| ObjectiveImmune thrombocytopenia is a common clinical hemorrhagic disease,which is directly related to the differentiation,maturation and platelet release of megakaryocytes regulated by bone marrow microenvironment.Between bone marrow mesenchymal stem cells as an essential division of the bone marrow microenvironment,have captured much attention in the research process of the pathogenesis and discourse of ITP.In this study,we used ITP cell model and ITP animal model to investigate the effect and mechanism of TFFHS in BMSCs treatment of ITP.Methods1.Rat bone marrow mesenchymal stem cells between the separation,purification and identificationThe whole bone marrow adherent culture method between the leave-taking and clarification of rat BMSCs.The purgative BMSCs were identified by flow cytometry,lipogenesis induction and osteogenesis induction,respectively,from two aspects of surface marker molecules and multidirectional differentiation ability,which laid the foundation for subsequent experiments.2.To study the effect of APS on BMSCs and the intervention effect and mechanism of TFFHS in the diseaseMTS method and flow cytometry to investigate the appropriate concentration of APS to interfere with BMSCs.The group was set up to explore intervention impact of various levels of TFFHS on BMSCs using chemiluminescence assay and finalize the experimental concentration.The BMSCs were grouped based on the determination of APS and TFFHS concentrations and set into control group,model group,TFFHS high(7.80 μg/ml),medium(3.90 μg/ml)and low(1.95 μg/ml)dose groups.Detection of interleukin 6,interleukin 11,stem cell factor,thrombopoietin and stromal cell-derived factor in BMSCs by enzyme-linked immunosorbent assay.Application of flow cytometry to probe the apoptosis of BMSCs.Detection of Bax,Bcl-2,Caspase-3,PI3 K,p-PI3 K,Akt,p-Akt expression by western blot.3.The role and mechanism of TFFHS in regulating BMSCs in ITP rat model through PI3K/Akt signaling pathwaySixty-six SD male rats were chosen as experimental subjects,normal group,model group,TFFHS high(94.5 mg/kg),medium(63.0 mg/kg)and low(31.5 mg/kg)dose groups,and prednisolone acetate group were set up.Eleven rats were set up in each group.The SD rat ITP model was established by intraperitoneal injection of APS and treated with TFFHS and prednisolone acetate by gavage,respectively.Monitoring platelet changes in rats.After successful modeling,the spleen of the rat was dissected and weighed,and the splenic index was calculated.The femur and tibia were fixed in 4 % paraformaldehyde and decalcified.Detection of pathological changes in bone marrow of rats by HE staining and CD90 expression of BMSCs in rat bone tissue by immunohistochemistry.Bone marrow of femur and tibia were extracted,and BMSCs were isolated and purified,morphological differences of BMSCs were recorded.Detection of apoptosis in BMSCs by AO/EB.WB test Bax,Bcl-2,Caspase-3,PI3 K,p-PI3 K,Akt,p-Akt expression in BMSCs.ELISA for interleukin 4,IL-6,IL-11,SCF,TPO in BMSCs.Results1.After isolation and purification by whole bone marrow culture,the cells showed a uniform long spindle shape,with strong growth ability and rapid proliferation.As cell density increases,cells grow in swirling clusters.The consequences of flow cytometry revealed that positive rates of CD90,CD44 and CD29 were 99.4 %、99.3 % and 99.2 %,positive rates of CD45 and CD34 were 8.1 % and 9.4 %.Red circle or oval lipid droplets were visible after lipogenesis induction,and as magnification increased,the lipid droplets became clearer,indicating that the cultured cells had the ability to differentiate adipogenesis.After osteogenesis induction,large orange-red calcified nodules were visible,which confirmed the existence of mineralized matrix deposition,indicating that the cultured cells had osteogenic differentiation ability.2.Application of MTS method and flow cytometry to screen the optimal concentration of APS for intervention in BMSCs to construct ITP models was 0.5 %.Application of chemiluminescence method to screen TFFHS concentrations of 1.95,3.90 and 7.80 μg/ml as experimental drug concentrations.Flow cytometry results demonstrated that apoptosis percentage in model group was more than normal group(P<0.01),TFFHS group was less than model group(P < 0.01).ELISA results showed that IL-6,IL-11,SCF and TPO concentrations decreased in model group compared to normal group(P<0.05,P<0.01),while IL-6,IL-11,SCF and TPO concentrations increased in TFFHS group compared to model group(P<0.05,P<0.01).WB demonstrated expression of Bax,Caspase-3,p-PI3K/PI3 K and p-Akt/Akt in model group was higher than normal group(P<0.05,P<0.01),TFFHS group was lower than model group(P<0.05,P<0.01),expression of Bcl-2 in model group was lower than normal group(P < 0.05,P < 0.01),TFFHS group was higher than model group(P<0.05,P<0.01).3.The weight of model group was lower than normal group(P<0.01).Decreased platelet count in model group(P<0.01).TFFHS group and positive drug group were more than model group(P<0.05,P<0.01).Spleen weight and spleen index in model group were higher than normal group(P<0.01),TFFHS group and positive drug group were lower than model group(P<0.05,P<0.01).Cells in model group grew slowly and had a disorganized short morphology,while cells in TFFHS group and positive drug group grew rapidly and had a regular stretching morphology.AO/EB staining showed that apoptosis in model group was more than normal group,TFFHS group and positive drug group were less than model group.HE staining results showed that compared with the normal control group,the bone marrow cavity of the model group was significantly empty,and the number of intact cells was significantly reduced.The bone marrow cavity of TFFHS group and positive drug group were significantly full,and the number of intact cells increased.Immunohistochemical showed that CD90 in model group was less than normal group(P<0.01),TFFHS group and positive drug group were more than model group(P<0.01).WB demonstrated expression of Bax,Caspase-3,p-PI3K/PI3 K and p-Akt/Akt in model group were higher than normal group(P<0.05,P<0.01),TFFHS group and positive drug group were lower than model group(P<0.05,P<0.01),expression of Bcl-2 in model group was lower than normal group(P<0.05,P<0.01),TFFHS group and positive drug group were higher than model group(P<0.05,P<0.01).ELISA showed IL-6,IL-11,SCF and TPO concentrations in model group were lower than normal group(P<0.05,P<0.01),TFFHS group and positive drug group were higher than model group(P<0.05,P<0.01),concentration of IL-4 in model group was higher than normal group(P<0.05,P<0.01),TFFHS group and positive drug group were lower than model group(P<0.05,P<0.01).Conclusions1.BMSCs are isolated and purified by whole bone marrow adherent culture.Using flow cytometry,lipogenesis induction and osteogenesis induction,the phenotype and multidirectional differentiation ability of our cells were verified to have mesenchymal stem cell-related characteristics.It lays a solid foundation for the follow-up experiment.2.The experimental results of ITP cell model and ITP animal model showed that APS can significantly inhibit the proliferation of BMSCs.The TFFHS may be through PI3K/Akt signaling pathway regulate the apoptosis of BMSCs.And improve the ability of BMSCs to secrete factors related to megakaryocyte differentiation and maturation. |
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