| Part 1 Abnormal surface markers expression on bone marrow CD34~+ cells and correlation with disease activity in patients with systemic lupus erythematosusObjective: To explore the phenotypic characteristics of bone marrow (BM) CD34+ cells in patients with SLE and its relationship with SLE disease activity.Methods: 10 SLE patients and 10 healthy subjects were recruited, their BM CD34+ cells were analyzed by flow cytometric analysis with CD45/SSC gating for the expression of CD90, CD95, CD117, CD123, CD164, CD166, FAS-L and HLA-DR.Results: The percentage of BM CD34+ cells was significantly decreased in active SLE patients (1.48±0.41%, n=7) compared to healthy controls (2.31±0.75%, n=10, p＜0.01), but no significant difference was found between the inactive patients (2.04±0.44%, n=3) and the controls. The expression of CD95, CD123 and CD166 on BM CD34+ cells were significantly increased in SLE patients (48.31±10.59%, 44.9±21.5%, 30.9±19.54%, n=10) when compared with control subjects (24.33±11.1%, 19.5±4.4%, 10.7±5.5%, respectively, n=10, p＜0.05). The increased CD123 expression was negatively correlated with the number of peripheral white blood cells (r=-0.700, p＜0.05, n=10). The percentage of CD166 expression was found significantly correlated with the index of SLE disease activity (r=0.472, p＜0.05, n=10), and 24 hour proteinuria (r=0.558, p＜0.05, n=10), but negatively with serum C3 level (r=-0.712, p＜0.01, n=10).Conclusion: The expression percentage of CD95, CD123 and CD166 on BM CD34+ cells were significantly increased in patients. This supports the hypothesis that there are abnormalities of hematopoietic stem cell (HSC) in SLE. Since CD166 and CD123 correlated with the overall lupus activity, their role as a biomarker of inflammatory disease activity also requires further study. Part 2 The therapeutic effect and mechanism of human bone marrow mesenchymal stem cells transplantation in MRL/lpr miceObjective: To investigate the therapeutic effects and mechanism of human bone marrow mesenchymal stem cells(MSC) transplantation in MRL/lpr mice.Methods: Human MSCs were expanded ex vivo. MRL/lpr mice were divided into cyclophosphamide (CTX) group, MSC group, CTX+MSC group, and control group. At the age of 16 weeks, the mice in CTX group and MSC+CTX group received intraperitoneal injection of CTX 100mg/kg×2d. 24 hours after injection, the MSCs were transplanted in MSC group and CTX+MSC group through tail vein. The 24-hour proteinuria was detected with Coomassi brilliant blue method. Indirect immunofluorescence was used to detect the antinuclear antibody (ANA) and anti-double-stranded DNA (ds-DNA) antibody. Flow cytometry was used to detect the changes of CD4~+ T cells and Thl/Th2 subpopulation. The expression of BAFF mRNA was analyzed by RT-PCR. The expression of fibronectin (FN), transforming growth factor-β(TGF-β), vascular endothelial growth factor (VEGF), complement C3 of the murine kidney was estimated by immunohistochemistry and immunofluorescence.Results:①At 24, 28, 32 weeks, the 24 hours proteinuria in MSC group (4.3±0.8mg, 5.4±0.8 mg, 5.8±0.8 mg) and MSC+CTX group (2.8±1.0mg, 3.9±0.4mg, 4.5±1.0mg) were significantly decreased than in control group (9.0±1.3mg, 9.3±0.6mg, 10.0±1.6mg) (P＜0.05), and they were also significantly decreased in MSC+CTX group (2.8±1.0mg, 3.9±0.4mg, 4.5±1.0mg) than in CTX group (5.1±1.0mg, 7.7±1.1mg, 9.6±1.9mg) (P＜0.05).②At 28 weeks, the anti ds-DNA antibodies were significantly decreased in MSC+CTX group (2.7±0.8) than in control group (4.6±0.9) (P＜0.05).③At 32 weeks, the bodyweight of MSC group (38.6±3.3g) and MSC+CTX (35.8±3.7g) group increased significantly than the control group (32.8±3.0g) (P＜0.05).④At 32 weeks, serum creatinine decreased significantly in MSC group (13.5±2.2μmol/L) and MSC+CTX (12.7±4.2μmol/L) group than in control group (20.8±5.1μmol/L) (P＜0.05).⑤The number of CD4~+ T cells and Th2 subpopulation decreased significantly in MSC group (76.4%±1.9%, 58.4%±1.8%) and MSC+CTX group (73.9%±3.2%, 57.6%±3.2%) than in control group (82.7%±2.9%, 64.2%±2.8%) (P＜0.05).⑥The expression of BAFF mRaNA in spleen was significantly decreased in MSC and MSC+CTX group.⑦The expression of TGF-β, FN, VEGF and the deposition of complement C3 in renal tissue were decreased significantly in MSC and MSC+CTX treated group. Conclusion: human MSC transplantation is effective treatment for MRL/lpr mice. The regulation of Th1/Th2 balance and inhibition the antibodies of B cells, mediated by human MSCs may be the mechanisms of effective treatment with MSC transplantation. The decrement of glomerulosclerosis probably due to its suppressive effect on the expression of local TGF-β, FN, VEGF and ameliorated the renal histological injury. |
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