| BackgroundGraft-versus-host disease(GVHD)is a major cause of complications and non-relapse mortality following allogeneic hematopoietic stem cell transplantation(HSCT)GVHD is a multi-organ syndrome characterized by tissue inflammation and/or fibrosis predominantly affecting the skin,gastrointestinal tract,liver,lungs and mucosal surfaces.Currently,Acute GVHD is characterized by donor T-cell mediated allogeneic responses,including uncontrolled T-cell activation and proliferation,and the release of pro-inflammatory cytokines.The first-line treatment for GVHD is glucocorticoids combined with immunosuppressant drugs such as methotrexate,cyclosporine or mycophenolate mofetil.However,this treatment regimen is effective only in 30% to 50% of GVHD patients.Mesenchymal stem cells(MSC)are non-hematopoietic stem cells with self-renewal and multi-lineage differentiation capabilities.MSC express CD73,CD90 and CD105,but not CD45,CD34,CD14,Human Leukocyte Antigen(HLA-DR,CD11 b,CD79a,or CD19).MSC could be induced to differentiate into various tissue cells such as osteocytes,adipocytes,and chondrocyte progenitor cells.MSC primarily regulate target cells,suppressing inflammatory responses and maintaining immune balance.Given the immunomodulatory properties of MSC,many researchers are dedicated to exploring their use in treating GVHD.MSC exhibit the following immunoregulatory functions:(1)Inhibit T-cell proliferation,cytokine secretion,reduce cytotoxicity,and regulate the balance between helper T cells(Th1/Th2).(2)Influence the function of regulatory T cells(Tregs).(3)Regulates B cell development and function.(4)Inhibit the maturation,activation and antigen presentation function of dendritic cells(DCs).(5)Inhibit interleukin(IL-2)induced activation of natural killer(NK)cells.MSC exert these immunoregulatory functions through direct cell-to-cell contact or paracrine secretion of soluble cytokines.Numerous studies have demonstrated that the MSC dose and the route of MSC administration significantly influence the therapeutic effects of MSC on aGVHD.Three dimensional microcarriers are capable of transport MSC,and improve the maintaining time and regenerative efficacy of MSC in vivo.However,few reports about the effects and underlying mechanisms of MSC on aGVHD are available so far.In the present study,the MSC were loaded on microcarriers and intraperitoneally given to aGVHD mice,which yield improved protective effects on intestinal injuries.ObjectivesTo explore the effects and mechanisms of 3D microcarrier combined with MSC in treating mouse aGVHD.Methods1,Establish a mouse aGVHD model after Allo-HSCT;2,Isolate and culture mouse MSC and construct 3D microcarrier-MSC complexes;3,Compare the therapeutic effects of MSC and 3D microcarrier-MSC complexes on mouse aGVHD;4,Compare the protective effects and mechanisms of MSC and 3D microcarrier-MSC complexes on the pathological damage of mouse intestinal organoids in aGVHD;5,Perform mixed transcriptome sequencing analysis to understand the protective mechanism of 3D microcarrier-MSC complexes and verify their function;6,Statistical analyses: The software of GraphPad Prism 8.0 version was used to perform the statistical analysis of this study,with results presented as mean ± standard deviation(X?± SD).T-test was used for comparisons between two groups,and differences were considered statistically significant when p < 0.05.Results1.Establish a mouse model of aGVHD:(1)Survival status: The survival time of the recipient mice in the BM group was longer than 25 days,and long-term survival could be obtained after transplantation.The median survival time of the mice in the GVHD group was significantly shorter than that of the BM group,only 15.5 days(p<0.05),and the death peak appeared around 10 days after transplantation;Symptoms of back and diarrhea,and then as the weight of the recipient rats increased,the manifestations of diarrhea and other symptoms were slightly relieved.After +10 days,hair loss gradually appeared,activity decreased,back arched,diarrhea appeared again,and even bloody stools.However,the activity,body weight and spirit of the rats in the BM group recovered gradually after +4 days,and there was no clinical manifestation of aGVHD.The clinical and pathological scores of the mice in the GVHD group were higher than those in the BM group(p<0.001),and the pathology of the intestinal tissue in the GVHD group showed necrosis and shedding of small intestinal villi,and inflammatory cell infiltration was seen in the base.2.Mouse-derived MSC showed a swirl shape under the light microscope after culture,and could be induced to differentiate into adipocytes,chondrocytes and bone cells by three lines.The appearance of the 3D microcarrier is sheet-like,soluble in water,and consists of multiple micropores when observed under an electron microscope.Comparing the difference in the proliferation ability of 2D-MSC/3D-MSC,it was found that the proliferation ability of 3D-MSC was better than that of 2D-MSC,which was statistically significant(p<0.001)3.Study on 2D-MSC/3D-MSC treatment of aGVHD mice:(1)Study on MSC treatment strategy: GVHD mice treated with MSCs(1× 106cells/mouse)survived significantly longer than those treated with MSCs(2×105cells/mouse)of GVHD mice(p<0.05).Therefore,within a certain range,the curative effect of the high-dose MSC group on aGVHD is better than that of the low-dose group;in addition,there is no significant difference in the survival period of GVHD mice treated with MSCs in the low-dose group after transplantation + 3 days compared with the high-dose MSC group.(p>0.05);+7 days after transplantation,the survival time of mice treated with MSCs was significantly prolonged(p<0.05),and the survival time of mice treated with high doses of MSCs at +14 days was prolonged,and the survival time of the control group treated with lower doses of MSCs was also statistically different(p<0.05)(2)The effect of 2D-MSC/3D-MSC in the treatment of aGVHD: After receiving intraperitoneal injection of 2D-MSC/3D-MSC in the aGVHD recipient mice on the second day,the symptoms of aGVHD were improved,and the clinical scores of the 3D-MSC treatment group Significantly lower than 2D-MSC treatment group(p<0.05).The recipient mice in the GVHD group began to have bowed back and diarrhea symptoms +4 days after transplantation,and the symptoms of aGVHD aggravated after +10 days.The 2D-MSC/3D-MSC group received MSC infusion,the aGVHD symptoms were improved,and the 3D microcarrier-coated MSCs had a better therapeutic effect in the aGVHD mouse model,and the clinical score of the3D-MSC group was significantly lower than that of the aGVHD mouse model.In the group not treated with 2D-MSC(p<0.05),the survival rate of mice in the 3D-MSC group increased after +7 days(p<0.05).The pathological staining results of the small intestine showed that the damage was milder than that of the 2D-MSC group,and the damage of intestinal villi was less than that of the 2D-MSC group.The results of statistical analysis were significantly different(p<0.001).The proportion of Th1,Th17 was decreased(p<0.05),the proportion of Lgr5 and Treg was increased compared with the 3D-MSC group(p<0.05)..Organoid results showed that 3D-MSC cell culture supernatant had a better treatment effect,the number of aGVHD small intestinal organoids cultured with 3D-MSC cell supernatant sprouted 154±24.53/field,and the small intestine cultured with 2D-MSC cell supernatant The number of budding organoids was 96±22.33 per field of view,and the small intestinal organoids cultured in 3D-MSC supernatant were significantly more than those cultured in2D-MSCs(p<0.05).4.Bioinformatics analysis to explore the mechanism of 3D microcarriers:(1)RNA sequencing was performed,and a total of 3,418 significantly different genes were obtained by sequencing,of which 2,355 differential genes were up-regulated,and1,063 differential genes were down-regulated.The enrichment results of GO analysis showed that the differentially expressed genes were mainly enriched in three aspects:immune regulation,intestinal protection and mesenchymal development.A comparative analysis of the gene sets of the three types of GO functions revealed that among the two genes at the intersection(Tnfaip3 and Sfrp1),the extracellular matrix gene set was more closely related to the expression of Tnfaip3 in 3D-MSCs,namely A20.(2)Activation of A20 in 3D-MSC: The synergistic effect of 3D-MSC cultured with YAP inhibitor was partially canceled,and the small intestinal organoids cultured with 3D-MSC+YAP inhibitor cell supernatant had less budding than 3D-MSC group,which was 105 ±20.43 cells/field,compared with the 3D-MSC group,the synergistic effect was partially canceled(p<0.05);compared with the 3D-MSC group,the expression of A20 in the 3D-MSC+YAP inhibitor group was down-regulated compared with the 3D-MSC group(p<0.05),the expression of A20 in the3D-MSC+YAP inhibitor group decreased in western blot;through intraperitoneal injection of mice,it was found that the intestinal protection effect of the mice in the3D-MSC+YAP inhibitor group was partially canceled,and the pathological score Compared with the 3D-MSC group(p<0.05),the intestinal Th1,Th17 were increased(p<0.05),and the proportion of Lgr5 and Treg was decreased compared with the3D-MSC group(p<0.05).Conclusion1.C57BL/6 mice as donor mice and BABL/c mice as recipients of allogeneic myeloablative doses of mouse aGVHD model were successfully established;the clinical manifestations and pathological changes of the established mouse aGVHD model In line with expectations.2.Isolate mouse-derived MSC and 3D microstem cell gels were successfully constructed.3.By constructing aGVHD mouse model,MSC and 3D microcarrier-coated MSC were injected intraperitoneally to treat aGVHD to treat intestinal injury,and found that 3D microcarrier-coated MSC had a stronger therapeutic effect in the aGVHD mouse model.Organoid results showed that 3D-MSC cell culture supernatant treatment effect is better.4.Excavated the mechanism of 3D microcarriers through bioinformatics analysis,and found that A20 factor played an important role in it.Through in vivo and in vitro verification,it was found that the synergistic effect of 3D-MSC was canceled after the addition of YAP inhibitors,which proved that 3D stem cells combined with microcarriers played a synergistic effect by activating the YAP pathway and up-regulating the expression of A20. |
PDF Full Text Request