected which were immobilized by 10% formaldehyde solution and blood samples were treated using 12.9mM /L citrate as the anticoagulant. 3. pathology diagnosis of the grafts: the immobilized kidneys were treated with paraffin section, HE staining, then acute rejection was diagnosed according to the Banff 97 classification. 4. analysis the quantity and phenotype of EMPs: the relation was analyzed between the quantity and phenotype of circulating endothelial microparticles and the acute allograft rejection after kidney transplantion in rats by flow cytometry. 5. Generation and analysis of EMPs in vitro: EAhy926 cells monolayers, a con?uent Human umbilical vein endothelial cells strain, were incubated for 48 hours with 100 ng/mL TNF-α. The supernatant was ultracentrifuged , then EMPs were collected. The acquired EMPs were analyzed by flow cytometry. 6. THP-1 cells were incubated with various amounts of EMPs(ratio:1:10,1:50,1:100,1:150 THP-1-EMP). The positive control was 10μg/ml LPS, and the negative control was PBS. The supernatant of separated EMPs was also as control. IL-6 and TNF-αwhich generated by THP-1 cell were measured by ELISA. 7. Analysis of MCP-1 and HLA-DR mRNA expression: the MCP-1 and HLA-DR mRNA levels of THP-1 cell were analyzed by RT-PCR.Results: 1. Optimization and establishment a stabile model of rats orthotopic kidney transplantation. The time of donor kidney anastomosis is 45±5min, the average time of operation is 150±10min. The state of rats is fine. 2. As examined from tissue morphology, there were lots of inflammatory cells infiltration in periglomerular and around small artery, scattered in tubules matrix and the number of cells in glomerulus increased in allo-groups on day 5 post-transplantation. Lots of inflammatory cells infiltrated into glomerulus along saccule, the endothelial cells of small artery shed, hemorrhage, thrombosis and small artery walls fibrinoid necrosis, lots of inflammatory cells infiltrated into renal interstitium widely, renal tubular cell degeneration, necrosis. The Banff score of the syn-groups on day 5 post-transplantation was (0.78±1.09), another was (5.22±0.83). The Banff score of the syn-groups was lower than that of allo-groups with independent-samples T test (P<0.01). 3. analysis the quantity and phenotype of EMPs and the correlations between the EMPs levels, the EMPs phenotype and the acute allograft rejection after kidney transplantion in rats: the numbers of Annexin V+MPs , CD144+EMPs and CD144 + CD62E + EMPs in allo-groups were higher than those in syn-groups with independent-samples T test(P < 0.05), and We found significant relationships between Annexin V+ MPs, CD144+EMPs, CD144+ CD62E + EMPs levels and the degree of acute allograft rejection with Spearman rank correlation analysis. (rs=0.84,P<0.01; rs=0.81,P<0.01; rs=0.65,P<0.05). 4. Generation and analysis of EMPs in vitro: EAhy926 cells monolayers were incubated for 48 hours with 100 ng/mL TNF-α. The supernatant was ultracentrifuged and EMPs were collected. Immediately, the acquired EMPs were analyzed by flow cytometry. The percentage of CD144+ EMPs was 88.82%. And the percentage of CD144+CD62E+EMPs was 29.97%. This indicated that the EMPs was suitable for experiment. 5. THP-1 cells generated IL-6 and TNF-αwhen EMPs activated human mononuclear cell strain THP-1 cells: one-way ANOVA tests were used for in vitro analysis of EMPs effects on THP-1 cells generating IL-6 and TNF-α. The IL-6 and TNF-αlevels of 1:50 group, 1:100 group, 1:150 group and supernatant group were obviously higher than the PBS group (P<0.05) , but lower than that of the LPS groups (P<0.05). 6. The information of THP-1 cells expressing MCP-1 mRNA, HLA-DR mRNA when EMPs activated the cells: Compared with PBS group, the express levels of MCP-1 mRNA, HLA-DR mRNA in 1:50group, 1:100group, 1:150group and supernatant group were increased (P<0.05). Compared with LPS group, the express levels of MCP-1 mRNA and HLA-DR mRNA in 1:10 group were decreased (P<0.05).Conclusions: The experimental result indicated CD144+ EMPs and CD144+ CD62E+EMPs levels of blood plasma in allograft rejection rats were elevated. Moreover, there were significant relationships between EMPs levels and acute allograft rejection. It confirmed EMPs levels could be regarded as a marker of acute allograft rejection reaction. The mechanism of EMPs participating in immune response possibly was EMPs as external antigen could induce monocyte activation.EMPs also could intensify the ability of monocyte presenting the antigen, and make monocyte secrete many kinds of cytokines which taking part in immune cytotoxicity effect.
|
PDF Full Text Request