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Objective:To investigate the therapeutic effects of a small interfering RNA (siRNA) targeting hypoxia inducible factor-1α (HIF-1α) on collagen-induced arthritis (CIA) in mice and to analyze the possible mechanism at the macrophage level.Methods:DBA/1 mice were injected with bovine type Ⅱ collagen to establish the CIA model. Then, they were injected with HIF-1α-siRNA adenovirus or negative control adenovirus through tail vein once a week for four weeks. This study included four groups: control group, CIA model group, negative control group and HIF-1α-siRNA group. Mouse bone marrow-derived macrophages (BMDMs) were isolated and cultured. The relative expression of CD206 and arginine (Arg) at mRNA level in mouse BMDMs was detected by RT-PCR. The proportions of F4/80 + CD16/32 + M1 and F4/80 + CD206 + M2 macrophages in spleen and thymus were detected by flow cytometry. Pathological changes in the ankle joint of mice were observed using HE staining. Immunohistochemistry was used to detect the expression of macrophages and the subsets in mouse synovial tissues. Results:(1) Compared with the control group, the CIA model group showed decreased expression of CD206 at mRNA level in BMDMs, but increased expression of Arg at mRNA level ( P<0.01). HIF-1α-siRNA increased the expression of CD206 at mRNA level ( P<0.05) and reduced the expression of Arg at mRNA level in BMDMs of mice with CIA ( P<0.01). (2) Compared with the control group, the mice in the CIA model group had increased proportions of F4/80 + CD16/32 + M1 macrophages in splenocytes and thymocytes ( P<0.05), but decreased proportions of F4/80 + CD206 + M2 macrophages in thymocytes ( P<0.05). HIF-1α-siRNA could down-regulate the proportions of F4/80 + CD16/32 + M1 macrophages in splenocytes and thymocytes ( P<0.05), and up-regulate the proportions of F4/80 + CD206 + M2 macrophages in thymocytes of CIA mice ( P<0.01). (3) CIA mice had synovial hyperplasia and macrophages infiltration, especially M1 macrophages, in the ankle joint. HIF-1α-siRNA could alleviate the synovial hyperplasia and macrophage infiltration. Conclusions:HIF-1α-siRNA could alleviate macrophage infiltration and synovial hyperplasia in CIA mice through reducing the proportions of M1 macrophages in thymocytes, BMDMs and synovial tissues and increasing the proportion of M2 macrophages, suggesting that HIF-1α-siRNA could treat CIA mice by regulating the differentiation of M1 and M2 macrophages.
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Objective:To investigate the effects of a eukaryotic expression plasmid for IL-6 and B-cell activating factor (BAFF) fusion protein on the histopathological changes in salivary and lacrimal glands of non-obese diabetic (NOD) mice with Sj?gren′s syndrome and to elucidate the possible therapeutic mechanism of IL-6/BAFF fusion protein eukaryotic expression plasmid in NOD mice.Methods:The eukaryotic expression plasmid for IL-6/BAFF fusion protein was constructed. After transfecting CHO cells with the plasmid, the expression of IL-6/BAFF fusion protein was detected by Western blot. BALB/c mice were injected with the plasmid every two weeks for three times and the titers of anti-IL-6 and anti-BAFF antibodies were measured by ELISA. Twenty-one NOD mice were randomly divided into three groups (control group, empty vector group and therapy group) by numerical table method. The mice in the therapy group were injected with the IL-6/BAFF fusion protein eukaryotic expression plasmid once a week for six times and the mice in the empty vector group were injected with empty plasmid. The levels of anti-IL-6 and anti-BAFF antibodies as well as cytokines (IL-6, BAFF, INF-γ, IL-10 and IL-17A) in mouse serum samples were detected by ELISA. The proportions of Th17, Treg, Th1 and Th2 cells in mouse splenocytes were measured by flow cytometry. Focal lymphocyte infiltration and pathological changes in the lacrimal and salivary glands of mice were observed under light microscopy after HE staining.Results:The eukaryotic expression plasmid for IL-6/BAFF fusion protein increased the levels of anti-IL-6 and anti-BAFF antibodies in the serum of BALB/c mice ( P<0.05). The levels of anti-IL-6 and anti-BAFF antibodies in the serum of NOD mice in the therapy group increased ( P<0.01), while the expression of IL-6, BAFF, INF-γ, IL-10 and IL-17A in NOD mice in the therapy group was lower than that in the control group and the empty vector group ( P<0.05). The percentages of Treg and Th2 cells in the splenocytes of NOD mice increased after treatment ( P<0.05). Moreover, the eukaryotic expression plasmid for IL-6/BAFF fusion protein significantly improved the irregular size and morphology of glandular vesicles in the lacrimal and salivary glands, reduced the ductal dilatation and decreased the focal lymphocyte infiltration in NOD mice. Conclusions:The eukaryotic expression plasmid for IL-6/BAFF fusion protein induced the production of anti-IL-6 and anti-BAFF antibodies, decreased the expression of inflammatory cytokines, regulated the balance of Th17/Treg and Th1/Th2 cells, improved the irregular alveolar structure and ductal dilation in the lacrimal and salivary glands and reduced the focal lymphocyte infiltration in NOD mice. This study showed that eukaryotic expression plasmid for IL-6/BAFF fusion protein might serve as a potential target for therapeutic targeting of T and B cells.
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To investigate the effect of co-expression of sleeping beauty (SB) transposon and IL-10 mediated by adenovirus on the balance of Th17/Treg cells in the splenocytes of the non-obese diabetes (NOD) mice, and to illuminate the possible mechanism of IL-10 in the treatment of NOD mice. Methods: The splenocytes were extracted from spleen of the C57BL6 mice and cultured. The splenocytes were divided into control group, empty vector group and therapy group. The cells in control group didn't receive any treatment, the cells in empty vector group were co-infected with the adenovirus vector without IL-10 gene containing transposon sequence and the adenovirus vector without transposase, and the cells in therapy cells were co-infected with the adenovirus vector containing IL-10 gene and transposon sequence and the adenovirus vector with transposase. After 48 h of infection, the expression leves of IL-10 mRNA in the splenocytes of the mice in various groups were assessed by RT-PCR. The cells of above three groups were subcutaneously injected into popliteal space in right hind leg of the NOD mice in control group, empty vector group and therapy group; there were six mice in each group; once a week and six times. The mice were killed 4 weeks after injection, the expression levels of IL-10 in serum of the NOD mice in various groups were assessed by ELISA, and the proportions of CD4 +IL-10+, CD4 + IFN-γ+, Th17 and Treg cells in the splenocytes were detected by flow cytometry. Results: Compared with control group and empty vector group, the expression level of IL-10 mRNA in splenocytes of the C57BL6 mice in therapy group was increased (F=72.71, P0.05). Conclusion: The co-expression of SB transposon and IL-10 can significantly increase the IL-10 level in serum of the NOD mice, increase the proportion of CD4 + IL-10+ cells, decrease the proportion of Th17 cells and increase the proportion of Treg cells in the splenocytes, which can regulate the balance of Thl/Th2 and Th17/Treg cells and play a therapeutic effect in the NOD mice.
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Objective:To investigate the effect of co-expression of sleeping beauty(SB)transposon and IL-10 mediated by adenovirus on the balance of Th17/Treg cells in the splenocytes of the non-obese diabetes(NOD)mice, and to illuminate the possible mechanism of IL-10 in the treatment of NOD mice.Methods:The splenocytes were extracted from spleen of the C57BL6 mice and cultured.The splenocytes were divided into control group,empty vector group and therapy group.The cells in control group didn't receive any treatment,the cells in empty vector group were co-infected with the adenovirus vector without IL-10 gene containing transposon sequence and the adenovirus vector without transposase,and the cells in therapy cells were co-infected with the adenovirus vector containing IL-10 gene and transposon sequence and the adenovirus vector with transposase.After 48 h of infection, the expression leves of IL-10 mRNA in the splenocytes of the mice in various groups were assessed by RT-PCR. The cells of above three groups were subcutaneously injected into popliteal space in right hind leg of the NOD mice in control group,empty vector group and therapy group;there were six mice in each group;once a week and six times.The mice were killed 4 weeks after injection,the expression levels of IL-10 in serum of the NOD mice in various groups were assessed by ELISA,and the proportions of CD4+IL-10+,CD4+IFN-γ+,Th17 and Treg cells in the splenocytes were detected by flow cytometry.Results:Compared with control group and empty vector group,the expression level of IL-10 mRNA in splenocytes of the C57BL6 mice in therapy group was increased (F=72.71,P<0.05);the expression level of IL-10 in serum of the NOD mice was increased(F=8.89,P<0.05);the proportions of CD4+IL-10+ cells and Treg cells were significantly increased(F=72.09,P<0.05;F=12.98,P<0.05);the proportion of Th17 cells was decreased(F=6.39,P<0.05).The proportion of CD4+IFN-γ+ cells had no significant differences between various groups(F=2.72,P>0.05).Conclusion:The co-expression of SB transposon and IL-10 can significantly increase the IL-10 level in serum of the NOD mice,increase the proportion of CD4+IL-10+ cells,decrease the proportion of Th17 cells and increase the proportion of Treg cells in the splenocytes,which can regulate the balance of Th1/Th2 and Th17/Treg cells and play a therapeutic effect in the NOD mice.
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Objective To analyze the clinical characteristics in elderly onset rheumatoid arthritis(RA) patients combined with interstitial lung disease(ILD).Methods Four hundred and four cases of elderly onset RA patients were summarized.They were divided into two groups according to whether combined with ILD.Its clinical manifestations,laboratory examinations and combined diseases were analyzed.Results (1)Male elderly patients with onset of RA were more likely to occur ILD than female(χ2=6.251,P=0.012).There was no significant difference in duration analysis of two groups(t=1.750,P=0.081).(2)The elderly onset patients in RA with knee pain were more likely to happen ILD(χ2=7.048,P=0.008),the difference was significant.And there was no significantly statistical difference in the shoulder joint pain(χ2=0.028,P=0.866),elbow pain(χ2=0.022,P=0.882),hand joint pain(χ2=2.041,P=0.153),hip pain(χ2=0.129,P=0.720),joint deformities(χ2=0.013,P=0.908),morning stiffness(χ2=0.000,P=0.984) and joints rheumatoid nodules(χ2=0.349,P=0.555) of two groups.(3)The elderly onset RA patients with a high level of serum RF were more likely to happen ILD(t=3.325,P=0.001),the difference was significant.And there was no significantly statistical difference in serum ANA antibodies(χ2=0.004,P=0.948),anti SSA(χ2=0.718,P=0.397),SSB antibody(χ2=0.638,P=0.424),AKA antibody(χ2=0.949,P=0.330),CCP antibody(χ2=0.500,P=0.479) and the level of ESR(t=0.582,P=0.561),CRP(t=0.381,P=0.703) and PLT(t=-1.246,P=0.213).(4)The elderly onset RA patients with ILD were more likely to appear osteoporosis(χ2=7.467,P=0.006),the difference was significant.And there was no significantly statistical difference in the occurrence of high blood pressure(χ2=0.403,P=0.526) and diabetes(χ2=0.180,P=0.671) in the two groups.Conclusion Male patients,the elderly onset RA patients with knee pain and associated with high level of serum RF more often occur with ILD.And the elderly onset RA patients with ILD are more likely to appear osteoporosis.
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Objective:To investigate the clinical and laboratory characteristics of lupus nephritis(LN) patients by detecting the anti-nucleosome antibodies, anti-C1q antibodies and anti-double stranded antibodies(anti-ds DNA), and to clarify the risk factors of LN in the patients with systemic lupus erythematosus(SLE),and the significance of three kinds of antibodies in diagnosis of LN.Methods:A total of 120 SLE patients were selected and divided into LN group(n=60) and non-LN group(n=60).The ANAS data of 120 patients were retrospectively analyzed,the levels of anti-C1q antibodies were measured.The clinical symptoms and laboratory data of the patients with positive anti-dsDNA,-nucleosome and-C1q antibodies (3-pos group)and negative three kinds of antibodies(non 3-pos group) were analyzed in LN group.Results:The positive rate of anti-C1q antibody of the patients in LN group (40.00%) was higher than that in non-LN group (21.67%) (χ2=4.728, P=0.03).The positive rate of anti-dsDNA antibody in LN group was 66.67%, and it was 46.67% in non-LN group;the positive rates of the patients had significant difference between two groups (χ2=4.887, P=0.027).The positive rate of anti-nucleosome antibody in LN group was 58.33%, and it was 40.00% in non-LN group;the positive rates of the patients had significant difference between two groups (χ2=4.034, P=0.045).The positive rates of U1-snRNP, SmD1 and other antibodies Jo-1, SSA/Ro60kD, SSA/Ro52kD, SSB, ScL-70, CENP-B,and P0 had no significant differences between two groups(P>0.05).The levels of C3 and C4 and hemoglobinin of the patients in 3-pos group were higher than those innon 3-pos group (P0.05).The clinical symptoms were not statistically significant in 3-pos and non 3-pos groups (P>0.05).Conclusion:The anti-nucleosome, anti-C1q and anti-dsDNA antibodies are the risk factors of SLE complicated with LN;the positive antibodies can improve the diagnostic rate of LN.The 3-pos patients have more severe damage in complements and blood system with higher renal disease activities.
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Objective To observe the effect of α‐Fodrin siRNA on human salivary gland(HSG)cells and to discuss its thera‐py on sj?gren′s syndrome(SS) .Methods The vectors expressing siRNA againstα‐Fodrin of human were transfected into HSG cells of 10μg α‐fodrin siRNA1 group andα‐Fodrin siRNA2 group ,while pGFP‐V‐RS vector were transfected into the cells of empty vec‐tor group ,there was no handling in HSG cell of control group .The efficiency was observed by fluorescence microscope after trans‐fection of 24 ,48 ,72 and 96 h by lipofectamine 2000 .The expression levels of α‐Fodrin mRNA and protein of HSG were detected by real‐time(RT)‐PCR and immunohistochemistry method respectively .The expression levels of IFN‐γ and IL‐10 in supernatant of cells were detected by ELISA .Results The efficiency was highest on 48 h after transfection .The level of α‐Fodrin mRNA and pro‐tein was lower in α‐fodrin siRNA1 group and α‐fodrin siRNA2 group than control group and empty vector group on 48 h after transfection (P0 .05) .The levels IFN‐γ in α‐fodrin siRNA1 group and α‐fodrin siRNA2 group were lower than of control group and empty vector group on 48 h after transfection ,but there were no significant differences in the four groups (P>0 .05) .Conclusion Theα‐fodrin siRNA1 andα‐fodrin siRNA2 can suppress the levels of α‐fodrin mRNA and protein of HSG cells ,at the same time;they can elevate the expression of IL‐10 and decrease the level of IFN‐γ.Soα‐Fodrin siRNA reduce the levels of inflammatory cyto‐kines and provide experimental basis to therapy of SS .
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Objective:By analyzing the effect of deacetylase inhibitors on macrophage polarization process of histone modification,and the influence of the process of macrophage polarization ,analysis deacetylase inhibitors whether have the effect on the activity of the macrophage polarization by altered histone modification of macrophages , in order to provide a new perspective for the treatment of autoimmune diseases .Methods:Using lipopolysaccharide ( LPS) and interferon-γ( IFN-γ) to stimulate J774.1 cells for 24 h,and interleukin-4 ( IL-4 ) to stimulate J774.1 cells for 24 h.And 2 mmol/L valproic acid ( VPA ) was added in the induction process.Collecting J774.1 cells,fluorescent quantitation PCR assay and ELISA assay was used for the detection of specific markers of gene expression in macrophage polarization , flow cytometry and immunofluorescence assay for the detection of histone modifications.Results:J774.1 cells were polarized into M1 macrophages which were stimulated by LPS and IFN-γfor 24 h;and also J774.1 cells were polarized into M2 macrophages which were stimulated by IL-4 for 24 h.The degree of acetylation of H 3K9 for M1 phenotype was increased after VPA treatment , the expression of interleukin-6 (IL-6 ) , inducible nitric oxide synthase ( iNOS ) , and chemotactic factor(CCL-2) was decreased,and the expression of CD86 was increased.The degree of acetylation of H3K9 for M1 phenotype was also increased after VPA treatment ,and also the expression of Arginase,Fizz-1,mannose receptor(CD 206) and Ym1 were increased.Conclusion:The polarization state of the macrophages and histone modification had a certain relevance .VPA could induce the transformation of M1 phenotype to M2 phenotype in the induction system of the M1 macrophages,however,the expression of specific genes in M1 phenotype was inhibited in the induction system of the M 2 macrophages.
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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 observe the effect of CD40 siRNA on the changes in kidney,urinary protein and complement C3 of MRL/Lpr mice and explore its therapy on lupus nephritis.Methods: 16 female MRL/Lpr mice were randomly divided into control group,empty vector group,CD40-siRNA1 group and CD40-siRNA2 group.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.There were six times injection and every one day.The 24 hours urine output was gathered 24 hours before mice were killed.Fourteen days following administration,these mice were killed,tissue sections of kidney were observed if the signal of siRNA were expressed in kidney.The expression levels of CD40 mRNA and protein in kidney tissue of MRL/Lpr mice were detected by RT-PCR and immunohistochemistry methods respectively.At the same time,the pathological changes of the kidney were observed by haematoxylin-eosin ( HE) staining method.The 24 h urinary protein content was detected using the method of coomassie brilliant blue and the expression levels of complement C3 in serum were detected by Immunoturbidimetric assays.Results:The vector of CD40-siRNA was expressed in kidney of MRL/Lpr.The expression levels of CD40 mRNA and protein in kidney were lower in CD40-siRNA1 group and CD40-siRNA2 group than control group and empty vector group on the 14th day after last injection ( P<0.05).The inflammatory cells infiltration of kidney and some glomerular volume were significantly reduced in CD40-siRNA1 group and CD40-siRNA2 group than control group and empty vector group.The renal tubular swelling was alleviated in CD40-siRNA1 group and CD40-siRNA2 group than control group and empty vector group.The levels of 24 hours urinary protein were lower and the levels of complement C3 were higher in CD40-siRNA1 group and CD40-siRNA2 group than control group and empty vector group ( P<0.05). Conclusion:CD-40 siRNA can suppress the expression levels of CD40 mRNA and protein and decrease inflammatory cells infiltration in kidney of MRL/Lpr.Meanwhile after suppressing expression of CD40 mRNA and protein, it can reduce the content of 24 hours urinary protein and elevate the level of complement C3 in serum,which CD-40 siRNA can delay progress of the disease and protect kidney,so that it has therapy effect on lupus nephritis.
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Objective To explore the clinical significance of dyslipidemia in patients with systemic lupus erythematosus (SLE).Methods By independent-samples t test,serum lipid level was compared between 326 SLE patients and 300 healthy controls.The total cholesterol (TC),triglyceride (TG),lowdensity lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) levels were partially compared in subgroups of SLE patients.The correlation of serum TC,TG,LDL-C and HDL-C with clinical manifestations and laboratory findings in SLE was analyzed by Pearson or Spearman correlation analysis.Results ①The serum levels of TC [(3.8±1.5) mmol/L],TG [(2.1±1.6) mmol/L] and LDL-C [(2.1±0.9) mmol/L] were significantly higher in SLE group than those of the control group [(3.4±0.6),(0.8±0.4),(1.9± 0.5) mmol/L],and the serum level of HDL-C [(1.2±0.9) mmol/L] was significantly lower in SLE group than that of the control group [(2.0±0.5) mmolFL] (t=4.953,P=0.000; t=14.569,P=0.000; t=3.204,P=0.001; t=-14.335,P=0.000].② The serum levels of TC [(4.0± 1.7) mmol/L],TG [(2.5± 1.7) mmol/L] and LDL-C [(2.2±1.0) mmol/L] were significantly higher in LN group than those of the non-LN group [(3.6±1.0),(1.6± 1.0),(1.9±0.7) mmol/L; t=2.646,P=0.009; t=6.292,P=0.000; t=3.261,P=0.001].③ The serum level of TG [(2.2±1.6) vs (1.8±1.4) mmol/L] was significantly higher in SLE patients with hypocomplementemia than that of the normal ones (t =2.098,P=0.038).The serum level of HDL-C [(1.1 ±0.4) vs (1.6± 1.7) mmol/L] was significantly lower in SLE patients with hypocomplementemia than that of the normal ones (t=-2.375,P=0.020).④ The serum level of TG [(2.3±1.7) vs (2.0±1.4) mmol/L] was significantly higher in anti-dsDNA antibody positive patients than that of negative ones (t=1.989,P=0.048).The serum level of HDL-C [(1.5± 0.4) vs (1.4±1.2) mmol/L] was significantly lower in anti-dsDNA antibody positive patients than that of negative ones (t=-2.979,P=0.003).⑤ The lipid level was correlated with the clinical manifestations and laboratory findings in SLE patients.Conclusion Dyslipidemia exists in patients with SLE and has close correlation with LN,hypocomplementemia and positive anti-dsDNA antibody.
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BACKGROUND:Recent studies have found that adipose mesenchymal stem cells can inhibit immune responses of T cells, B cells and dendritic cells. But it is unclear whether adipose mesenchymal stem cells are able to regulate the immune responses of macrophages. OBJECTIVE:To observe the expression of interleukin-6 and tumor necrosis factorαin J774.1 cells co-cultured with adipose mesenchymal stem cells co-cultured. METHODS:Adipose mesenchymal stem cells at a density of 2×107 per wel were inoculated into the upper chamber of the transwel and 24-wel plates. Then, J774.1 cells were suspended with cellculture medium containing 1 mg/L lipopolysaccharide and planted into the lower chamber of the transwel and 24-wel plates containing adipose mesenchymal stem cells as co-culture group. Meanwhile, inactive J774.1 cells to lipopolysaccharide-activat J774.1 cells served as negative and positive control groups, respectively. After 48 hours, the J774.1 cells were col ected to extract the RNA samples. Then, mRNA expressions of interleukin-6 and tumor necrosis factorαwere measured by real-time quantitative PCR. RESULTS AND CONCLUSION:Compared with the positive control group, direct-contact co-culture of adipose mesenchymal stem cells and J774.1 cells significantly reduced expressions of interleukin-6 and tumor necrosis factorαin J774.1 cells, but non-contact co-culture only reduced interleukin-6 expression. These findings indicate that adipose mesenchymal stem cells can inhibit the immune response of J774.1 cells activated by lipopolysaccharide.
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Objective To investigate the therapeutic effect of tumor necrosis factor (TNF)-αsiRNA on typeⅡcolla-gen induced arthritis (CIA) in rats. Methods The expression vectors of siRNA against TNF-αgene were constructed suc-cessfully and were injected by tail veil into CIA rats. Twenty-four CIA rats were randomly divided into 4 groups including model group, empty vector group, TNF-α-siRNA1 group and TNF-α-siRNA2 group. CIA rats were injected with the same dose of phosphate buffered sodium (PBS) and pGFP-V-RS vector respectively in model group and empty vector group, while TNF-α-siRNA1 group and TNF-α-siRNA2 group were injected with TNF-α-siRNA1 eukaryotic expression vector and TNF-α-siRNA2 eukaryotic expression vector respectively. Another 6 rats, which were not established CIA model, were in-jected with PBS (blank control group). The serum expression levels of IL-1 were detected by ELISA on day 1, 5, 9 and 13 af-ter injection. The expression level of TNF-αmRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) on day 13. Results The expression level of IL-1 was significantly higher on day 1, 5, 9 and 13 in model group than that of blank group (P＜0.05). The expression levels of IL-1 were significantly lower on day 1, 5 and 9 in TNF-α-siRNA1 group and TNF-α-siRNA2 group than that of model group and blank group (P ＜ 0.05). The expression level of TNF-αmRNA was significantly higher on day 13 in model group than that of blank group (P＜0.05). The expression levels of TNF-αmRNA were significantly lower in TNF-α-siRNA1 group and TNF-α-siRNA2 group than those of model group and emp-ty vector group (P＜0.05). Conclusion TNF-αspecific siRNA can suppress the levels of TNF-αmRNA and IL-1, which provides experimental basis for gene therapy of rheumatoid arthritis.
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Objective To construct two vectors of small interfering RNA (siRNA) expressing α-Fodrin and investigate its therapeutic effects on mice model with primary Sj(o)gren's syndrome (non-obese diabetic mice,NOD mice).Methods Sixteen 8-week-old NOD mice were randomly divided into four groups:the control group,the vector group,the α-Fodrin-siRNA1 group and α-Fodrin-siRNA2 group,4 mice in each group.Four template DNA of α-Fodrin siRNA were chemically synthesized and annealed to two double stranded (dsDNA),then digested by BamH Ⅰ and Hind Ⅲ.The digested double strands oligos were inserted into the downstream of U6 promoter of linearized pGFP-V-RS vector.Recombinant were confirmed by restrictive enzyme digestion and sequencing.Then the vectors were injected throughtail veil once a week,two times in total,while mice in the control group were injected with the same dose of phosphate buffer saline (PBS)and the vector group were injected with the same dose of vector vehicle.pGFP-V-RS was labeled by green fluorescent protein(GFP) and lacriminal glands underwent pathological examination.In addition,the expression of α-Fodrin mRNA in lung were detected by reverse transcription-polymerase chain reaction (RTPCR),and α-Fodrin protein in lacriminal glands and lung were detected by immuno-histochemistry.Serum interferon (IFN-γ),interleukin-17 (IL-17) concentrations in each group were detected by enzyme linked immunosorbent assay (ELISA) in order to observe changes in cytokine levels.At the same time,the pathological changes of the lacriminal glands and organs with hematoxylin-eosin (HE) staining were observed.The repeat ANOVA was used for statistical analysis.Results ① We constructed two siRNA eukaryotic expression vector successfully; ② α-Fodrin-siRNA could target to the lacriminal glands.③ Compared with the control group and vector vehicle group,the expression of α-Fodrin mRNA and protein were significantly decreased in the treatment groups.④ Compared with the control group [(11.73±2.73) pg/ml] and vector vehicle group [(15.40±1.99) pg/ml],serum IL-17 levels in the treatment groups were [α-Fodrin-siRNA 1 group (4.38±1.02) pg/ml; α-Fodrin-siRNA 2 group (4.55±0.06) pg/ml] significantly decreased (P＜0.05),but IFN-γ levels in the αt-Fodrin-siRNA group were not decreased significantly (P＞0.05).⑤ Compared with the control group and vector vehicle group,lymphocyte infiltration of lacriminal gland and inflammatory cell infiltration of alveolar and interstitial were significantly reduced in α-Fodrin-siRNA groups.Conclusion Specific α-Fodrin siRNA can inhibit the inflammation,and suppress the inflammatory infiltration of lacriminal glands and lung in mice with primary Sj(o)gren's syndrome.So the constructed vectors may slow the progression of pSS.
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Objective To explore the relationship between cardiac involvement and laboratory indicators in patients with rheumatoid arthritis(RA). Methods Cardiac echocardiography and ECG performance of 184 patients with RA were analyzed retrospectively. Results Among the 184 patients with RA, the pulmonary hypertension detection rate was 8. 3%, valvular disease 38. 9%, arteriosclerosis 27. 8%, wall to reduce the exercise 13.9%, myocarditis 5.6% and pericardial effusion 5.6%, according to the echocardiography examinations;Sinus tachycardia was evidenced in 15. 22% patients, ST-T changes in 39. 13%, electric axis left side in 8. 70%, branch block in 13.04%, left ventricular hypertrophy in 4. 35%, atrial fibrillation in 4. 35%, premature in 8.70%, early repolarization syndrome in 2. 17% and electric-axis right side in 4. 35% patients by ECG examinations. The serum level of CRP (46. 77 ±5. 87) mg/L was significantly higher in RA patients with cardiac involvement than that in the non-cardiac involvement patientsm (28. 45 ±3. 21) mg/L (P ＜0.05) ;While the serum level of ESR,RF,IgG,IgA,IgM, PLT showed no statistically significant differences between the two groups (P ＞ 0.05); Within RA patients withcardiac involvement, the serum level of CRP showed no significant difference among different sub-groups , which were classified according to the echocardiography performance (P ＞ 0.05). Conclusions Cardiac involvement occurred frequently in patients with rheumatoid arthritis. The valvular disease, arteriosclerosis, reducing of the wall motion and pericardial effusion are the main manifestations by echocardiography examination; Sinus tachycardia, ST-T changes,branch block and premature beats are the main ECG abnormalities. The serum level of CRP is significantly higher in RA patients with cardiac involvement than that with non-cardiac involvement patients. The higher level of CRP in patients with RA may indicate the cardiac involvement presence.