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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 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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BACKGROUND: With the improvement of biomaterial and further recognition in aetabular revision, the main tendency is using biologic prosthesis for mild or moderate acetabular bone defect in total hip revision. OBJECTIVE: To observe the clinical effect of simple particulate bone grafting on mild or moderate acetabular bone defect in total hip revision. DESIGN, TIME AND SETTING: A retrospective case analysis. All cases were from Department of Orthopedics, Shanghai East Hospital Affiliated to Tongji University from June 2003 to June 2008. PARTICIPANTS: A total of 10 patients with mild or moderate acetabular bone defect who received total hip revision were from Department of Orthopedics, Shanghai East Hospital Affiliated to Tongji University. Among them, there were 5 male and 5 female, with an average of 58.8 years old from 43 to 77 years old. Their last replace time were 3-10 years, with an average of (7.0?0.5) years. Two of them in the last replacement used cement components, three used cementless, four used mixture, and one used dipolar artificial thigh bone. According to AAOS typing, there were 6 cases of type Ⅰ and 4 cases of type Ⅱ. The average preoperative Harris hip joint score was 40.6. METHODS: Simple particulate bone grafting combined with biologic prosthesis was used to treat the 10 patients with mild or moderate acetabular defects. All operations were at the posterolateral approach. The acetabular cup and screw fixation were performed after grafting. The postoperative effect should be evaluated by clinical and X-ray manifestations. MAIN OBJECTIVE INDEX: The bone grafting time, bleeding volume, Harris score and complications in the perioperative period were recorded. RESULTS: All patients were adopted simple particulate bone grafting combined with biologic prosthesis to reconstruct the acetabular bone defects. One acetalum cup was found loose after one month. The titanium plate combined with particulate bone grafting was used in the second revision. At 3 months after operation, the patient could walk without crutch. No complication was found in other patients. An average of 3 years following-up was obtained. The average Harris score was 87.0. Except one acetalum cup was found loose, the X-ray showed that the bed of acetabulum and particulate bone was good healing and there was no complete transparent zone. There was no patient with displacement of acetalum cup. No osteolysis in any patient. The particulate bone and host bone had a good healing. CONCLUSION: The simple particulate bone grafting is simple and effective for mild or moderate acetabular bone defects with the good follow-up result in early period.