Effects Of Rapamycin On Biological Characteristics Of Bone Marrow Mesenchymal Stem Cells Derived From Patients With Aplastic Anemia

Background:Bone Marrow Mesenchymal stem cells(BMSCs) are multipotent stem cells with the capacity for self-renew and multi-lineage differentiation. They can be typically differentiated into a variety of mesenchymal cells, including osteoblasts, adipocytes, chondrocytes and neural cells. As an important part of the body,bone marrow microenvironment, Mesenchymal stem cells not only differentiate into osteoblasts,fat cells within the bone, support hematopoiesis,but also have close connections with endothelial cells,macrophages,reticular cells and stromal cells, which will no doubt play an important role in bone marrow hematopoietic cell niches and adipocytes differentiation balance. SAA is a kind of immune disorder characterised by decreased red bone marrow capacity substituted by the fatty marrow and resulting in the hematopoietic microenvironment abnormalities. Thus the evaluation of the influence of the SAA therapeutic drugs on the bone marrow mesenchymal stem cells is considered to be especially important. Rapamycin is a novel macrolide immunosuppressive agent which supresses the immune activity by blocking the G1to S cell cycle phase process of T lymphocytes. In the clinical application, the rapamycin is used as a treatment for organ transplantation rejection reaction and autoimmune diseases. Its immunosuppressive activity is ten times more than cyclosporin with low toxicity, and it also has great synergistic effect with cyclosporin. So an assumption is proposed that when the rapamycin is added into the thrapeutic regime, it may improve the treatment of SAA combined with cyclosporin by supressing the T cells activity. However, there are also reports on the faliure of the elevation on the survival rate in newly dignosed SAA patients by rapamycin, but it still takes effect in some refractory and relapsed patients. In a word, it still cannot reach an agreement on whether rapamycin would be applied in clinical treatment for severe aplastic anemia.Objective:At present,treatment strategies for the Refractory and relapse of SAA remain to be current top issues in the field of bone marrow failure.Studies have shown that abnormal mTOR signal activation occurs among refractory and replapsed SAA patients,in addition,rapamycin is not only a new macrolide immunosuppressant,but also a kind of mTOR inhibitors. Effects of rapamycin on biological characteristics of bone marrow mesenchymal stem cells derived from patients with Refractory and relapse of SAA were investigated in our study, so as to provide experimental basis for the clinical treatment of severe aplastic anemia with rapamycin. This study aimed at observing roles of rapamycin in cell apoptosis, proliferation,cell cycles,and adipogenesis, further explore whether these phenomena may be caused by autophagy activation and mTOR inhibition.Methods:(1) SAA-MSCs were induced for adipocytes in the specific-induction medium. Oil red O staining and Alizarin red staining were performed to examine lipid droplet and mineralization.(2) Cells were treated with different concentrations of rapamycin(0,10,50,100nmol/L) for48h in the following.Cell apoptosis of SAA-MSCs cells which induced by rapamycin for48h was tested by the Annexin V/PI apoptosis detection kit In addition,related genes Bcl-2and Bax were tested by real-time PCR (3) The cell cycle was analyzed by PI staining in a flow cytometer. cell cycle related genes p21was tested by real-time PCR.(4)The proliferation of SAA-MSC stimulated with rapamycin was detected by cell counting kit-8.(5) SAA-MSC and human peripheral blood mononuclear cells (hPBMCs) were cocultured in vitro,IFN-y secretion was detected by ELISA kits to assess the effect of rapamycin on the immunosuppressive potential of SAA-MSC.(6) Oil red O were stained after AA-MSCs induced with adipogenesis media, in addition,the related genes for adipocytes (LPL,CFD,PPARy) were tested by real-time PCR.(7)Transmission electron microscopy(TEM) and western blot were used to identify the cell autophagy and the expression of LC3B.(8)After treated with different concentrations of rapamycin (0,10,50,100nmol/L) for48h, the expression of mTOR was detected by Western blot and real-time PCR was used to identify the expression of4EBP1and p70S6K.Results:(1) SAA-MSC possess multi-lineage differentiation potential.(2) rapamycin induced the apoptosis of SAA-MSCs in time and dosage-dependent manner.(3) Cell cycle was arrested in G0/G1after pretreated with rapamycin.(4) Proliferation of SAA-MSCs were inhibited by rapamycin.(5)After SAA-MSC and human peripheral blood mononuclear cells (hPBMCs) were cocultured in vitro,Rapamycin did not affect the immunosuppressive potential of SAA-MSC on IFN-γ production inhibition.(6) Adipogenesis of SAA-MSCs were inhibited by rapamycin in a dose-dependent way.(7) Transmission electron microscopy(TEM) and western blot identified the cell autophagy and the expression of LC3B.(8)After treated with different concentrations of rapamycin (0,10,50,100nmol/L) for48h, the expression of mTOR、4EBP1and p70S6K were inhibited accordingly.Conclusions:Rapamycin played an critical role in cell proliferation,cell cycles,and adipogenesis, these may be caused by autophagy activation and mTOR inhibition.