Molecular Genetical Analysis Of The Bone Marrow Mesenchynal Stromal Cells In Myelodysplastic Syndrome Patients And The Study On Cyclosporine's Effects On Their Proliferation And Apoptosis

Background Marrow stromal cells(MSCs) not only have the properties of common stem cells such as self-replenishing, multilineage differentiation potential and proliferation, they also play a role in immune regulation; these cells have relatively low immunogenicity because they only express a low level of HLA-1, with no HLA-2 expressed at all. Owing to these qualities, MSCs are of great research and application value in many fields of the medical science, which have already found their uses in many a department of the hospital. MSCs have become a hot spot of stem cell research.Myelodysplastic syndrome (MDS) is a group of malignant clonal diseases, its genetic instability leads to its disposition to developing into acute myeloid leukemia. The etiological mechanism of leukemia has been studied on the basis of researches in into the etiological mechanism of MDS, thus the research into MDS has become a hot spot of research into hematopathy. Allo-HSCT is the only way to cure MDS at present and it is generally believed that patients with progressive MDS, dependent on transfusion and without 5q- should undergo transplantation as soon as possible. But severe post-transplantational GVHD is still the main cause of death of MDS patients after trasplantation.The BMSCs cultured in vitro, however, after reinfusing, has the effects of promoting the recovery of marrow hematopoiesis after radiotherapy and chemotherapy, reconstructing hematopoiesis and reducing the occurrence of GVHD. The interaction between malignant clones and the microenvironment of bone marrow plays an important role in the development of hematological malignancies. However, studies on the matrix cells of marrow are rare. The etiological mechanism of MDS is not clear yet, which might involve such factors as the proliferation of hematopoietic stem and progenitor cells, hematopoiesis microenvironment, the participation of immune activities, and the methylation of genes. As an important part of hematopoiesis microenvironment, MSCs play the important role of regulating the proliferation and differentiation in hematopoiesis regulation. MSCs abnormities in MDS patients may promote the occurrence and development of MDS. MDS are a group of heterogeneous malignant clonal diseases of stem cells. It is still controversial about whether there are corresponding abnormal clonal genetic changes in MDS patients.Apoptosis, together with cell proliferation, maintains the stasis of human organism, and the inhibition of apoptosis and the disorder in genes regulating apoptosis are another important mechanism of the genesis of hematological malignancies. The various hematopathies caused by abnormal apoptosis have attracted increasing attention. The treatment of hematological malignancies can be realized through inducing apoptosis of tumor cells. As an immune inhibitor, CsA has been widely used clinically in countering rejection reaction and treating autoimmune diseases. Meanwhile CsA has also been used in treating MDS patients. It has been discovered that CsA can induce the apoptosis of HL-60 and K562 cells in recent years. It is worthwhile to study whether the clinical application of CsA affects marrow MSCs in MDS patients, though there are rare reports on the effects of CsA on the proliferation and apoptosis of MSCs in MDS patients. Objective By using cytogenetic technique and interphase fluorescence in situ hybridization (FISH) this study analyzes the number and structure of mononuclear cell (MNCs) chromosome in MDS patients and studies the biological property and karyotype of marrow MSCs in MDS patients. It also analyzes whether there exist corresponding abnormal clonal genetic changes in MSCs in MDS patients as seen in patients'mononuclear cells. It also investigates the effects of CsA on the proliferation and apoptosis of MSCs in MDS patients, which provide a basis for the MSCs treatment of MDS patients.Methods (i) Analyze the MNCs in 42 MDS patients by using cytogenetic technique and/or FISH. (ii) The culture and identification of MSCs in MDS patients. We used the adherence screening method to separate, culture, and purify MSCs, and examined their growth characteristics under microscope; we also induced MSCs to become insulin-secreting cells, and tested the immune expression type (CD34, CD45 and CD105) of the cells thus acquired. Most of the cells were determined to be MSCs. (iii) Collect the marrow sample of the 17 MDS patients who were first diagnozed to have definite clonal abnormity using cytogenetic technique and/or FISH, culture their MSCs, and analyze the MSCs using interphase FISH. (iv) Collect the marrow of 15 untreated MDS patients who were categorized into the groups of RAEB-I and RAEB-II, and collect the marrow samples of 15 patients with malnutritional anemia as control group at the same time. Culture marrow MSCs, divide the MDS patient group and control group into 3 groups respectively, namely no-drug group, 1×103ng/mlCsA group, and 1×104ng/mlCsA group. Determine the effects of CsA on the apoptosis rate, the Caspase-3 mRNA expression and the enzymatic activity of Caspase-3 of MSCs in MDS patients using flow cytometer, PT-PCR and other techniques.Results (i) Tests showed definite clonal abnormities in the MNCs of MDS patients, such as 5q-, +8, 7q- and 20q-, i(17), +15 and +22. (ii)The culture and identification of MSCs in MDS patients showed the MSCs grew in the shape of shuttle or whirlpool. After the process of insulin-producing cell induction, the MSCs showed the characteristics of insulin-producing cells. Flow cytometer surface marker tests showed CD29 and CD105, the important markers of MSCs were highly expressed, while no hematopoietic cell surface antigen such as the marker antigen of hematopoietic precursors CD34 and leukocyte marker antigen CD45 were not expressed. The ability of MSCs in MDS patients to be induced into islet-like cells was the same as that of the control group. The culture duration of primary MSCs in MDS patients of RCUD and RCMD types was similar to but a little longer than that of the healthy control group. While the culture duration of primary MSCs in MDS patients of RAEB-Ⅰand RAEB-Ⅱtypes was a little shorter than the control group, but was similar to but a little longer than that of chronic granulocytic leukaemia and AA patients. (iii) For those MDS patients whose MNCs were tested to have definite clonal abnormities, their MSCs showed no corresponding clonal abnormity. (iv) The mean absorption rate of MSCs in MDS patients was higher than that of the control group, and the absorption rates did not vary with the increase of concentration. CsA did not promote the proliferation of MSCs in both MDS patient group and control group. (v) The Caspase-3 mRNA expression, the enzymatic activity of Caspase-3 and the apoptosic rate of the MSCs in MDS patients gradually increased with the increase of concentration of CsA. And CsA did not promote the apoptosis of the MSCs in control group.Conclusion: (i) The MNCs had definite abnormal clonal genetic changes in MDS patients, such as 5q-, +8, 7q- and 20q-, i(17), +15 and +22. But tests on the MSCs of these patients did not show corresponding markers for genetic abnormity. (ii) The shape, immune expression type and ability to be induced into islet-like cells of MSCs in MDS patients were the same as that of control group. Compared with the culture duration of primary MSCs in control group, the culture duration of primary MDCs in patients of different types of MDS were different. (iii) The proliferation activity of MSCs in MDS patients was significantly higher than that of the control group. CsA did not have the effect of promoting the proliferation of MSCs in both patient and control groups. (iv) CsA did not promote the apoptosis of the MSCs in control group. But it promoted the apoptosis of MSCs in MDS patients through improving The Caspase-3 mRNA expression and the enzymatic activity of Caspase-3 of MSCs in MDS patients.