Research Of The Effect Of Amniotic Mesenchymal Stem Cells On Acute GVHD

Objective:Mesenchymal stem cells (MSC) is a kind of stem cells which is in Capacity of hematopoietic support, immune regulation and self-renewal, and it is pluripotent, furthermore. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the the most effective treatments of blood diseases. But during and after the transplantation, we inevitably face some tough problems, such as the destruction of the hematopoietic microenvironment, stem cell homing and graft-versus-host disease(GVHD). aGVHD is a common and tough problem after transplantation. So the successful prevention and treatment of aGVHD is of great significance to improve the success rate of HSCT. It has been confirmed that MSC can play its role in immune regulation, by suppressing the generation of dendritic cells, maturing T cell proliferation and reducing the formation of cytotoxic T cells and other mechanisms. It is safe and effective treatment of aGVHD by infusing of bone marrow-derived MSC. Thus, MSC is a cellular therapy for the prevention and treatment of aGVHD. In vitro studies, it showed that amniotic mesenchymal stem cells (AMSC) has the similar immune phenotype, multiple differentiation potential and hematopoietic support effect with the bone marrow mesenchymal stem cell. It is expected to become a more ideal MSC new source of clinical and scientific research. Our earlier in vitro studies have confirmed that AMSC has low immunogenicity, which can inhibit the transformation of human peripheral blood lymphocytes, and this inhibition is dose dependent. The first part of this research is to study the application of human peripheral blood mononuclear cells(PBMC) in NPG mice to construct the aGVHD model of the xenograft. In the second part, on the basis of constructing the aGVHD model of mouse xenograft, we have a further study on the effect of human AMSC on the aGVHD of the xenograft. In order to lay a theoretical foundation for the clinical application of AMSC, and hope to provide experimental basis for the improvement of MSC transplantation in clinical.Methods:In the first part, in order to establish the X-GVHD model of NPG mice, we injected PBMC into NPG mice, which were irradiated by sub lethal dose. The PBMC were isolated from the peripheral blood of healthy people with fresh and stock sources respectively. After injection, mice were observed daily activities, weight, hemogram, to do the relevant pathological examination of the organs of mice by H&E staining technique. Through the above indicators, compare the difference of the x-aGVHD models established by the two sources of human PBMC. In the second part, we isolated and cultured AMSC from human amniotic membrane tissue by method of tissue block, and injected the mice with AMSC on the basis of X-GVHD models established. After injection, also in mice were observed daily activities, weight, hemogram, to do the relevant pathological examination of the organs of mice by H&E staining technique, and detecting the infiltration of human T cells in peripheral blood, tissue and organs of mice by Flow cytometry and Immunofluorescence technique. Thus, determining the effect and function of AMSC on aGVHD by comparing the differences between various indicators.Results:1. Compared to the human PBMC, which is the source of blood in stock, the use of fresh blood sources of human PBMC tail vein iniection in NPG mice is more likely to successfully establish a X-aGVHD model, and the modeling effect is more significant.2. AMSC intravenous infusion in the X-aGVHD model of mouses can reduce aGVHD symptoms(burnout, arched, towering hair, weight loss, etc) and improve the pathological damage of target organs (lung, liver, spleen, kidney, intestine). AMSC combined infusion significantly reduced the proportion of human T lymphocytes (CD3+, CD45+) in mice, and increased the ratio of CD4+/CD8+T cells.Conclusions:To use fresh blood sources of human PBMC tail vein injection in NPG mice is more likely to successfully establish a xenograft-aGVHD model, and the modeling effect is more significant; Intravenous injection of human AMSC in the xenograft-aGVHD model of mouse has inhibitory effect on aGVHD.