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Objective:Preliminary study on therapeutic effects of adipose tissue derived stem cells (ADSCs) on MRL/lpr mice and the effect on imbalance of Th17/Treg.Methods:Fifteen 12-week-old MRL/lpr mice were randomly divided into 3 groups by using random number table,including ADSCs group,control group and cyclophosphamide (CTX) group,with 5 in each group.ADSCs group and control group were injected with 1 × 106ADSCs or phosphate buffered solution (PBS) via tail vein respectively,once a week,a total of eight times.CTX group was injected CTX at a dose of 15 mg/kg body weight,once a week for 2 weeks,and then repeated after 2 weeks' rest,a total of four times.The 24-hour proteinuria was measured before and after treatment.All the mice were sacrificed after treatment for 8 weeks.Th17 cells and Treg cells in splenic were examined by flow cytometry.Results:(1) The 24-hour proteinuria in the three groups had no significant difference before treatment (P ＞ 0.05).After therapy for 4 weeks,the 24-hour proteinuria in the ADSCs and CTX groups was much lower than those in control group,and the difference was significant [(5.02 ± 1.61) g/L vs.(7.10 ± 1.63) g/L,(4.90 ±0.71) g/L vs.(7.10 ± 1.63) g/L,P ＜ 0.05],and the longer the duration of treatment (8 weeks),the more obvious effect [(2.24 ± 0.73) g/L vs.(10.36 ± 1.64) g/L,(3.80 ± 1.45) g/L vs.(10.36 ± 1.64) g/L,P ＜0.01].There was no significant difference in 24-hour proteinuria between ADSCs group and CTX group (P ＞ 0.05).(2) Percentage of Treg cells/CD4 + T cells in the spleen lymphocytes:The percentages in ADSCs and CTX groups were higher than that in control group.The levels were 13.62％ ± 1.87％,14.14％ ± 1.29％,10.71％ ± 1.23％,respectively,but there was no significant difference (P ＞ 0.05).(3) Percentage of Th17 cells/CD4 +T cells in the spleen lymphocytes:The percentages in ADSCs and CTX groups were significantly lower than that in control group.The levels were 1.43％ ± 0.20％,1.63％ ± 0.65％,6.37％ ± 1.64％,respectively,with statistical significance (P ＜ 0.01).Conclusion:Transplantation of ADSCs can reduce the 24-hour proteinuria in MRL/lpr mice.To prolong the time of treatment,the effect is more significant.Transplantation of ADSCs can up-regulate Treg cells and down-regulate Th17 cells.ADSCs have the ability to regulate the immune balance of Th17/Treg in MRL/lpr mice,suggesting that ADSCs play the role of anti-inflammatory and immune regulation by regulating the Treg and Th17 cells.
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BACKGROUND:The non-specific immune suppression method is generaly used for treatment of systemic lupus erythematosus, but poor prognosis, such as infection and high recurrence rate, exists. OBJECTIVE:To evaluate the therapeutic effect of bone marrow mesenchymal stem cel transplantation on systemic lupus erythematosus in mice. METHODS:Sixteen mice with systemic lupus erythematosus were equivalently randomized into control and experimental groups, or then subjected to passage 3 bone marrow mesenchymal stem cel transplantation or the equal volume of normal saline via the tail vein, respectively. Mouse urine samples were colected to detect urine protein levels by Bradford method. Blood samples from the tip of the mouse tail were extracted to detect serum anti-ds-DNS antibody concentration by radioimmunoassay. Mouse kidney tissues were taken and observed pathohistologicaly through hematoxylin-eosin staining and immunohistochemistry staining under microscope. Flow cytometry was used to detect the expression of CD4+CD25+T cels in the inner canthus blood, fresh spleen and thymus. RESULTS AND CONCLUSION:Within 10 weeks after cel transplantation, the urine protein levels in the two groups were gradualy increased, and the rising velocity was higher in the control group than in the experimental group. From the 4th to 10th week, the urine protein levels in the experimental group were significantly lower than those in the control group (P 0.05). The serum anti-ds-DNA antibody concentration in the experimental group was significantly lower than that in the control group (P < 0.05). Taken together, bone marrow mesenchymal stem cel transplantation can improve the pathological damage in systemic lupus erythematosus mice, and has a certain therapeutic effect on systemic lupus erythematosus.
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Objective:To observe the effect of CD40 siRNA on expression of IFN-γ,IL-17,IL-4 and anti-dsDNA antibody of systemic lupus erythematosus (SLE)animal model MRL/Lpr mice and to discuss its therapy on MRL/Lpr mice.Methods:In the study,16 female MRL/Lpr mice were randomly divided into control group (n =4),empty vector group (n =4),CD40-siRNA1 group (n =4)and CD40-siR-NA2 group (n =4).The vectors expressing siRNA against CD40 were injected by tail veil into MRL/Lpr mice,while MRL/Lpr mice in control group and empty vector group were injected with the same dose of PBS and pGFP-V-RS vector respectively.The injection was given six times and every one day.The mice were sacrificed 14 d after injection,and the spleen tissue was weighed.The pGFP-V-RS was labeled by green fluorescent protein(GFP)and the tissue sections were observed whether siRNA expressed in the spleen.The expression levels of IFN-γ,IL-17,IL-4 and anti-dsDNA antibody in the sera were detected by ELISA method on the 1st day before the first time and the 2nd,5th,8th,11th,and 14th days after last injection,and the expression levels of CD40 mRNA in spleen tissue of MRL/Lpr mice were detected by RT-PCR and the expression levels of CD40 protein in spleen tissue of MRL/Lpr mice were detected by immunohistochemistry method.Results:The expression vector of CD40-siRNA could express in the spleen of MRL/Lpr.The spleens in CD40-siRNA1 group [(78.85 ±5.61 )mg]and CD40-siRNA2 group [(80.25 ±4.07)mg]were lower than those in control [(141.88 ±7.81)mg]and empty vector group [(153.10 ±7.60)mg].The levels of IL-17,IFN-γand anti-dsDNA antibody were lower and the levels of IL-4 was higher in CD40-siRNA1 group and CD40-siRNA2 group on the 2nd,5th and 8th days after last injection than on the 1st day before the first time (P 0.05). Though the levels of anti-dsDNA antibody in CD40-siRNA1 group and CD40-siRNA2 group on the 11th day was higher than on the 8th day,there was more significance than those in control group and empty vector group (P <0.05).There was no significance between the 4 groups on the 14th day.The levels of CD40 mRNA and protein were lower in CD40-siRNA1 group and CD40-siRNA2 group than in control group and empty vector group on the 14th day after last injection (P <0.05).Conclusion:CD-40 si-RNA can reduce the concentration of IL-17,IFN-γand of anti-dsDNA antibody in serum,and at the same time,it can elevate the concentration of IL-4 and suppress CD40 mRNA and protein of spleen in MRL/Lpr.Meanwhile after suppressing CD40 mRNA and protein,it can reduce inflammatory response of the mice and the disease activity of MRL/Lpr,suggesting that CD-40 siRNA has therapy effect on SLE.
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Objective To investigate the effects of artesunate (ART) on interstitial pneumonia and sialadenitis in MRL/lpr mice.Methods A total of 18 MRL/lpr mice were randomly allocated to a hydroxychloroquine sulfate (HCQ) group,a ART group and a control group.At the age of 18 weeks,the mice in the HCQ group and ART group were given HCQ 150 mg/kg daily and ART 50 mg/kg daily for 12 weeks,respectively.The histopathological changes of pneumonitis and submaxillaritis were assessed by hematoxylin and eosin staining.The levels of monocyte chemoattractant protein-1 (MCP-1) in the serum and urine were measured by the enzyme-linked immunosorbent assay.Results At the age of 30 weeks,the index of peribronchiolar lesion (1.62 ± 0.19,1.52 ± 0.30 vs.1.95 ± 0.34;all P＜0.05),the index of perivascular lesion (1.23 ± 0.18,1.28 ± 0.12 vs.1.57 ± 0.33;all P＜0.05),the alveolar lesions index (1.35 ± 0.16,1.05 ± 0.15 vs.1.72 ± 0.34;all P＜0.05) and the submaxillaritis index (1.48 ± 0.22,1.43 ± 0.15 vs.1.84 ± 0.34;all P＜0.05) in the HCQ group and the ART group were significantly decreased than those in the control group.The MCP-1 levels in the serum (1 103.02 ± 185.56 pg/ml,1 072.37 ± 242.43 pg/ml vs.1 490.67 ± 329.43 pg/ml;all P＜0.05) and urine (189.16 ± 70.85 pg/ml,198.79 ± 113.47 pg/ml vs.446.79 ± 192.31 pg/ml;all P＜0.05) in the HCQ group and the ART group were significantly lower than those in the control group.Conclusion ART can decrease the MCP-1 level,and ameliorate interstitial pneumonitis and sialadenitis in MRL/lpr mice.
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Objective To detect the expression of Blimp1 gene in MRL/lpr mice,so as to provide new ideas for plasma cell-targeting therapy of systemic lupus erythematosus (SLE).Methods Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation analysis (ChIP) were performed to evaluate the regulatory effect of Blimp1 on B cell maturation antigen (BCMA),and fluorescence-based quantitative PCR was carried out to detect the expression of Blimp1 mRNA in spleen and lymph node tissue of MRL/lpr mice and normal control mice.The intergroup difference in Blimp l mRNA expression was assessed by rank sum test.Results Blimp1 could bind to the BCMA gene promoter.Increased Blimp1 mRNA expression was observed in both the spleen and lymph node tissue of MRL/lpr mice compared with the normal control mice (Z =2.609,3.402,respectively,both P ＜ 0.01).Conclusions BCMA appears to be the target gene of Blimp1,and Blimp1 may plays a certain role in the maintenance of plasma cell survival and antibody production via directly regulating BCMA gene expression.
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Objective To investigate the efficacy of umbilical cord mesenchymal stem cells (UC-MSCs) transplantation in the treatment of the MRL/lpr mice. Methods Twenty four 18-week-old MRL/lpr female mice were divided into 3 groups:group 1 (G1) were transplanted with 1×106 UC- MSCs through caudal vein, group 2 (G2) were transplanted with 1×106 UC- MSCs three times and group 3 (G3) were treated with 0.5 ml normal saline as controls. Enzyme linked immunosorbent assay (ELISA) was used to measure the levels of serum anti-dsDNA antibodies. Twenty-four hours proteinuria and body weight were assessed every two weeks. The histopathology changes of the kidneys and lungs were observed. Results ① At the 25th weeks, the 24 hours proteinuria in group G1 (2.3±1.9) mg and G2 (1.8±1.4) mg was decreased than that in the control group (3.8±2.1) mg (P<0.05), and at the 27th weeks, that of groups G1 (2.5±1.5) mg and G2 (1.9±1.2) mg was also significantly decreased than in the control group (5.4±2.4) mg (P<0.01); ② From the 24th week, the body weight of groups G1 and G2 increased significantly than that of the control group (P< 0.05). At week 29, serum creatinine decreased significantly in both groups G1 (7.2±3.2) μmol/L and G2 (6.2±2.8) μmol/L than in the control group (12.5±2.3 ) μmol/L (P<0.05); ③One week after transplantation, the levels of anti-dsDNA antibodies in group G1 (46±11)×102 U/ml and G2(49×43)×102 U/ml were bothsignificantly decreased than those of the control groups (99±42)×102 U/ml (P<0.05) and the difference between group G2 (36±15)×102 U/ml and the controls (68±32)×102 U/ml was statistically significant; ④The nephron crescent formation in group G1 (0.12±0.07) and G2 (0.08±0.02) was significantly lower that of the control group (0.20±0.06) (P<0.05) and that of group G2 was significantly less that of froup G1 (P<0.05); ⑤ The interstitial pneumonitis was singnificantly milder in group G1 than group G2. Conclusions UC- MSCs is very effective in treating MRL/lpr mice. It is safe and free of rejection reactions.
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Objective To investigate the expression levels of DNA methyltransferases 1, 3A and 3B in CD4+ T cells of MRL/lpr mice, and explore their relationship with the expression levels of methylation-sensitive genes (ITGAL, CD70). Methods CD4+ T ceils were isolated from spleens of 16-week-old MRL/lpr and BALB/c control mice by anti-CIM antibody labeled magnetic beads. Transcription levels of DNA methyhransferases 1, 3A and 3B and methylation-sensitive genes(ITGAL, CD70) were measured by real-time mice when compared with BALB/c control mice and the difference was significant (P<0.05), while the expression of DNMTI and DNMT3A showed a tendency of decrease (P>0.05). The mRNA expression of CD70 was significantly higher (P<0.01), but the expression of ITGAL had no significant difference between the two P<0.01 ). Conclusion Decreased expression of DNMT3B may attribute to the elevated expression of methylation-seusitive gene CD70, thus lead to the dysfunction of CD4+ T cell and play an important role in the pathogenesis of SLE.
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Objective CD4+ T lymphocytes have been shown to play an important role in the pathogenesis of systemic lupus erythematosus (SLE). To identify the commonly accumulated T cell clones and to investigate its role in murine lupus models, it was analyzed that the T cell clonality infiltrating in different tissues derived from (NZB?NZW)F1 as well as MRL/lpr mice. Methods The expressions of T cell receptor (TCR) V? gene were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) combined with single-strand conformation polymorphism (SSCP) study. The phenotype of T cells expanded in different organs was determined by magnetic cell sorting (MACS). Results RT-PCR and SSCP study of TCR V? chain demonstrated that there were some identical T cell clonotypes expanded and accumulated in different organs in aged diseased mice. Most of these identical clonotypes were CD4+ T cells in both of the two strains. In contrast, young mice exhibited little accumulation of common clone in different organs. The TCR V? usage of these identical clonotypes was limited in V?2, V?6, V?8.1, V?10, V?16, V?18 in MRL/lpr mice and V?6, V?7 in (NZB?NZW)F1 mice respectively. Conclusion The results suggest that activated and clonally expanded CD4+ T cells commonly accumulated in different tissues in aged murine lupus models. These CD4+ T cell clonotypes may be involved in specific immune responses of SLE, thus playing a pathogenic role in these lupus mice.
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Objective There were some identical T cell clonotypes expanded and accumulated in different organs in aged diseased (NZB?NZW)F1 and MRL/lpr mice. The amino acid motifs in the third complementarity determining region (CDR3) from the T cell receptor (TCR) V? chain was analyzed to demonstrate the antigenic specificity of these identical clones. Methods The TCR V?6 gene in kidneys and brains from different individuals of aged diseased lupus mice were amplified by reverse transcriptase polymerase chain reaction (RT-PCR) respectively. The amino acid motifs in CDR3 loops of TCR V?6 chain were demonstrated by DNA cloning and DNA sequence analysis. The amino acid motifs from the identical T cell clones accumulated in different organs were identified by single-strand conformation polymorphism (SSCP). Results Some conserved amino acid motifs such as isoleucine (I) and aspartic acid (D) were observed in CDR3 loops of TCR V?6 from these identical T cell clones in different individuals of aged diseased (NZB?NZW)F1 mice. Likewise, I, glycine (G) and D or glutamic acid (E) were also found in MRL/lpr mice. Conclusion These identical T cell clones may recognize restricted T cell epitopes on autoantigens and are involved in specific immune responses in SLE.
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Objective To explore the biological characteristics and to compare the different regulation mechanisms of bone marrow mesenchymal stem cells(MSCs)derived from both lupus(MRL/Ipr)and normal (C57BL/6)mice on T iymphoeytes in vitro. Methods MSCs from MRL/lpr and C57BL/6 mice bone marrow were isolated and cultured. The surface phenotypes were detected by flow cytometry(FCM). The morphologi- cal changes of MSCs were evaluated in primary and passage cultures. The growth curves were assayed. CD3~+T lymphocytes from spleen of C57BL/6 mice were isolated by nylon wool columns and stimulated by ConA and co-cultured with or without the two strains of MSCs or supernatant for 72 h. The proliferation of T cells stained by CFSE and activation of T cells were detected. The apoptosis was assessed by now cytometry using Annexin V/PI. Results The growth of lupus MSCs was faster than normal controls(P