| Objective To investigate the function of genes involved in OAZ signaling pathway in the patients with systemic lupus erythematosus (SLE)Methods Part one:The expression levels of OAZ, BMP6, BMP4, Id3, Smad6, EHZF,LY6E genes were valuated in bone marrow progenitor cells of 5 SLE patients and 5 normal subjects and replicated in peripheral blood cells of 30 SLE patients and 20 normal individuals by real-time quantitative PCR technique. Relationships of the expression levels of OAZ, BMP6, BMP4, Id3,LY6E mRNA with disease activity and other clinical index were analyzed.Part two:Peripheral blood mononuclear cells (PBMC) from SLE patients were collected. Each sample was equally divided into four groups for the cell culture in 96 well plates. Specific siRNA for OAZ and GAPDH were concordantly added to experimental group and positive control group, while nonspecific siRNA was added to negative control group and only culture medium was added to Mock control group. Cells and supernatant were harvested after culturing for 72 hours, then RNA was extracted and reverse transcripted to cDNA. OAZ, Id1, Id2, Id3, Id4, LY6E mRNA expression levels were analyzed by real-time PCR. Levels of IFN-y, IL4, IL10, IL12, IL21, CCL2, ANA in supernatant were tested by ELISA. Relationships of the expression levels of OAZ mRNA with levels of cytokines and ANA were analyzed.Part three:Human MSCs isolating and expanding from bone marrow cells or umbilical cord of healthy donors were infused into SLE patients. Peripheral blood cells were collected from 10 patients pre-MSCT as well as 1 week and 4 week post-MSCT, and RNA was extracted and reverse transcripted to cDNA. mRNA expression levels of OAZ and Idl-3 were measured by real-time PCR. Serum levels of IL10, IL12, IL21, CCL2 and ANA were tested by ELISA. Relationships of the gene expression levels with the levels of cytokines and ANA were analyzed.Results Part one:The expression levels of OAZ, Id3, LY6E mRNA(ACt) in the bone marrow (10.6±0.5,5.8±3.2) and peripheral blood (14.1±2.7,7.5±1.8) of SLE patients were significantly increased than those observed in normal controls (16.5±0.9,10.4±2.6, 16.1±2.2,9.5±1.7), which was found to negatively correlate with SLEDAI score, renal lesion index, titers of anti-dsDNA and anti-RNA antibodies, but positively correlate with serum complement C3. Expression levels of BMP4 and BMP6 were differentially expressed in peripheral blood cells but not bone marrow progenitor cells.Part two:OAZ, Id1, Id2, Id3, LY6E gene mRNA expression levels (△Ct:12.5±1.4, 8.9±1.5,4.3±0.8,8.0±1.1) in experimental group were significantly decreased comparing to those in negative control group (△Ct:10.2±1.1,6.5±1.2,2.4±1.3,6.2±1.2 respectively, p< 0.05). Levels of IFN-y, IL10, IL12, IL21 and ANA in experimental group were significantly lower than those in negative control group (p< 0.05); while level of CCL2 was higher than the negative control group (p<0.05). Difference of OAZ mRNA expression levels (△△Ct) between experimental group and negative control group were negatively correlated with changes of ANA, IL21 levels, but positively correlated with changes of Thl/Th2, CCL2.Part three:mRNA expression levels of OAZ, Idl and Id3 gene in patients with SLE were significantly decreased 1 week (△Ct:12.4±1.1,9.7±1.9,9.7±1.9,2.1±1.0) and 4week (△Ct: 13.3±1.2,10.4±1.5,10.8±1.2,2.1±1.2) after MSCT comparing to those pre-MSCT (△Ct: 11.0±0.9,7.4±2.1,7.8±2.1,0.1±1.5 respectively, p all< 0.05). Levels of IL10, IL21 and ANA were significantly lower 4 week post-MSCT than those before (p< 0.05); while level of CCL2 was higher than pre-MSCT (p<0.05). Cytokines and ANA levels 1 week after MSCT were not differentially changed comparing to those pre-MSCT. Alteration of OAZ mRNA expression levels pre-and post-MSCT were negatively correlated with changes of ANA, IL21 levels and positively correlated with changes of IL12/IL10 and CCL2 levels.Conclusion 1. OAZ pathway is involved in the pathogenesis of SLE.2:OAZ siRNA can effectively reduce the expression of genes involving in the OAZ signaling pathway in SLE. OAZ may lead to abnormal production of ANA via regulating Id genes and cytokines.3:The expression of genes involving in the OAZ signaling pathway was effectively reduced along with the alteration of several cytokines and ANA after allogeneic MSCT in SLE patients. OAZ signaling pathway could play an important role in MSCT treatment for SLE.
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