The Study On The Haematopoiesis Support And Immunosuppression Of Mesenchymal Stem Cells From The Patients With Aplastic Anemia

【Background】Mesenchymal stem cells (MSC) are the main source of the bone marrow hemopoietic inductive microenvironment (HIM). MSC can produce many hemopoietic growth factors and mediate the interactions between hemopoietic stem cell (HSC) and HIM to support hematopoiesis. The stem cell factor (SCF), one of the factors, plays an important role in the differentiation and proliferation of HSC, and represents the hemotopiesis regulation of bone marrow stromal. Some studies have also reveald that MSC possess immunosuppressive function, in which the hepatocyte growth factor (HGF) and transforming growth factor -β1 (TGF-β1) are the effective factors. Aplastic anemia(AA) is characterized by cytopenia and bone marrow failure. The abnormality of HIM, immunity and HSC are involved in the pathophysiology of AA.【Objective】The aim of the study is to compare the hemotopiesis support and immunosuppressive function of MSC from the patients with AA with those from the controls in vitro. 【Methods】Bone marrow samples were collected from the patients with AA at diagnosis[n=24, 12 from severe aplastic anemia(SAA), 12 from chronic aplastic anemia(CAA)] and the patients with idiopathic thrombocytopenic purpura (ITP), infectious mononucleosis, lymphadenitis, as the controls (n=20). MSCs from bone marrow samples were isolated, cultured and expanded. Morphology, proliferative activity and colony forming unit-fibroblast (CFU-F) were measured and analyzed. The ability of bone marrow MSC to adhere hemopoietic cells was assayed by MTT. SCF secreted from MSC was tested by enzyme linked immunosorbent assay (ELISA). Mononuclear cells (MNC) of bone marrow were plated onto a feeder layer formed by MSC. Cells count and BFU-E, CFU-GM, CFU-GMME productions were counted. MSCs were tested for the function of lymphocytes suppression by mixed lymphocyte reaction (MLR). The HGF and TGF-β1 mRNA expression was detected by RT-PCR. HGF, TGF-β1 secreted from MSC was assayed by ELISA.【Results】1. The cultured MSC from the patients with AA showed a typical fibroblast-like morphology, the same as the controls before the 3rd passage. The first and third passage time of MSC from the patients with AA were longer than that from the controls. The numbers of CFU-F from the patients with AA (15.70±5.78) were fewer than those from the controls (21.73±5.74), t=-2.94, P<0.05. 2. The adheresion rate of MSC from the patients with AA [(59.65±15.52)% ] didn't show any significant difference from that in the controls [(68.18±15.53)%], t=-1.48, P>0.05.3. The concentration of SCF in MSC supernatants from the patients with AA [(30.69±16.82)pg/ml] was significantly lower than that from the controls [(50.74±14.83) pg/ml], t=-3.28, P<0.01.4. The total MSC count and the numbers of BFU-E, CFU-GM, CFU-GMME yielded in the support of MSC from the patients with AA [(1.35±0.32)×106, (61.88±10.32), (122.88±14.76), (5.41±2.81)] were significantly lower than those in the support of MSC from the controls [(1.70±0.29)×106, (87.00±10.95), (160.00±24.02), (9.33±3.08)] respectivly, P<0.01.5. The immunosuppressive rate of MSC from the patients with AA [(49.78±20.36)% ] was significantly lower than that from the controls [(76.42±8.80) %] t=-4.56,P﹤0.01;6. The mRNA expression of TGF-β1 in MSC from the patients with AA was significantly lower than that from the controls, t=-4.14,P﹤0.01; and the mRNA expression of HGF in MSC from the patients with AA was significantly lower than that from the controls, t=-2.86,P﹤0.05;7. The concentration of TGF-β1 in MSC supernatants from the patients with AA [(415.09±108.10) pg/ml] was significantly lower than that from the controls [(630.00±115.85) pg/ml], t=2.83, P﹤0.01. The concentration of HGF in MSC supernatants from the patients with AA [(340.72±40.44) pg/ml] was significantly lower than that from the controls [(518.12±32.00)pg/ml], t=-3.02,P﹤0.01.【Conclusion】1. The hematopoiesis support of MSC from the patients with AA in vitro was significantly decreased. The mechanism may be related to the decreased proliferative capacity and the decreased SCF scretion.2. The immunosuppression of MSC from the patients with AA in vitro was significantly decreased. The mechanism may be related to the decreased proliferative capacity, decreased TGF-β1 and HGF mRNA expression in MSC and decreased TGF-β1, HGF scretion. .