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Objective@#To study the distribution of natural killer (NK) cells and the CD38 positive subpopulations in different tissues in rheumatoid arthritis (RA) patients and the mechanism of CD38 agonist.@*Methods@#The levels of NK cells, CD38 positive subpopulation and NK cell surface chemokine receptors (CXCR3, CCR5), as well as granzyme B and perforin were detected by flow cytometry in peripheral blood and knee synovial fluid. The isolated cells were then cultured in vitro and divided into 3 groups, namely blank control group, IB4 stimulation group (Anti-CD38 mAb), IL-2 and IB4 co-stimulation group. And the concentration of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in culture supernatants was determined by enzyme linked immunosorbent assay (ELISA). Liposome CD38-siRNA was transfected into peripheral blood lymphocytes of RA patients, and transfection efficiency was detected by reverse transcription-polymerase chain reaction (RT-PCR). The effect of CD38 and CD38-siRNA silencing on the expression of phospholipase C-gamma 1 (PLC-γ1) protein in the peripheral blood lymphocytes of RA was detected by Western blot.@*Results@#The expression of CCR5 and CXCR3 in NK cells in peripercal NK cells of RA was significantly higher than that in normal healthy subjects (P<0.05). The two chemokine receptors (CXCR3, CCR5) were mainly expressed in CD38+ NK cell subsets. The levels of granzyme B and perforin in synovial lymphocytes cells were significantly higher than those in peripheral blood lymphocytes (P<0.05). The secretion of IL-6 and TNF-α in synovial and peripheral blood lymphocytes cells existed significant difference only in IL-2 and IB4 co-stimulation group (P<0.05). The levels of PLC-γ1 protein in the peripheral blood lymphocytes of RA patients was significantly decreased than that in the blank control group after the treatment with CD38-siRNA (P<0.05).@*Conclusions@#The synovial NK cells are more lethal than the peripheral NK cells in the RA patients. CD38 might mediate the phosphorylation of intracellular proteins via the Protain kinase C (PKC) pathway. Blocking CD38 molecules can reduce the phosphorylation level of intracellular proteins. CD38 maybe involved in the mechanism of chemotaxis and killing capability of NK cells.
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Objective To study the distribution of natural killer (NK) cells and the CD38 positive subpopulations in different tissues in rheumatoid arthritis (RA) patients and the mechanism of CD38 agonist.Methods The levels of NK cells,CD38 positive subpopulation and NK cell surface chemokine receptors (CXCR3,CCRS),as well as granzyme B and perforin were detected by flow cytometry in peripheral blood and knee synovial fluid.The isolated cells were then cultured in vitro and divided into 3 groups,namely blank control group,IB4 stimulation group (Anti-CD38 mAb),IL-2 and IB4 co-stimulation group.And the concentration of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in culture supernatants was determined by enzyme linked immunosorbent assay (ELISA).Liposome CD38-siRNA was transfected into peripheral blood lymphocytes of RA patients,and transfection efficiency was detected by reverse transcription-polymerase chain reaction (RT-PCR).The effect of CD38 and CD38-siRNA silencing on the expression of phospholipase C-gamma 1 (PLC-γ1) protein in the peripheral blood lymphocytes of RA was detected by Western blot.Results The expression of CCR5 and CXCR3 in NK cells in peripercal NK cells of RA was significantly higher than that in normal healthy subjects (P ＜ 0.05).The two chemokine receptors (CXCR3,CCRS) were mainly expressed in CD38 + NK cell subsets.The levels of granzyme B and perforin in synovial lymphocytes cells were significantly higher than those in peripheral blood lymphocytes (P ＜0.05).The secretion of IL-6 and TNF-α in synovial and peripheral blood lymphocytes cells existed significant difference only in IL-2 and IB4 co-stimulation group (P ＜0.05).The levels of PLC-γ1 protein in the peripheral blood lymphocytes of RA patients was significantly decreased than that in the blank control group after the treatment with CD38-siRNA (P ＜ 0.05).Conclusions The synovial NK cells are more lethal than the peripheral NK cells in the RA patients.CD38 might mediate the phosphorylation of intracellular proteins via the Protain kinase C (PKC) pathway.Blocking CD38 molecules can reduce the phosphorylation level of intracellular proteins.CD38 maybe involved in the mechanism of chemotaxis and killing capability of NK cells.
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OBJECTIVE:To investigate the effects of triptolide(TP)on the proliferation of fibroblast-like synoviocytes(FLS) from patients with rheumatoid arthritis(RA)in vitro. METHODS:5 RA patients received knee arthroplasty or synovectomy to ob-tain synovial tissue. FLS was isolated,cultured and identified,and then incubated in the presence of TP [0 (blank control),50, 100 and 200 nmol/ml] for 24,48 and 72 h. The effects of TP on FLS was evaluated by MTT,and then proliferation inhibitory rate was calculated;flow cytometry was used to detect the apoptosis and cell cycle of FLS. RESULTS:The inhibitory rates of TP(50, 100 and 200 nmol/ml)on the proliferation of FLS were 17.46%-52.56%,which was positively correlated with drug concentration. Compared with blank control group,100 and 200 nmol/ml TP could increase the percentage of cells in G0/G1 phase and decrease the percentage of cells in S phase,with statistical significance(P<0.05);200 nmol/ml TP could induce cell apoptosis. CONCLU-SIONS:TP could inhibit the proliferation and also could induce the apoptosis of FLS in RA patients in vitro,which may be one of its mechanism for treating RA.
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Objective To investigate the clinical characteristics and related pathogenetic factors in Takayasu arteritis with myocardial involvement.Methods We retrospectively analyzed clinical data of Takayasu arteritis patients with myocardial involvement of Peking Union Medical College Hospital from 1987 to 2013.Results Myocardial involvement was diagnosed in 23 patients from 86 Takayasu arteritis patients.Sixty-five percent (15/23) of patients presented with congestive heart failure.Fifteen patients were without coronary artery involvement.Sixty-five percent (15/23) of patients with mild to moderate hypertension.Seventyfour percent(17/23) patients had pulmonary hypertension.Left ventricular ejection fraction was (34.70 ±0.09)％.Glucocorticco costeroid and immunosuppressive therapy could significantly increase left ventricular ejection fraction (t=4.302,P＜0.05).Conclusion We emphasize that myocardial involvement is common in Takayasu arteritis.It is an independent risk factor that impacts the prognosis.Early detection of myocardial involvement and effective therapy can improve the prognosis.
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Objective To investigate in vitro possible mechanisms by which low frequency electrical stimulation may stimulate peripheral blood stem cells' proliferation and differentiation into Schwann cells (SCs).Methods The original generation of peripheral blood stem cells was cultured using Sprague-Dawley (SD) rats.The third passage stem cells were divided into a low frequency stimulation group,an extracellular signal-regulated kinase (ERK) group,a combination group and a control group.All 4 groups were cultured in DMEM containing 2％ fetal bovine serum by adding the SC supernatant.The low frequency electrical stimulation group was given 1 h of continuous low frequency stimulation.For the ERK group 50 mmol/L of the inhibitor PD98059 was added.The combination group was given the inhibitor plus 1h of sustained low frequency electrical stimulation.The control group received no special intervention.Before and after induction the 3-(4,5-dimethyl-thiazol-2)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to detect the absorbance value A of 4 cells at 570 nm (A750),and after induction Western blotting was used to determine cyclin D1 (cyclin D1) and cyclin-dependent kinases (CDK4).Results Before the intervention peripheral blood stem cells in each group had no significant differences in their A750 values.After the intervention,the A750 values of the low frequency electrical stimulation,the ERK group,the combination group and the control group were (1.051 ±0.058),(0.363 ±0.343),(0.894 ±0.343) and (0.758 ±0.047),respectively.This showed a statistically significant increase for all groups except the ERK group.The differences among the groups were also statistically significant.The expression of S-100,glial fibrillary acidic protein (GFAP) and P75 was highest in the low frequency electrical stimulation group,and in the ERK group they were the lowest.S-100,GFAP and P75 protein expression also was highest in the low frequency electrical stimulation group and lowest in the ERK group,and the inter-group differences were statistically significant.ERK expression showed no significant difference among the groups.Compared with the control group,the expression of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2),cyclin D1 and CDK4 protein in the low frequency electrical stimulation group and the combination group were all significantly higher and the protein expression in the ERK group was significantly lower.The p-ERK1/2,cyclin D1 and CDK4 protein levels in the combination group were significantly lower than in the low frequency electrical stimulation group and higher than in the ERK group.Conclusions Low frequency electrical stimulation can promote peripheral blood stem cell proliferation and induce cell differentiation into Schwann cells,at least in vitro.The ERK signaling pathways are part of the signaling pathways of the proliferation and differentiation.
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Objective To explore the mechanism of combined treatment with methotrexate (MTX) and Ga-Al-As laser irradiation for rheumatoid arthritis (RA) and to assess the effectiveness of Ga-Al-As laser therapy for RA. Methods Twenty-two patients with RA were randomly and evenly divided into two groups: the treatment group treated with Ga-Al-As laser irradiation combined with MTX and the control group treated with MTX only. Ten age-matched normal subjects were observed as normal controls. The amount of CD4 + CD25 + regulatory T cells in peripheral blood (PB) of the normal controls and that in the PB and synovial fluid (SF) of the 22 patients before and after therapy were counted by flow cytometry. Meanwhile, the amount of prostaglandin E2 (PGE2) in synovial fluid of the patients was measured before and after treatment by enzyme-linked immunosorbent assay(ELISA). Results After combined treatment the clinical symptoms of the patients were improved significantly, and the amount of PGE2in SF decreased significantly. The count of CD4 + CD25 + regulatory T cells in PB of RA patients was ( 3.84 ±3.20) % , compared to ( 10.05 ± 7.04) % in healthy individuals. The count of CD4 + CD25 + regulatory T cells in SF of RA patients was ( 14.89 ± 12.30) % , much higher than that in PB. The count of CD4 + CD25 + regulatory T cells in SF decreased significantly in treatment group compared to control group (P ＜0.05). Conclusion Ga-Al-As laser irradiation eombined with MTX can effectively improve the clinical symptoms of RA patients. It may be related to the decrease of amount of PGE2 and count of CD4 + CD25 + regulatory T cell in PB and SF.