| Objective:1.Adipose-derived mesenchymal stem cells(A-MSCs)were cultured in three dimensions,and their morphological and surface molecular identification was performed to detect their immunomodulatory functions.2.A mouse model of acute graft-versus-host disease(GVHD)was established and treated with two-dimensional and three-dimensional cultured A-MSCs,and the difference in the therapeutic effect of the two on acute GVHD mouse liver tissue injury was compared and further explored its mechanism.Methods:1.The primary A-MSCs was extracted and performed phenotypic identification and detection of three-line differentiation ability2.A 3D culture system of A-MSCs was established by soluble microcarriers and cell viability and migration capacity were compared with two-dimensional cultured A-MSCs.3.The proliferation activity of A-MSCs in 2D and 3D cultures was compared.4.Apoptosis kit and flow cytometry were used to detect the apoptosis ratio of A-MSCs cultured in two and three dimensions.5.The expression of immunoregulation related proteins in A-MSCs cultured in 2D group and 3D group was detected by PCR and Western blot.6.T lymphocytes were co-cultured with 2D and 3D A-MSCs to detect the proliferation and apoptosis of CD3+CD8+T cells and the level of IFN-y in supernatant.7.Mouse GVHD models were established by irradiation and spleen lymphocyte transplantation,and the GVHD model was treated with 2D and 3D cultured A-MSCs,and then the weight,survival,activity,coat color,arch back and diarrhea was measured,Elisa was used to detect the levels of TNF-α and IFN-γ in serum,and the proportion of Th1 cells in peripheral blood was analyzed by flow cytometry.At the same time,livers of target organs were taken for HE staining.8.The expression of JAK2,STAT1,p-JAK2,p-STAT1 and T-bet in GVHD liver after treatment was detected by Western Blot.Results:1.The extracted primary A-MSCs express surface markers of mesenchymal stem cells and have the ability of adipogenesis,osteogenesis and chondrogenesis.2.A-MSCs with 3D culture have higher cell viability and migration ability,and the proliferation ability is not significantly different from that of the 2D group.At the same time,the proportion of apoptosis is slightly increased,but the expression of TLR3,TLR4 and IDO1 is increased.3.Compared with the two-dimensional culture group,after co-culture of T lymphocytes with three-dimensionally cultured A-MSCs,the proportion of CD3+CD8+T lymphocytes decreased,the proliferation ability decreased and the proportion of apoptosis increased.The level of IFN-y decreased in three-dimensional culture group.4.Compared with the 2D group,GVHD mice treated with 3D A-MSCs recovered faster in body weight,better survive,higher mobility,reduction in symptoms of arch back and diarrhea,and HE staining showed less liver damage.5.Compared with the 2D group,the levels of TNF-α and IFN-γ in serum of GVHD mice treated with 3D A-MSCs decreased,and the proportion of Th1 cells decreased significantly.6.Compared with the 2D group,the expression levels of JAK2,STAT1,p-JAK2,and p-STAT1 in the liver of GVHD mice treated with 3D A-MSCs decreased.Conclusion:1.3D A-MSCs have multi-directional differentiation ability.Compared with the two-dimensional culture mode,the apoptosis ratio is increased,the migration ability is enhanced,and the proliferation ability is not significantly different.The A-MSCs improve the homing ability of injured tissue.2.The expression of TLR3,TLR4 and IDO1 are up-regulated in 3D A-MSCs,which means stronger immune regulation function.3.There is stronger immune regulation function in 3D A-MSCs,and it can reduce the level of IFN-y secreted by Th1 cells in the serum of GVHD mice and inhibit the expression of JAK2/STAT1 pathway in GVHD liver to reduce liver tissue fibrosis.in addition,it can also significantly down-regulate the transcription factor T-bet,inhibiting the transformation of Th0 cells into Th1 cells in peripheral blood,which can reduce systemic inflammation,protect liver tissues,and reduce liver damage in GVHD. |
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