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Objective To establish collagen‐induced arthritis (CIA) model used of DBA/1J mouse ,a preliminary study of the expression levels of BM Ps in mononuclear cells of peripheral blood and the joints tissue of CIA mice .Methods We induced DBA/1J mice and developed arthritis pathology by using of bovine type Ⅱ ;then ,we detected mRNA and protein expression levels of BMPs by using quantitative PCR and immunohistochemical staining .Results We successfully established CIA mouse model .The expres‐sion of BMP4 and BMP9 mRNA was significantly down‐regulated in peripheral blood mononuclear cells and in the pathogenesis of joint tissues of CIA mice (P<0 .05) .It showed that BMP9 protein significantly decreased in joint tissues of CIA mice by immuno‐fluorescence technique (P=0 .002) .Conclusion At the genetic level ,the expression of BMP4 and BMP9 could be significantly down‐regulated in the CIA mouse .At the protein level ,BMP9 could be significantly down‐regulated in the CIA mouse .
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Objective To investigate the inhibiton effect of 4‐styrenesulfonic acid‐co‐maleic acid(PSM ) in HIV‐1 .Methods The inhibition effect of different doses of PSM on HIV‐1 in susceptible cells GHOST (3) X4/Hi5 was observed by Luciferase ,and so did the inhibitory effect of PSM on JR‐FL、HXB2、CNE6 ,CNE30 ,CNE50 ,CNE55 .The cellular toxicity of PSM on the VK2/E6E7 was also evaluated by CCK8 kit .The transcript level of tight junction proteins (ZO‐1 ,E‐cadherin and Occludin) of HEC‐1‐A were analyzed by qRT‐PCR .And then observed the effect of PSM on expression of genitourinary epithelial cells HEC‐1‐A ,so we could e‐valuated the effect of integrity of local mucosal indirectly .Results The results showed that PSM exhibited potent antiviral activity against a broad spectrum of HIV‐1 major isolates with different genotypes and biotypes (EC50 value of JR‐FL ,HXB2 ,CNE6 , CNE30 ,CNE50 ,CNE55 were 5 .78 ,0 .77 ,1 .85 ,3 .15 ,1 .70 ,2 .27 μg/mL respectively) .Meanwhile ,it had less cytotoxicity on VK2/E6E7 .qRT‐PCR showed that no obvious restrain effect on expression of ZO‐1 was observed and PSM increased the level of tran‐scription of E‐cadherin and Occludin .Conclusion PSM may be a potential agent for the prevention of HIV‐1 infection .
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Suspension array technology,a bead-based multiplex assay,allows a fast and automatic detection of multi-analytes and batch samples in limited sample volume,and it is great potential clinical application in the determination of autoimmune antibodies,muhitumor markers,the phenotype of Human Leukocyte Antigen and the genes of infectious pathogens.However,currently,there are various limitations which hinder the clinical application of this technology broadly.The globally accepted panel of multi analytes,performance criteria,and quality control programs should be established before we get benefit from this high throughput,sensitivity and repeatability platform,which will help to provide scientific and accurate laboratory data for the clinical diagnosis,treatment and prevention.
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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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Objective To investigate the effect of sodium valproate in suppression of cell proliferation and arrest of cell cycle of in human hepatoma cell line SMMOL/LC-7721 and pancreatic cancer cell line PaTu8988.Methods Hepatoma cell line SMMOL/LC-7721 and pancreatic cancer cell line PaTu8988 were inoculated on the culture plate,cultured in the DMEM medium,they were intervened with sodium valproate in concentration of 0.2 mmol/L (0.2 mmol/L group),1.0 mmol/L (1.0 mmol/L group),5.0 mmol/L (5.0 mmol/L group) and dimethyl sulfoxide (control group) for 48 h respectively.Absorbance was measured by enzymelinked immunosorbentassay equipment,and inhibition ratio was calculated.Cell cycle was detected by flow cytometry.Results The absorbance of hepatoma cell line SMMOL/LC-7721 and pancreatic cancer cell line PaTu8988 in 5.0 mmol/L group were significantly lower than those in control group and 0.2 mmol/L group (0.569 ±0.059 vs.0.706 ±0.033 and 0.760 ±0.020,2.068 ±0.178 vs.2.793 ±0.144 and 2.663 ± 0.078,P ＜ 0.05),the absorbance of pancreatic cancer cell line PaTu8988 in 1.0 mmol/L group (2.432 ± 0.084) was significantly lower than that in control group and 0.2 mmol/L group (P ＜ 0.05).With the sodium valproate concentration increased,inhibition rate of tumor cell increased gradually,the inhibition rate of hepatoma cell line SMMOL/LC-7721 and pancreatic cancer cell line PaTu8988 in 5.0 mmol/L group was 23.5％ and 25.9％ respectively.Compared with control group,with the sodium valproate concentration increased in 0.2,1.0,5.0 mmol/L group,the proportion of G1 phase cell increased gradually in hepatoma cell line SMMOL/LC-7721 [(49.25 ± 1.63)％,(65.26 ± 2.34)％,(83.13 ± 1.78)％ vs.(49.22 ± 4.35)％],the proportion of S phase cell decreased gradually [(26.84 ± 2.30)％,(17.76 ± 3.90)％,(3.38 ± 0.65)％ vs.(29.21 ± 2.35)％],cell cycle showed G1 phase arrest,there was significant difference (P ＜ 0.05).Compared with control group and 0.2 mmol/L group,the proportion of G2 phase cell increased in pancreatic cancer cell line PaTu8988 in 1.0 and 5.0 mmol/L group [(26.80 ± 1.50)％,(36.58 ± 3.78)％ vs.(12.00 ± 4.62)％,(16.54 ± 2.26)％],cell cycleshowed G2 phase arrest,there was significant difference (P ＜ 0.05).Conclusion Sodium valproate mightsignificantly suppress the cell proliferation in hepatoma cell line SMMOL/LC-7721 and pancreatic cancercell line PaTu8988 and induce cell cycle arrest,it is clinically promising antitumor drug.
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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 detect the levels of Th17 and regulatory T(Tr)cells in Imripheral blood mononuclear and tumor tissue from patients with cervical cancer and analyze the relationship hetween Th17 and Tr cells in cervical cancer progression.In addition.the significance of the Th17/Tr cells ratio in cervical cancer pathogenesis was discussed.Methods The expression levels of Th17 and Tr cells were determined by flow cytometry from 32 patients with cervical cancer and 15 health people.The mechanism of involvement of Th17 and Tr cells proportionality in cervical cancer pathogenesis and the correlation between Th17 and Trwas assessed by bivariate correlation analysis.Results The expression levels of both Th17 and Tr in patients were higher than control groups,especially in late stage patients Th17 and Tr proportionality lower than early group,and there was a negative correlation between them.Conclusion Th17 and Tr cells proportionality may be involve in the development of cervical cancer so as to provide novel strategies for tumor immunotherapy.
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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 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 distribution of TH17 cells in peripheral blood of patients with rheumatoid arthritis(RA). Methods Intracelluar flow cytometirc detection of TH17 cells in peripheral blood was establised using PHA or PMA + Ion as stimulators in vitro. Thirty-five active and 30 stable RA pa-tients and sex and age paired healthy controls were enrolled in this study. Results The stimulating effect of PMA + Ion was better than that of PHA alone. Under PMA + Ion stimulation, the percentage of TH17 cells in peripheral blood from RA patients and healthy controls was increased significantly(P<0.05). With or with-out PMA + Ion stimulation, such percentage in active group was higher than that of stable group and both of them were higher than that of heathy controls(P<0.05). The distribution profile of TH1 cells was similar to that of TH17 cells. Conclusion There is a distribution abnormality of TH17 cells in peripheral blood of RA patients, which could be used as a new marker for the assessment of immunological condition in RA.
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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.
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Objective To investigate the percentage of CD5-positive B cells in peripheral blood mononuclear cells(PBMC) of patients with chronic HCV infection and its clinical significance.Methods The expression of CD5 molecule on B cell surface was detected by flow cytometry and HCV RNA copies were detected by real-time PCR.Results The percentage of CD5+-B cells significantly increased in the patients with chronic HCV infection(58.4%?9.8%) compared with healthy controls(22.5%?5.9%)(P