The Role And Mechanism Of Mouse Bone Marrow Mesenchymal Stem Cells In Immune Thrombocytopenia Mice Model

Objective: Immune thrombocytopenic purpura(ITP)is a common autoimmune hemorrhagic disorder by isolated thrombocytopenia.Patients with chronic refractory ITP have the higher risk of death and disease-related or therapy-related complications Despite large research efforts and clinical trials,the optimal treatment for patients with chronic ITP remains to be furthered,Among them,mesenchymal stem cell may be a new approach to ITP treatment.This study is to establish a stable and efficient method for the isolation and expansion of mouse bone marrow mesenchymal stem cell(BM-MSCs).By studying the basic biological characteristics,immunophynotypes and multiple differentiation potentials of BM-MSCs,and their use to treat immune thrombocytopenic purpura(ITP)using ITP mice model,we will gain deep insinght about the physical and chemical properties of BM-MSCs and provide theoretical basis for the clinical application of BM-MSCs used to treat ITP.Methods: The study was divided into three parts: 1.MSCs were separated from mouse boneMethods: The study was divided into three parts: 1.MSCs were separated from mouse bone marrow by the whole bone marrow adherence method and cultured to expand in vitro.Morphology was observed under inverted phase contrast microscope and flow cytometry was performed to detect cell surface markers.BM-MSCs were induced towards ostegenic and adipogenic differentiation to observe their multidirectional differentiation potentials.2.Platelets of male WTC57BL6 mice were isolated and purified.Then CD61 KO mice were injected with 1×10^8 WTC57BL6 platelets via the tail vein weekly for 3 weeks.Their serum was collected and tested weekly for the presence of anti-CD61 antibodies using flow cytometry and the platelets-immuned mice were killed by cervical dislocation and their spleens were collected.Then the SCID mice were first γ-irradiated(200 c Gy)and infused with 5 × 10^4 spleen cells intraperitoneally.Platelet counts were measured weekly in order to establish a stable ITP mouse model.3.Male SCID mice were devided into three groups:negative control group,ITP group,ITP + BM-MSCs group.ITP + BM-MSCs group were treated with BM-MSCs(1 × 106 cells/mouse)by IV injection and the other groups were given the same volume of saline infusion.The platelet counts,peripheral Treg levels and serum cytokines(IL-2,IFN-γ,IL-4,IL-10)were detected at Days7,14 and 21 after BM-MSCs injection。Results: 1.MSCs were separated from mouse bone marrow by the whole bone marrow adherence method and the scccess rate was 100%(10/10).The method to separate,culture and expand mouse BM-MSCs was successfully established.BM-MSCs were spindle-shaped or polygonal with uniform size according to the morphological observation under inverted microscope.The adherent cells displayed positive expression of CD105,CD90,and CD73,and did not express hematopoietic markers(CD34,CD45 and HLA-DR)based on flow cytomety results.Following osteogenic and adipogenic induction,cells were positive for oilred O staining and alizarin red staining.2.In CD61 KO mice,the concentration of Ig G against CD61 increased from the 14 th day to the highest level(39.14%±3.68%)after infusion of purified platelets from WT C57BL6 mice.The ITP mice model was established using the irradiated SCID mice infused with the splenocytes from platelets-immuned CD61 KO mice.The platelets in ITP mice continued to decline and decreased to the lowest level on the 7th day(401.25×109/L±65.34×109/L).We found that BM-MSCs were effective in elevating the PLT count in the ITP plus BM-MSCs group.Although the PLT count did not reach the normal range,compared with the ITP controls,a significant increase in PLT count(d4:(606.11±76.16)×109/L;d8: 611.11±67.85)×109/L;d12:(608.56±47.59)×109/L;d16:(600.44±64.68)×109/L).3.We found that BM-MSCs were effective in elevating the PLT count in the ITP plus BM-MSCs group.Although the PLT count did not reach the normal range,compared with the ITP controls,a significant increase in Tregs count was observed after the administration of BM-MSCs,no matter in peri blood(d7: 1.84%±0.07%;d14:2.46%±0.31%;d21:3.23%±0.23%)or spleen(d7: 2.49%±0.03%;d14: 2.67%±0.04%;d21:3.00%±0.02%).Compared to the normal control group,serum IL-2(42.33±5.78pg/m L,t=2.50,P<0.01)and IFN-γ(153.22±9.18pg/m L,t=2.49,P<0.01)in the ITP group were significantly higher(P<0.01),while serum IL-4(39.33±2.12 pg/m L)and IL-10(48.00±4.82 pg/m L)were considerably lower(all p<0.01)in the ITP group compared to the normal control group.After BM-MSCs treatment,serum IL-2 and IFN-γ decreased(P<0.05),whereas IL-4(42.22±2.91pg/m L,t=-2.83,P < 0.01)and IL-10 were significantly higher(55.11±6.74pg/m L,t=-2.58,P<0.01)(all P<0.05)in the ITP plus BM-MSCs group than the ITP group.Conclusions: 1.Whole bone marrow adherent culture to isolate mouse BM-MSCs is a simple,low cost,stable and efficient separation method.BM-MSCs will be worthy of further study and research.2.The successful establishment of the ITP mouse model provides a powerful tool for further studying the pathogenesis of ITP and evaluating the effect of BM-MSCs used to treat ITP.3.BM-MSCs can improve PLT levels and effectively improve the differentiation status of Treg and Th cells in ITP mice,which is a safe and effective method for the treatment of ITP.The related mechanism remains to be determined and is worthy of further study and research.