| Backgroud:Immune thrombocytopenia(ITP)is an autoimmune disorder characterized by both reduced platelet counts and the suppression of megakaryocytes.Abnormalities of cell-mediated immunity are known to contribute to the pathological process.Like many autoimmune diseases,ITP has a T helper cell type 1 bias and a reduced activity of T-regulatory cells.Mesenchymal stem cells(MSCs)show immunomodulatory properties and have been utilized extensively for the treatment of autoimmune diseases.The impaired immunosuppressive capacity of MSCs in patients with ITP might contribute to disease development.Based on in vitro cytological experiments,MSCs significantly inhibit IL-2 and IFN-y secretion by Th1 cells and promote the release of IL-4 and IL-10 by Th2 cells in ITP;this regulates the balance between Thl and Th2 activity and up-regulates CD4+ CD25+ T cells,inducing immunologic tolerance in ITP.Accordingly,MSCs could improve platelet counts in mice with ITP.A small number of clinical trials have shown that MSCs transfusions are effective for the treatment of ITP.MSCs immunosuppress IL-22 in patients with ITP via soluble cellular factors.Moreover,MSCs inhibit the proliferation of activated CD4+T cells.The immunomodulatory function of MSCs is positively correlated with the dose;however,high-dose MSCs are associated with an increase in adverse reactions,such as increased acute pulmonary embolism.Therefore,it is essential to improve immune regulation by individual MSCs.The advantages of three-dimensional(3D)cell culture are increasingly recognized by cell biologists.For example,3D culture can increase the expression of the pluripotency gene NANOG in MSCs by relaxation of cytoskeleton tension.Under 3D conditions,there are multi-directional cell-matrix interactions.The spatial conformation of 3D scaffolds can promote interactions between cells and enhance the secretory function of MSCs.Furthermore,3D cultured MSCs exhibit lower immunogenicity and higher immunosuppressive capacity than those of 2D cultured MSCs.These results suggest that MSCs cultured in 3D conditions may have beneficial effects in immune-related cell therapy.We speculate that ITP can be treated more effectively using 3D-MSCs than using 2D-MSCs.To evaluate this hypothesis,we prepared mouse platelet antigen for guinea pig immunization and used it to construct a chronic ITP mouse model by serum injection,resulting in a persistent reduction in platelets.Finally,the effect of MSCs was evaluated.Objective:1.Primary culture umbilical cord mesenchymal stem cells(UC-MSCs)and study the basic biological characteristics of three-dimensional cultured MSCs.2.Compare the immunosuppressive ability,anti-inflammatory environmental ability and extracellular matrix secretion ability of two-dimensional and three-dimensional cultured MSCs.3.Construct ITP experimental model mice that can meet the clinicopathological characteristics.4.Compare the general physical condition,peripheral blood platelet count and inflammatory environment of two-dimensional and three-dimensional cultured MSCs after treating ITP model mice,and compare and analyze the survival time of MSCs in vivo.Methods:1.Three dimensional culture and biological characteristics of umbilical cord mesenchymal stem cells1.1 Primary culture of human umbilical cord mesenchymal stem cells:take the human umbilical cord of full-term cesarean section in normal pregnancy,cut it,digest and adhere to the wall with collagenase?trypsin,observe the cell morphology,calculate the cell doubling time,and detect the expression of CD29,CD31,CD34,CD44,CD45,CD73,CD90 and CD 105 by flow cytometry.1.2 Three dimensional culture of UC-MSCs:three dimensional culture of UC-MSCs was carried out with polyvinylidene fluoride(PVF)scaffold for routine inverted microscope observation,he staining,electron microscope and cell histochemical staining.The expression levels of immune regulation related genes and ECM related genes in MSCs cells were detected by quantitative PCR.1.3 Lymphocyte proliferation inhibition test:mononuclear cells(MNCs)isolated from cord blood samples were activated by anti-CD3 monoclonal antibody.The 2D-MSCs group and 3D-MSCs group were cocultured with activated lymphocytes in a certain proportion,and the cells were counted by CCK-8 colorimetry.The inhibition rate of mesenchymal stem cells on lymphocyte proliferation was calculated.1.4 In vitro cell anti-inflammatory stress test:MSCs in 2D group and MSCs in 3D group were added with 50 ng/mL IFN-? At 12h,the cells without inflammatory factor treatment were used as the control group.The proportion of apoptotic cells was detected by flow cytometry.2.Treatment of ITP model mice with three-dimensional cultured MSCs.2.1 Construction of ITP mouse model:40 BALB/c mice were randomly divided into 4 groups:normal group(nor),model group(MOD),2D-MSCs treatment group(2D)and 3D-MSCs treatment group(3D),with 10 mice in each group.In order to prepare guinea pig anti mouse platelet serum(GP-APS),platelets were obtained from the venous blood of BALB/c mice and immunized guinea pigs for many times.After 5 weeks,GP-APS was prepared by taking blood from guinea pig heart,and the titer was tested ? The ITP mouse model was established by intraperitoneal injection of GP-APS(dilution 1:4)from BALB/c mice.GP-APSs was injected on days 1,3,5,7,9,11 and 13 to maintain low platelet levels.Observe the general condition of the animals,and record the platelet count and spleen/body mass index.2.2 Stem cell therapy:for 2D and 3D groups,2 mice were infused from the caudal vein to each mouse on days 15 and 17 2×10(6)/200 ?L 2D or 3D-MSCs for treatment.On day 21,all mice were sacrificed to measure spleen index and platelet concentration.IL-10 and IFN in peripheral blood serum were detected-?Level.2.3 Homing and survival time of mesenchymal stem cells in vivo:female BALB/c mice(20ą2 g)were randomly divided into 2D-MSCs group and 3D-MSCs group,with 6 in each group.UC-MSCs from male infants were then injected intravenously into each mouse 2×10(6)/200 ?L?At 6,12,24,48 and 72 hours after injection,three BALB/c mice were killed at each time point,and bone marrow,lung,liver,spleen and kidney samples were collected for DNA amplification.Genomic DNA Extraction Kit(beyotime)is used to isolate total genomic DNA from different tissues.Then,human sex determining region Y(SRY)and mouse actin in different tissues were detected by qPCR.The SRY value in the liver of each mouse was set to 1.3.Statistical Analysis:All data are presented as meansąSEM of three separate experiments.Data were analyzed by the Student's t-test for comparison between two groups or ANOVA for comparisons among multiple groups,as appropriate.Differences were considered statistically significant at P<0.05.Statistical analyses were performed using SPSS Statistics 17.0.Results:1.Three dimensional culture and biological characteristics of umbilical cord mesenchymal stem cells1.1 The primary seed cell culture system of UC-MSCs was successfully established and the identification of UC-MSCs was completed:in this study,DMEM/F-12 medium and 10%fetal bovine serum were used as basic culture conditions.Without the addition of foreign cytokines,the success rate of primary culture of MSCs from umbilical cord was as high as 100%(20 successful,20 successful).We used the double enzyme digestion system of collagenase+trypsin,After adherent culture in polystyrene based Petri dishes,fibroblast like cells with uniform cell morphology can be quickly obtained.The multiplication time of the fourth generation UC-MSCs is about 32H.Flow cytometry showed that UC-MSCs expressed CD29,CD44,CD73,CD90 and CD 105 positively and CD31,CD34 and CD45 negatively,which met the international standards for MSCs.1.2 The three-dimensional culture system of UC-MSCs was established and its biological characteristics were observed:we established the three-dimensional culture system of MSCs by using three-dimensional nested quality inspection of polyfluoroethylene.The cells adhered well on the scaffold,but the doubling time of cell growth was longer than that of two-dimensional culture.In addition,the ratio of CD31 and CD34 positive cells in three-dimensional mesenchymal stem cells was higher than that in two-dimensional mesenchymal stem cells,while the ratio of CD105 positive cells was lower than that in two-dimensional culture group(P<0.05).1.3 The immune regulation ability of 3D-MSCs is stronger:after 3D culture,the expression of all 8 immune regulation related genes detected after 3D culture is higher than that in 2D culture group.Among them,HLA-G,idol,PTGS2 and TGF-? The expression of four genes was significantly up-regulated(P<0.05).We further evaluated the inhibitory effect of UC-MSCs on the proliferation of activated lymphocytes by mixed culture with activated lymphocytes in vitro.Regardless of the co culture ratio,UC-MSCs cultured in 3D could more effectively inhibit the proliferation of activated lymphocytes(P<0.05).1.4 The extracellular matrix formation ability and anti stress ability of 3D cultured MSCs were enhanced:the 3D cultured UC-MSCs expressed higher levels of type? collagen,fibronectin and integrin(P<0.05).When MSCs are infused into ITP mice,they must first face the severe inflammatory environment,especially the environmental test of inflammatory factors.Therefore,we evaluated whether 3D culture could improve the resistance of MSCs to inflammatory factor IFN by flow cytometry apoptosis detection kit-? Ability.According to the pre experimental results,50NG/ml IFN was directly selected-? MSCs were intervened.Compared with the control group,this concentration of IFN-? It increased the proportion of late apoptotic cells(P<0.05),but had little effect on early apoptosis.In the 3D group,IFN-? Stimulation did not increase the ratio of early and late apoptotic cells(P<0.05 compared with the control group).2.Treatment of ITP model mice with three-dimensional cultured MSCs.2.1 ITP mouse modeling:we induced ITP in the mouse model by injecting guinea pig antiplatelet serum(GA-APS).After the first injection of gp-aps,the activity of ITP model group mice began to decrease,accompanied by the decrease of food and water intake.The mice in the control group developed normally,responded flexibly,had smooth fur and took the initiative to look for food;In the ITP model group,the agility of mice was also significantly reduced,accompanied by stool relaxation and final weight loss(P<0.05).After infusion of MSCs,the spleen index in 3D-MSCs group decreased significantly(P<0.05 compared with model group).No mice died during the experiment.2.2 Three dimensional cultured MSCs can quickly restore the platelet count of ITP model mice:24 hours after injection of gp-aps,the platelet count of ITP model mice decreased significantly and remained at a low level throughout the 2-week modeling period(P<0.05 compared with the normal group).After two MSCs treatments,the platelet count of the two MSCs groups increased significantly on the 21st day(P<0.01 compared with the model group),especially in the 3D-MSCs group(P<0.05 compared with the model group),However,there was no significant difference between 3D-MSCs group and 2D-MSCs group.2.3 Three dimensional cultured MSCs have better anti-inflammatory effect:in order to explore the anti-inflammatory effect of MSCs,we detected cytokines(IFN)in mouse peripheral serum-? And IL-10).The level of serum IL-10 in ITP model group was lower than that in normal group,while IFN-? The level was higher than that in the normal group(P<0.05).Compared with the model group,the expression of IL-10 increased after MSCs treatment,while IFN-? The expression of was significantly decreased(P<0.05),especially in 3D-MSC s group,but there was no significant difference between 3D-MSCs and 2D-MSCs groups.2.4 3D-MSCs survived longer in vivo:UC-MSCs from male infants were used for intravenous treatment.We detected the male specific SRY gene by PCR to track whether the cells could survive in various tissues of female mice.At the early time point,cells were mainly concentrated in the lung,and there was no significant difference between 3D-MSCs group and 2D-MSCs group.However,the bone marrow migration rate in 3D-MSCs group was significantly higher than that in 2D group(P<0.05).At 72h,SRY was still detected in bone marrow of 3D-MSCs group and not in 2D-MSCs group.Compared with 2D-MSCs group,3D-MSCs group had a slight delayed increase in spleen and kidney,but the difference was not significant.Conclusion:1.The primary culture system of UC-MSCs was successfully established by double enzyme digestion and adherent culture,which met the international standards for MSCs.2 The three-dimensional culture system of UC-MSCs was successfully established,and it was proved that the ratio of CD31 and CD34 positive cells in three-dimensional mesenchymal stem cells was slightly higher than that in two-dimensional mesenchymal stem cells.3 It is proved that the three-dimensional cultured MSCs have better immune regulation ability,including HLA-G,idol,PTGS2 and TGF-? The expression of four immune regulatory genes was significantly up-regulated,and MSCs cultured in 3D could more effectively inhibit activated lymphocytes.4 3D cultured MSCs have better extracellular matrix generation ability and anti stress ability.3D cultured UC-MSCs express higher levels of type ? collagen,fibronectin and integrin.It can better resist inflammatory factor IFN-? Induced apoptosis.5 Three dimensional cultured MSCs can quickly restore the platelet count of ITP model mice.After MSCs treatment,the expression of anti-inflammatory factor IL-10 increased,while the expression of inflammatory factor IFN increased-? The expression of was significantly decreased,especially in the 3D-MSCs group.6 The male specific SRY gene was detected by PCR,and the survival time of male MSCs cells in various tissues of female mice was tracked.It was observed that the survival time of 3D-MSCs in vivo was longer than that of 2D-MSCs. |
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