| Background:Aplastic anemia(AA)is a group of bone marrow failure diseases characterized by reduced bone marrow karyocyte hyperplasia and pancytopenia.The damage and functional defects of hematopoietic stem cells(HSCs)directly cause bone marrow failure,leading to the onset of AA.The immune dysfunction is an important cause of HSCs damage and functional defects,especially the dysfunction of regulatory T cells(Tregs),significant reduction in CD4+/CD8+T lymphocyte ratio,and the abnormal activation of cytotoxic T lymphocytes(CTL).Allogeneic hematopoietic stem cell transplantation(Allo-HSCT)is the first-line treatment option for severe aplastic anemia.When immune cells in the donor tissue(graft)recognize the recipient(host)as antigen,graft-versus-host disease(GVHD)occurs.GVHD is the most serious life-threatening complication of Allo-HSCT,which limits the clinical application of Allo-HSCT.Umbilical cord mesenchymal stem cells(UCMSCs)are heterogeneous cells isolated from the umbilical cord,and have the functions of immunoregulation,blood vessel growth promotion and injury repair.There is huge potential in the treatment of liver diseases,autoimmune diseases,lung injury and diabetes.Traditional cell culture in vitro is carried out on a two-dimensional(2D)plane.As the passage and expansion proceed,the mesenchymal stem cells(MSCs)gradually age and lose their original characteristics.Three-dimensional(3D)culture promotes the interaction between cells by simulating the 3D environment in vivo,so that the MSCs maintain their original characteristics and even improve the function of the cells.MSCs widely express Toll-like receptors(TLRs).In different inflammatory environments,corresponding TLR activation can result in different functions of MSCs.Our previous research results show that the 3D culture can increase the expression of TLR3 of MSCs.Studies have shown that activating TLR3 of MSC enhances the ability of MSCs to induce Treg differentiation and enhance their immunosuppressive function.We plan to improve the growth environment of UCMSCs cultured in vitro through 3D culture,enhance the function of secreting active factors and cell status,and provide better quality tissue engineering cells for clinical application.To study the therapeutic effect of TLR3 activated three-dimensional UCMSCs in bone marrow transplantation for aplastic anemia,the possible clinical value of UCMSCs is being preliminarily discussed.Objective:1.By improving the culture method of UCMSCs in vitro,to obtain better treatment seeds.2.To establish a 3D culture system,study the influence of 3D culture on UCMSCs and provide more theoretical basis for the clinical application of MSC.3.To study the advantages of modified UCMSCs combined with Allo-HSCT in the treatment of aplastic anemia,and further explore its mechanismMethods:1.UCMSCs were isolated by tissue block adherence.Cell surface markers were identified by flow cytometry.Cells were induced to differentiate into osteoblasts,chondrocytes and adipocytes to detect their multidirectional differentiation potential.2.Polyvinyl fluoride nested plates were used as the carrier of three-dimensional culture.Acridine orange fluorescence staining was used to observe UCMSCs in three-dimensional culture,and the proliferation function of UCMSCs was detected by CCK8 proliferation assay..3.The angiogenesis promoting ability of UCMSCs was detected by in vitro angiogenesis test.4.Soft AGAR colony formation was used to detect the tumorigenic tendency of UCMSCs.5.The expression levels of immune regulatory factors and blood-promoting factors of UCMSCs were detected by qRT-PCR.6.The aplastic anemia mouse models were established by irradiation combined with lymphocytes tail vein infusion.7.Allo-HSCs were isolated from bone marrow cells of C57 mice using the SCA1+sorting kit.8.3D cultured UCMSCs with TLR3 activated and Allo-HSCs were injected into mice by tail vein infusion,and the efficacy was evaluated.Results:1.UCMSCs were successfully isolated,expressed specific surface markers,had the potential of three-line differentiation,and the 3D culture system of UCMSCs was successfully established.2.The results of CCK8 proliferation assay showed that the proliferation of UCMSCs cultured in 3D was not active.3.Umbilical vein endothelial cells(HUVECs)were obtained by enzyme digestion,and the angiogenic ability of UCMSCs in 3D cultured was significantly stronger than in 2D.4.The results of in vitro tumorigenesis experiments showed that 3D culture did not increase the risk of UCMSCs tumorigenesis.5.After 3D culture,UCMSCs' expression of immunoregulatory factor PGE2 was significantly increased,but the expression of DL1,IDO and other factors were not significantly upregulated;the expression of hematopoietic related factors CSF1,CSF2,CSF3 was significantly upregulated,SCF,THPO,IL3,EPO expression were not significantly increased.6.After modeling,the mice in the modeling group showed the clinical manifestations of AA,the bone marrow showed obvious vacuolization,the number of karyocyte was significantly reduced,and the medullary cavity was filled with adipocyte.There were no obvious GVHD manifestations in the internal organs of the mice.7.In terms of therapeutic effect,the UCMSCs cultured in 3D May be superior to the UCMSCs cultured in 2D in terms of early survival and long-term efficacy of bone marrow transplantation.8.The improvement of symptoms of TLR3 activation+3D cultured UCMSCs+Allo-HSCT group and 3D cultured UCMSCs+ Allo-HSCT group may be better than that of 2D cultured UCMSCs group and Allo-HSCT group.9.The H&E staining results of bone tissue sections showed that the number of karyocyte in bone marrow of mice treated with UCMSCs cultured in 3D was significantly higher than that of other groups,and the number of karyocyte in bone marrow of mice treated with UCMSCs was more than that of mice treated with Allo-HSCT alone.No significant lymphocyte infiltration was observed in tissue sections of each group.10.In the hematopoietic progenitor cell colony formation experiment,the bone marrow cell colony formation ability of mice treated with 3D cultured UCMSCs was better than that of the 2D group,and the bone marrow cells of mice treated with UCMSCs after TLR3 activation formed CFU-GEMM's ability was superior to the rest of the group,and the bone marrow proliferation ability of mice just given Allo-HSCT treatment was the weakest.Conclusion:1.UCMSCs could be isolated according to tissue explants adherent method,and the polyvinyl fluoride nested 3D culture system of UCMSCs could be constructed successfully.In this system,3D culture does not promote the proliferation of UCMSCs or increase their tumorigenic tendency2.The 3D culture system can improve the function of UCMSCs in promoting angiogenesis3.3D culture can enhance the PGE2 expression of UCMSCs and affect its immune regulation function.4.3D culture of UCMSCs can enhance the expression of CSF1,CSF2 and CSF3,and enhance their hematopoietic function5.The AA mouse model is successfully established.The AA mice have typical clinical manifestations such as lethargy,decreased activity,sparse fur,weight loss,humpback,pancytopenia,and reduced bone marrow hyperplasia.6.UCMSCs can promote bone marrow reconstruction in AA mice after bone marrow transplantation.Activated TLR3 UCMSCs in 3D culture may promote the formation of leukocytes at the early stage of bone marrow transplantation in AA mice by increasing the expression of CSF1,CSF2 and CSF3,and boost immune,which may be beneficial to the early survival of bone marrow transplantation. |
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