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Objective To investigate the mechanism of tumor necrosis factor-α (TNF)-α inhibiting osteo blastdifferentiation of mesenchymal stem cells (BMMSCs) in the pathogenesis of osteoporosis in the mouse model of systemic lupus erythematosus (MRL/lpr). Methods The femurs of MRL / lpr and C3He/HeJ mice were isolated, the bone structure were examined by hematoxylin-eosin (HE) staining. The proteins of TNF-α, NF-κB P50, bone morphogenetic protein -2 (BMP-2) and PSmad1/5/8 were measured by immunohistochemical stain. Bone marrow mesenchymal stem cells (BMMSCs) were isolated. After BMMSCs grew on the cover slips, the proteins on top of it were evaluated by immunohistochemistry stain. Moreover, the alkaline phosphatase (ALP) staining was employed for the measurement of the early osteogenic differentiation. BMMSCs together with hydroxyapatite were embedded subcutaneously in the nude mice and eight weeks later, the ectopic bone formation was evaluated. The recombinant human tumor necrosis factor receptor type Ⅱantibody fusion protein (etanercept) or normal saline was subcutaneous injected to the mice with lupus. After four weeks, the expression of these proteins was observed and the ectopic bone formation was investigated. Image-Pro plus 6.0 software was employed for imagine analysis, and Studentˊs t-test was used to test the differences between 2 independent groups. Results MRL/lpr mice showed decreased volume of cortex and the percentage of cortex to the volume of bone of MRL/lpr mice was significantly lower compared to control groups and with C3He/HeJ mice (13.96±0.25 vs 23.61±0.71, n=3, P0.05). The expressions of TNF-α and NF-κB P50 in BMMSCs of MRL/lprl mice were higher than those of the C3He/HeJ (0.184±0.021 vs 0.136±0.013, 0.132±0.021 vs 0.097± 0.014, n=3, P0.05). The ectopic bone formation of BMMSCs of the etanercept injected MRL/lpr mice was higher than that of the normal saline injected mice, however, it was lower than that of the C3He/HeJ mice. Conclusion TNF-α inhibits osteoblast differentiation of mesenchymal stem cells by depressing Smad signaling which may contribute to the osteoporosis of the lupus mice.
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BACKGROUND:Systemic lupus erythematosus (SLE) is classified into four types, and the major treatment is to tonify kidney and nourishyin, clear blood stasis and toxin by the traditional Chinese medicine (TCM). Even though, there are stil many patients with poor efficacy. Mesenchymal stem cels have the capacity of multiple differentiation, hematopoietic support and immune regulation, thus having been used for the treatment of refractory, recurrent SLE and achieving good effects. OBJECTIVE:To investigate the therapeutic effect of umbilical cord-derived mesenchymal stem cel transplantation on SLE patients with different patterns of syndromes. METHODS: Twenty-one SLE patients were clustered to four syndrome types of TCM, including heat-toxin,yin deficiency of liver and kidney,yang deficiency of spleen and kidney, andqi stagnation and blood stasis. The changes in clinical and laboratory indicators were analyzed statisticaly before and after cel transplantation. RESULTS AND CONCLUSION:The level of 24-hour proteinuria and SLE disease activity index scores in SLE patients were significantly decreased at 1, 3, 6 months after cel transplantation (P < 0.01). Umbilical cord-derived mesenchymal stem cel transplantation could significantly reduce the 24-hour proteinuria in SLE patients withyin deficiency of liver and kidney at 1, 3 and 6 months (P < 0.01), while slightly reduce the 24-hour proteinuria in SLE patients with heat-toxin andqi stagnation and blood stasis at 1, 3 months (P < 0.05) as wel as in SLE patients withyang deficiency of spleen and kidney at 1 month (P < 0.05). Additionaly, umbilical cord-derived mesenchymal stem cel transplantation could increase the serum albumin levels in al the SLE patients (P < 0.01), although the changes in patients with heat-toxin were moderate (P < 0.05). Al the SLE patients of four types had an increasing trend of their platelet counting after cel transplantation, but there was no statistical difference before and after cel transplantation. Taken together, umbilical cord-derived mesenchymal stem cel transplantation is effective for treatment of SLE, but has different therapeutic efficacy on SLE patients with different syndrome types of TCM.
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Objective To investigate the change and significance of T follicular helper cells(Tfh) in the peripheral blood of patients with systemic lupus erythematosus (SLE).Methods The percentage of CD4+ CXCR5+ICOS+ Tfh cells and the expression of activation marker CD69 on the Tfh cells of peripheral blood from 60 SLE patients (30 in active and 30 in inactive) and 30 healthy subjects (control group) were determined by flow cytometry.The correlations between the percentage of Tfh cells of SLE patients and SLE disease activity index (SLEDAI),anti-nuclear antibody (ANA) titers and the levels of complement 3 (C3),complement 4 (C4) were analyzed.ANOVA and Pearson's correlation analysis were used for statistical analysis.Results The percentage of the Tfh cells in the peripheral blood of active SLE patients was higher than inactive patients and healthy controls [(0.80±0.17)％ vs (0.63±0.13)％ vs (0.57±0.08)％; P＜0.01],there was also statistical significance between inactive patients and healthy controls (P＜0.05).The expression of CD69 on the Tfh cells in the peripheral blood of active SLE patients was higher than that in inactive and healthy controls [(7.3±1.6)％ vs (5.9±1.3)％ vs (5.2±0.9)％; P＜0.01].There was no statistical significant difference between inactive patients and healthy controls (P＞0.05).The percentage of Tfh cells of SLE patients was significantly related with SLEDAI (r=0.680,P＜0.01) and C3 levels (r=-0.416,P＜0.05),butthere was no correlation between that and the ANA titer,C4 levels (r=-0.042,-0.204,P＞0.05).Conclusion Increased percentage and activity of the Tfh cells in the peripheral blood of patients might contribute to the pathogenesis and development of SLE.
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ObjectiveTo investigate the profile of Th17/Treg balance in the peripheral blood of patients with systemic lupus erythematosus (SLE).MethodsThirty-two SLE patients in active disease were selected and 30 SLE patients in remission were included in this study.The control group was consisted of 25healthy individuals.The expressions of IL-17 and FoxP3 on CD4+ T cells in the peripheral blood were assessed by flow cytometry and the mRNA levels of these two cytokines were examined by quantitative PCR respectively.ANOVA was used for statistical analysis.ResultsThe percentage of CD4+IL-17+ T cells and IL-17mRNA expression of the active group were significantly higher than those of the remission group and control group[(l.0l±0.22)％,(2.04±0.63)vs (0.48±0.16)％,(1.12±0.34) vs (0.41±0.12)％,1; P＜0.01].There was no difference between the remission group and control group(P＞0.05).However,the percentage of CI4+FoxP3 + T cells and FoxP3 mRNA expression of the active group were significantly lower than those of the remission group and control group [(2.36±t0.54)％,(0.42±0.16) vs (4.34±0.95)％,(0.87±t0.28) vs (5.09±11.17)％,1; P＜0.01 ],and there was also significant differencesbetween the remission group and control group(P＜0.05).ConclusionThl7/Treg balance shift may exist in the peripheral blood of patients with SLE and the degree of imbalance may be related to disease activity of SLE.
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Objective To summarize the experience of establishing the stable rat model of chronic allograft nephropathy. Methods We used Fisher rats as donors and Lewis rats as recipients.After the left kidney of the donor perfused in situ under hypothermic condition, the left renal vein,abdominal aorta and bladder flap of the donor was anastomosed with the left renal vein, renal artery and bladder of the recipient, respectively. The recipients were given cyclosporin oral solution 10 mg/kg every day by gavage for 10 days after transplantation. The blood and urine samples were collected 1 month, 2 months and 4 months after transplantation and renal function and total urine protein were examined. The pathological changes of the renal allograft were observed 2 and 4 months after transplantation. Results Forty-five rats received operation and achievement ratio was 85%. The renal transplantations were finished in 120 ± 20 min. The Scr, BUN, Cycs and total urine protein demonstrated a significant increase one month after transplantation. On the second and fourth month,with the exception of urine protein continued to increase, the other indicators did not change significantly. Two months after transplantation renal pathology demonstrated light to moderate interstitial fibrosis, infiltration of lymphocytes and plasma cells. At 4th month the renal allografts showed extensive interstitial fibrosis, a large number of infiltrating interstitial cells, thickening,hardening, occlusion of glomerular basement membrane, and renal tubular atrophy that were consistent with pathological changes of chronic allograft nephropathy. Conclusion Through adequate surgical training and improvement, and specification for rat nephrectomy, transplantation surgery,and postoperative management in every detail, the model with high success rate and stability can be achieved.
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Objective To study the expression of IL-17, RORγt in CD4+T cells from patients with ankylosing spondylitis (AS). Methods The specimens of venous blood PBMC were collected from 28 patients with AS and 15 healthy subjects. Intracellular flow cytometry detection of IL-17 was established after isolation of human CD4+ T cells from PBMC. The expression level of IL-17, RORγt mRNA in CD4+ T cells was determined from 28 AS patients and 15 healthy controls by real-time fluorescence quantitative RT-PCR using Anti -CD3/Anti -CD28 as stimulators or not. Analysis of variance and Pearson correlation were selected. Results The isolation of human CD4+ T cells from PBMC was effective and its purity reached 90%. The percentage of intracellular IL-17 in CD4+ T cells from AS pati-ents in the AS active group was higher than that of the AS stable group and healthy control group (P<0.01). The expression level of IL-17, RORγt mRNA in CD4+ T cells was significantly higher in patients with AS than in controls. After stimulated with anti-CD3/ anti-CD28 stimulation, the percentage of IL-17, RORγt mRNA was increased significantly (P<0.01). The percentage of IL-17, RORγt mRNA in the 12 h group was higher than that of the 24 h group, while both of them were higher than those without stimulation (P<0.05). Conclusion There is an abnormal expression of IL-17, RORγt in human CD4+ T cells from AS patients. Our results indicate that the abnormal expression of IL-17 might play a role in the development and progression of AS.
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Objective To investigate the immunoregulatory effect of thalidomide on the peripheral blood T-iymphocytes in systemic lupus erythematosus patients in vitro. Methods T-lymphoeytes were treated by thalidomide with different concentrations, then the proliferation of these T-lymphocytes proliferation was deteted by MTT while apoptosis and lymphocyte activation marker were analyzed by flow cytometry. The mRNA expression of IL-6, IL-10 and TNF-α was measured by real-time RT-PCR, One-way ANOVA was used for statistical analysis. Results In vitro, thalidomide inhibited the expression of CD3~+CD28~+ [500 μg/ml group vs the control group, (48±9)% vs (57±9)% P<0.05]. The pro-portion of apoptotic T-lymphoeytes in the 500 μg/ml group was (36±8)%, which was significantly higher than that in the control group [(23±5)%,P<0.05 ]. The values of A_(570nm) T-lymphocytes were significantly lower in the 100 μg/ml group, 300 μg/ml groupand 500 μg/ml group compared with those of the control group ( 100 μg/ml group vs 300 μg/ml group vs 500μg/ml group vs the control group, 0.39±0.05 vs 0.34±0.04 vs 0.30±0.03 vs 0.51±0.07, P<0.05), while thalidomide promoted the expression of CD8~+CD152~+ [ 100 μg/ml group vs 500 μg/ml group vs the control group, (5.0±0.6)% vs (7.8±0.7)% vs (4.2±0.6)%, P<0.05 ]. 500 μg/ml thalidomide inhibited the mRNA expression of IL-6, 2.5～500 μg/ml thalidomide inhibited IL-10, TNF-α mRNA expression of T-lymphocytes.Conclusion Thalidomide can inhibit the proliferative activities and CD28 expression of T-lymphocytes,reduce mRNA expression of IL-6, IL-10, TNF-α, stimulate CD28 expression and apoptosis of T-lymphocytes. These effects may play an important role in it's immune-suppressive effects on systemic lupus erythematosus.
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Objective To investigate the immunoregulatory effects of mesenchymal stem cells (MSCs) on the peripheral T-lymphocytes of lupus nephritis in vitro. Methods MSCs were isolated and expanded from human bone marrow cells. The purity of MSCs was identified by flow cytometry (FCM). The MSCs (4×104, 1×104, 2×103) were added into wells containing peripheral blood lymphocytes (2×105) from lupus nephritis in the presence of phytohemagglutinin [PHA). Samples were divided into the following groups: group A:T-lymphocytes alone; group B: MSCsl with T-lymphocytes(MSCsl:T=1:5); group C: MSCs2 with T-lymphocytes (MSCs2:T=1:20); group D: MSCs3 with T-lymphocytes (MSCs3:T=1:100). The proliferation of T-lymphocytes was assessed by MTT. FCM was used to analyze the apoptosis of T-lymphocytes and surface markers of CD28 and CD152. The gene expression of interferon γ (IFN-γ), interleukin-10 (IL-10), transforming growth factor-β1 (TGF-β1) were detected by real-time RT-PCR. Results In vitro, with the presence of MSCs, peripheral blood T-lymphocytes from lupus nephritis were statistically significantly decr-eased in their proliferative activities , apoptosis and CD28 expression in a dose-dependent manner. No inhibitory effects on CD152 expression of T-lymphocytes had been observed . MSCs promoted the gene expression of TGF-β1 and inhibited the gene expression of IL-10, IFN-γ. Conclusion MSCs can inhibit the proliferative activities, apoptosis and CD28 expression of peripheral blood T-lymphocytes of lupus nephritis, increase gene expression of TGF-β1 and lower the gene expression of IL-10, IFN-γ, which may play an important role in it's immunosuppressive effects on lupus nephritis.
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Objective To investigate the immunoregulatory effects of mesenchymal stem cells (MSCs)on peripheral blood T lymphocytes from patients with systemic lupus erythematosus(SLE)in vitro and their potential mechanism.Methods MSCs were isolated from the bone marrow of 3 healthy human volunteers,cultivated and identified.Under phytohemagglutinin(PHA)stimulating,peripheral blood T lymphocytes from 8 patients with SLE were treated with MSCs with the T lymphocyte/MSC ratio being 50:1 in group B and 5:1 in group C or without MSCs(group A).MTT assay was used to detect the proliferation of T lymphocytes.flow cytometry to analyze the expressions of surface markers CD152 and CD28 on T lymphocytes.and real time PCR to measure the mRNA expressions of interleukin-6 and interferon-γ,in the T lymphocytes.Results MSCs could markedly inhibit the proliferation of T lymphocytcs.The proliferation of T lymphocytes expressed as absorbance value at 570 nm was 0.484±0.032 in group B.0.308±0.025 in group C,significantly lower than that in group A(0.765±0.036,both P＜0.05),and significant difference was also observed between group C and B(P＜0.05).In the case of the percentage of CD28 positive T lymphocytes.group B and C were significantly lower than group A(60.39%±3.92%and 45.05%±3.46%vs 74.73%±3.74%,both P＜0.05),and group B significantly differed from group C(P＜0.05).MSCs had no obvious effect on the expression of CD152 on T lymphocytes,but significantly suppressed the mRNA expression of interleukin-6 and interferon-γ(both P＜0.05).and the suppressive effect was enhanced with the incrgase in MSC count.ConclusionsMSCs exert an immunosuppressive effect on T lymphocytes from patients with SLE,likely through inhibiting the proliferation,CD28 expression,interleukin-6 and interferon-γ mRNA expression of T lymphocytes.
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Objective To study the expression and significance of Th17 cells from peripheral blood of patients with rheumatoid arthritis (RA). Methods Intracelluar flow cytomete detection of IL-17/IFN-γ and IL-17/IL-6 was established using anti-CD3/Anti-CD28/IL-23 as stimulators after isolation of untouched human CD4~+T cells from PBMC. There were three groups in the present study: ①healthy controls group; ② RA stable group; ③RA active group. Results The isolation of untouched human CD4~+T cells from PBMC was effective and its purity was over 90%. The percentage of intracelluar IL-17 in CD4~+ T cells from RA patients was increased significantly. Such percentage in active group (1.54±0.41) was higher than that of stable group (0.70±0.21, P<0.01) and both of them were higher than those of healthy controls (0.42±0.12, P<0.01). Under anti-CD3/Anti-CD28/IL-23 stimulation, the percentage of intracelluar IL-17 was also increased significantly(P<0.01). The porcentage of intracellular IFN-γ was similar to that of IL-17, while that of IL-6 was not significantly different. There is an correlation between IL-17 and IFN-γ or IL-6. Conclusion There is an abnormal expression of IL-17 and IFN-γ in human CD4~+T cells in RA patients, which is related to disease activity . Th17 cells may be used as a new marker for the assessment of RA activity.
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Objective To investigate the immunoregulatory effects of bone marrow-derived mesenehy-real stem ceils (BMSCs) combined with leflunomide (LEF) on mice T-lymphocytes in vitro. Methods BMSCs from BALB/c mice were isolated and expanded. The purity of BMSCs was identified by flow cytometry (FCM). The BALB/c mice's spleen lymphocytes were isolated by using EZ-Sep<'TM> Mouse 1X. Under ConA stimulation, spleen lymphocytes were pretreated with LEF, then washed and co-cuhured with BMSCs. We set up four groups to investigate in this study: group A, spleen lymphocytes alone; group B, spleen lymphocytes with BM- SCs; group C, LEF-pretreated spleen lymphocytes alone and the group D, LEF-pretreated spleen lymphocytes with BMSCs. T-lymphocytes proliferation was assessed by MTT. FCM was used to analysis T-lymphocytes apoptosis and surface markers of CD69 and CD28. The mRNA expression of interleukin (IL)-2, IL-10 were detected by real-time RT-PCR. Results In vitro, the T-lymphocytes'values of A570 nm were significantly lower in group B and group C, compared with group A (group B vs group C vs group A, 0.578±0.042 vs 0.502± 0.040 vs 0.778±0.035, P<0.01), while the value of A<,570 nm>in group D was 0.218±0.033, which was also obviously lower than that in group B and group C (P<0.01). There were no suppressing effects on T-lympho-cytes'activation and expression of IL-2 had been observed. The proportion of apoptotic T-lymphocytes in group B and group D were (2.29±0.32)％ and (4.22±0.98)％, which was significandy lower than that in group A (8.08±1.20) (P<0.01). The expression of IL-10 in group B and C were also lower than that in group A (group B vs group C vs group A, 0.098±0.039 vs 0.054±0.022 vs 1.000, P<0.01 ). Either, the expression of IL-10 in group D was 0.023±0.015, which was obviously lower than that in group B and group C (P<0.01). Conclusion BMSCs combined with LEF show synergistic immunoregulatary effects on mice's T-lymphoeytes.
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Objective To study the immunoregulatory effects of thalidomide on the peripheral blood T-lymphocytes of rheumatoid arthritis patients.Methods MTr was used to detect the effects of different thalidomide concentrations on the proliferation of T-cells.Flow eytometry was used to analyze T-cells early apoptosis and the T-cells subsets in different concentration of thalidomide.The mRNA expression of IL-6,IL- 10 and TNF-α was measured by RT-PCR method.Results The level of thalidomide at 500 μg/ml inhibited the proliferation of T-ceils and the CD3+CD28+ expression of T-cell subsets,but promoted the early apoptosis and the CD8+CD152+ expression of T-cell subsets.Thalidomine at any concentration could inhibit the mRNA expression of IL-6,TNF-α.However,the level of thalidomide that could promote the mRNA expression of IL- 10 was 100 μg/ml and 500 μg/ml.Conclusion Thalidomide can inhibit the proliferation of T lymphocytes and the expression of CD3+CD28+ on T-cell subsets.It can promote the early apoptosis and the CD8+CD152+ expression of T-cell subsets.Thalidomide inhibits the mRNA expression of IL-6 and TNF-α but promote the mRNA expression of IL-10.Thalidomide has immuno-regulatory effects on rheumatoid arthritis T-cells.