Biological Characteristics Of Bone Marrow Mesenchymal Stem Cells And Expression Of P27^kip1/PTEN Protein In Patients With SLE

I Reseach backgroundSystemic lupus erythematosus (SLE), which involves multiple organs and systems, is a chronic autoimmune diseases with a diversity of clinical manifestations. At present, the recognized pathogenesis theory of SLE is that under the interaction of genetic and environmental factors, immune tolerance mediated by self-antigens is broken,resulting in abnormal activation of T and B lymphocytes and inflammatory cytokines and autoantibodies excessive production.At present, the treatment of SLE rely mainly on glucocorticoid and cytotoxic drugs, such as cyclophosphamide, newer immunosuppressive agents such as mycophenolate mofetil, anti CD20 monoclonal antibody in the treatment of patients with SLE also have certain curative effect. However, the mortality of refractory and severe SLE in 1 year and 5 years is still as high as 20% and35%, respectively. Many scholars consider that SLE is a kind of stem cells disease, not only an abnormity in hemopoietic stem cell but also in mesenchymal stem cells (BMSCs). In recent years, there is a great progress in transplantation of allograft BMSCs to treat refractory and severe SLE. In 2003, Lkehara etc reported firstly that MRL/lpr mice treated with transplantation of HSCs and BMSCs survived more than 2 years; Subsequently, it has been found that BM-MSCs and umbilical cord blood-derived MSCs from normal controls transplantation significantly decreased anti ds-DNA antibodies titers and 24 hour proteinuria and alleviated renal and lung injury in MRL/lpr mice with lupus nephritis and interstitial pneumonia. In 2009, Sun at al. reported that transplantation of allogeneic MSCs successfully treated 4 cases of the refractory SLE, which were resistance to cyclophosphamide or glucocorticoid, without any transplantation-related-complications. However, in 2010, Carrion el,t. reported that the transplantation of autogenous MSCs had little effects on 2 cases of patients. This suggests that BMSCs from SLE patients might have some defects.Mesenchymal stem cell(MSC) is a kind of non hematopoietic stem cell derived from mesoderm with highly self-renewal and expansion in vitro, multiple differentiation, weak immunogenicity, and the potential of supporting hematopoiesis, immunoregulation, inducing induction of immune-tolerance and tissue repairment and reconstruction.In recent years, the aging and immune dysfunction of MSCs have been paid more and more attention. General characteristics of aging BMSCs include thatl) Cell proliferation are slow, the number of cell colonies are decreased, with the irreversible stage G1 growth stagnation; 2) The multi-differentiation ability are markly declined; 3)The telomere is shortened with a downregulated expression of telomerase; 4) The activity of aging associated β-galactosidase is strengthened 5) Some changes occur in the ultrastructure,such as disordered F-actin, and chaotic stress fiber,and larger appearance; 6) The secretion of cytokines are abnormal, such as transfolling growth factor-β and bone morphogenetic proteins-2/4, interleukin IL-6; 7)abnormal expression of the genes regulating cell biology behavior (e.g. cell cycle, DNA damage repairment and mitosis, etc.).There are a variety of hypothesisabout the mechanism of cell senescence, the disorder of cell cycle is believed to play a key role in cell senescence. Meanwhile several studies have proved that the p53/p21Cip1, pl6INK4a/Rb, p27Kip1/pTEN proteins were involved in the program of cell aging.Based on the present researches about BMSCs on the pathogenesis of SLE patients, this paper focuses on abnormal biological behavior of the BMSCs from SLE patients and the expression of cell cycle regulatory proteinP27kipl/PTEN, to confirm that BMSCs from SLE patients may be aging. The study was divided to two parts as follows:II Chapter 1 The biological features of BMSCs from SLE patientsObjectives:By studying the differences of aging related biological behavior of BMSCs between the active stage of SLE patients and healthy controls, such as the surface antigen markers, cell proliferation, differentiation and migration, cell cycle and apoptosis. We would suggest that the BMSCs from SLE patients be a kind of aging cells, and further reveal potential pathogenesis of SLE and provide a theoretical basis for allogenic BMSCs transplantation in the treatment of SLE.Methods:Clinical materials Collection:We collect six SLE patients in active stage who received treatment in Department of Dermatology of Nanfang Hospital, Southern Medical University during January 2015 to July, all the SLE diagnosis are based on the criteria proposed in 1997, by American College of Rheumatology. The Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) published in 2012 is used to measure disease activity. Six SLE patients aged 21-27 (mean24±3years), including female (17-26years) and male (23-28years), are enrolled in this study. Eight healthy cases are used as healthy control group, and have no rheumatism, whose age distribution is similar (mean25±5 years), including female (23-33years) and male (15-28years). There is no markly difference between the age of the two groups (t=0.529, P= 0.605). All subjects give consent to the study, which is approved by the Ethics Committee of the Affiliated Nanfang Hospital of Soutnern Medical University.Islotion and cell culture:BM-MSCs are isolated and expanded from BM taken from the iliac crest of the whole SLE patients and healthy controls. The resuspended cells are layered over Ficoll solution (1.077 g/mL) by the method of density gradient centrifugation, and the characteristic of adherence. Cells are cultured in a37℃ 5% CO2 incubator, the medium is replaced and non-adherent cells are removed after 5 days and thereafter, every three days. When the BM-MSCs become nearly confluent, at the level of 80%-90%. The adherent cells are released from the dishes with 0.25 % trypsin-EDTA.After passages 4 (P4), cells are used for the following studies:indentification phenotype of BM-MSCs, such as CD29、CD44、CD 105、CD73、CD14、CD34、 CD45&HLA-DR,by low cytometry;Induction BMSCs to differentiate to osteogenic and chondrogenic; Oobservation BMSCs physiognomy; Proliferation assay of BMSCs to get the growth curve of the BMSCs; Migration assay of BMSCs; Flow cytometry analyses were performed to evaluate the cell cycle and apoptosis of BM-MSCs.Statistical analysis:The whole data are shown as the mean±standard deviation (SD) from at least three independent experiments. All statistical analyses are performed using SPSS 20.0 software; If variances is equal after levene’s test, and data are analyzed by Student’s t-test; If not, are analyzed by Mann-Whitney rank sum test; P< 0.05 is considered statistically.Result:1. Expression of surface markers are similar in MSCs from SLE and controls including D29+、CD44+、CD105+、CD73+、CD14、CD34-、CD45-�'� HLA-DR—.2. The BM-MSCs passage 4(P4) morphologically are shown in Figurel. BM-MSCs from SLE patients are larger, flattter, and have more and longer podia than the normal BM-MSCs with fibroblast-like appearance.3. Both BM-MSCs from SLE patients and normal controls could be induced to differentiate into adipocytes and osteoblasts respectively. But, the differentiation ability of BM-MSCs from SLE into adipocytes and osteoblasts are lower than controls.4. The growth curves are shown in Figure3. The curves of MSCs from SLE patients and normal subjects are " S " type. The MSCs have two days as an adaptive phase after replating, and then begin to expand rapidly and move into the phase of logarithm growth. Eight days later, MSCs counts reach the highest level, and then the cell growth is in plateau phase. On the basis of the growth curve, the population doubling time of MSCs from SLE patient is slower than normal controls.5. In Figure.4, by microscope, observing the migration distance of BM-MSCs during the 24 hours, it reveals the migration ability of the SLE is lower than that of healthy controls.7. In Figure.5, Annexin V-positive(Q2/Q4) cells in the BM-MSCs of SLE patients (17.98% ± 3.26%,16.80% ± 9.63%) show a significant increase compared to the control group (8.23% ± 3.25%,3.33% ± 2.21%). In summary, we confirm that the BM-MSCs from SLE patients are senescent and apoptotic cells, significantly(t Q2=-3.91, PQ2= 0.011; tQ4=-2.99, PQ4= 0.048).8. In Figure.6, Flow cytometry show that there are more MSCs restricted in the G1 phase harvested from the SLE patients (92.34% ± 5.80%) than in the MSCs from normal controls (78.65% ± 3.22%), with statistical significance markly (t=-3.635, P = 0.015); In the S phase,the number of BM-MSCs from SLE patients (0.86% ± 1.72%) significantly fewer than controls group (5.06% ± 1.874%). There are no significant differences (t= 3.084, P= 0.027).Conclusion:1. Expression of surface markers is similar in MSCs from SLE and controls;2. BM-MSCs from SLE patients are larger, flattter, and have more and longer podia. than the normal BM-MSCs;3. The differentiation, migration and proliferation ability of BM-MSCs from SLE are lower than that of controls.4. BM-MSCs from the SLE patients were restricted in the G1 phase, In the early and middle apoptosis stage, BM-MSCs from SLE patients were significantly increased, compared to the control group.In summary, we confirm that the BM-MSCs from SLE patients are senescent and apoptotic ones, significantly.Ⅲ Expression of P27kip1/PTEN protein in patients with SLEObjective:Our studies had confirmed that BMSCs from SLE patients were aging stem cells, but its aging mechanism is not known. The defective regulation of cell cycle progression which invovles in p53/p21Cipl, p16INK4a protein, plays a key role in aging mechanism. P27kip1 is an important negative regulator in the cell cycle progression. And the cell cycle suppressor PTEN, is an important tumor suppressor gene. In our study, we aim to investigate whether the expression of P27kipl/PTEN protein is different significantly between SLE patients and healthy controls.Materials and methods1. Collection of clinical materials, Isolation and cell cuiture BMSCs from SLE subjiects are same as the part two:the research of defective phenotype of mesenchymal stem cells in patients with systemic lupus erythematosus.2. Immunofluorescence detect the expression of P27kip1/PTEN in the BMSCs of SLE and control subjects; and then Image J software analysis cell fluorescence intensity to preliminary know about where is mainly the protein locate in the nucleus or cytoplasm.3. Western Blotting and Quantity one software test and analysis the expression of P27kip1/PTEN protein in BMSCs of SLE and healthy subjescts.Statistical analysis:The whole data are shown as the mean±standard deviation (SD) from at least three independent experiments. All statistical analyses were performed using SPSS 20.0 software; If variances is equal after levene’s test, and data are analyzed by Student’s t-test. If not, are analyzed by Mann-Whitney rank sum test; P< 0.05 is considered statistically.ResultBoth P27kip1 and PTEN proteins were found to express in the cytoplasm and nucleus of BMSCs, and the expression level in the nucleus was higher than that in the cytoplasm;In the BM-MSCs from SLE patients, the expression of p27kip1/PTEN are markedly up-regulated (p-actin is run as a reference protein). (tp27=-2.784, Pp27= 0.039; tpTEN=-4.812, Ppten= 0.041), compared with the normal controls.ConclusionThe anormal expression of P27kipl/PTEN, as the regulator of the cell cycle progression, invovles in senescent progression of BMSCs from patients with SLE.Innovation:1) This study is focus on the hot spot—cell senescence; Firstly, we reveal BMSCs of SLE patient is apoptosis stem cells, through the analysis of the biological characteristics of BMSCs from patients with SLE.2) It is the first time to analysis the expression of p27Kip1/pTEN, as the regulator of the cell cycle progression, invovles in senescent progression of BMSCs, in addition, promote to proceed of SLE.