Experimental Study On Swine Acute Liver Failure By Combined Therapy Of IL-1Ra Chitosan Nanopaticles And Mesenchymal Stem Cells Transplantation

In comparison with other pluripotent cells, Autologous bone marrow MSCs are accessible, safe, low immunogenicity and without ethical. Their high capability for self-renewal and differentiation make them an important cell source for acute liver failure. As the liver inflammation greatly suppress the viability, survival and transformation of MSCs, we prepared IL-1Ra-loaded nanopaticles made of lactosylated chitosan. In order to break the constraints for research of cell transplantation,we built Green fluorescent protein (GFP) transfection MSCs mediated by lentivirus vector to teste the biological behavior of the transplanted cells in vivo such as migration, distribution, proliferation, differentiation and survival. The combined therapy consist of IL-1Ra-loaded nanopaticles and MSCs transplantation carried out in sequence via ear veins or portal veins expecting to improve the homeostasis, increase the viability,the survival rate of the transplanted cells and promote liver regeneration.Part Ⅰ. Isolation, culture, labeling and identification of mesenchymal stem cells from bone marrowObjective:To built Green fluorescent protein transfection MSCs mediated by lentivirus vector and to teste the biological behavior of the transplanted cells in vivo.Methods:Mononuclear cells harvested from bone marrow aspirate of swines were isolated by density gradient centrifugation, and mesenchymal stem cells (MSCs) were purified by means of adherence incubation. Green fluorescent protein (GFP) was transfected into MSCs mediated by lentivirus vector. The cells were identified by flow cytometry with CD44, CD45and CD90respectively. The viability and proliferation of MSCs was determined by MTT method.Results:Cultured primary Mononuclear cells were circular and the BMSCs were adherent after several passages showing spindle-shaped, like fibroblast. The Flow cytometry identification showed that almost90%cells was CD44, CD90positive and CD45negative.It indicated that the GFP transfection did not change the characteristics of mesenchymal stem cells. The MTT method showed that there were no differences in the levels of the proliferation between GFP-MSCs and MSCs. Conclusions:GFP can be effectively transferred into MSCs under lentivirus vector mediation in MOI80.The GFP-MSCs is the best one for efficient and sensitivity in lots of methods for tracing in vivo.Part Ⅱ. Preparation and function testing of IL-1Ra-loaded nanopaticles made of lactosylated chitosanObjective:To prepare IL-1Ra-loaded nanopaticles made of lactosylated chitosan intended for improving the homeostasis of ALF.Methods:IL-1Ra-loaded nanopaticles made of lactosylated chitosan-FITC were prepared using an electrospraying technique. The active live targeting of thus nanopaticles were investigated by fluorescence microscope and flow cytometer (FCM); In vitro release of IL-1Ra in PBS from nanopaticles was analysed by ELISA.Results:The Infrared spectroscopy showed that the characteristic peaks of Lactobionic Acid (1742.66cm-1) and chitosan disappeared or moved. Nuclear magnetic resonance spectroscopy showed the characteristic peaks of the lactosylated chitosan(4.3ppm) meant that LA grafted to chitosan successfully. SEM results clearly demonstrated that the particles were approximately spherical with uniform size. The live targeting of the lactosylated hitosan-based nanoparticles was achieved by ligand-receptor specificity through FCM and ELISA method; The profiles in vitro showed that there was a steady-state release after a fast linear release (9h-30h). In other words,30%of cumulative release was achieved after30hours. Conclusions:Lactosylated chitosan-based nanopaticles can greatly enhance targeting abilities and slow release characteristics of IL-1Ra.Part Ⅲ. Experimental study on swine acute liver failure by combined therapy of IL-1Ra chitosan nanopaticles and mesenchymal stem cells transplantationObjective:To evaluate cooperative effects and mechanism on swine acute liver failure by combined therapy of allogeneic mesenchymal stem cell transplantation and IL-1Ra chitosan nanopaticles and to improve prospects of mesenchymal stem cells transplantation.Methods:The combined therapy was given and the Detailed Steps as follows: Chinese experimental mini swine were given D-galactosamine to build models of acute liver failure. Thirty-four pigs were randomly divided into five groups. In the control group(A),the normal saline was injected into liver via portal veins after24h; in IL-1Ra group(B), IL-1Ra was injected via ear veins6h before normal saline; In the MSCs transplantation group (C),8×107MSCs were injected into liver via portal veins after24h;IL-1Ra together with MSCs transplantation group(D) and nanopaticles group(E) as follows:on the one hand,8×107MSCs were injected into liver via portal veins after24h, on the other hand,rhIL-1Ra/IL-1Ra chitosan nanopaticles (1mg/kg) was injected via ear veins respectively6hours before. Liver function, serum inflammation and pathological changes were measured. The fate of MSCs was also observed.Results:The biochemical assay, the serum inflammation lever and pathological changes of the nanopaticles group were all greatly different from the other groups, the hepatocytes grow rate was significantly improved after1week(P<0.05); The liver engraftment was very low in group C and D with significantly higher numbers found in nanopaticles group, but the differentiation of MSCs after2weeks relatively rare; western blot showed that there was more HGF and VEGF secreted in nanopaticles group.Conclusions:The combined therapy showed great synergistic effects through improving homeostasis of liver failure contributed to increased hepatic engraftment and secretion of cytokines.