RESUMEN
Objective To investigate the suppressive effect of prostaglandin E2 (PGE2),hepatocyte growth factor (HGF) and indoleamine 2,3-dioxygenase (IDO) whose secretion was promoted by human umbilical cord mesenchymal stem cells(MSCs) on the activated peripheral blood CD4+ T cells in primary Sj(o)gren syndrome (pSS) in vitro.Methods Primary cultured umbilical cord MSCs were identified by flow cytometry,and peripheral blood CD4+ T cells were sorted in pSS patients.CD4+ T cells were cultured with CD3,CD28 antibody for 72 h to be the activated(control group);the activated CD4+ T cells were co-cultured with MSCs for 72 h(MSCs group) or MSCs were pre-stimulated with interferon-γ (IFN-γ),then the activated CD4+ T cells were co-cultured with pre-stimulated MSCs for 72 h (pre-stimulated group).The suspension of CD4+ T cells were collected and counted.PGE2,HGF and IDO in the supernatants were detected by ELISA.Mean in groups were compared using ANOVA,and multiple comparisons were used with LSD method.Results The concentrations of PGE2 in the supernataut of the control group,MSCs group and pre-stimulated group were (111 ±4) pg/ml,(2 814±6) pg/ml and (2 716±8) pg/ml (F=167 292.12,P＜0.01) respectively.The concentrations of HGF in the above groups were (597±9) pg/ml,(383±9) pg/ml and (727±12) pg/ml(F=878.61,P＜0.01) respectively.The concentrations of IDO in the above groups were (143±4) pg/ml,(835±5) pg/ml and (588±3) pg/ml (F=21 104.41,P＜0.01) respectively.Compared with the control group,levels of PGE2 significantly increased in the MSCs group and the pre-stimulated group that CD4+ T cells were co-cultured with MSCs (t=509.88,P＜0.01 and t=491.48,P＜0.01),and levels of IDO also significantly increased (t=202.69,P＜0.01 and t=130.39,P＜0.01),while the activation and proliferation of CD4+T cells were inhibited (t=-16.20,P＜0.01 and t=-31.48,P＜0.01).Compared with MSCs group,levels of PGE2 and IDO significantly decreased in pre-stimulated group (t=-18.40,P＜0.01 and t=-72.30,P＜0.01),and levels of HGF significantly increased in pre-stimulated group(t=41.51,P＜0.01),while the activation and proliferation of CD4+ T cells were further inhibited (t=-15.28,P＜0.01).Conclusion MSCs can inhibit the activation and proliferation of CD4+ T cells in pSS in vitro.The suppressive effect of MSCs may be achieved by promoting secretion of cytokines such as PGE2,HGF and IDO.HGF plays a more important role in the suppressive effect of MSCs pre-stimulated with IFN-γ.Too much PGE2 or IDO propably results in negative feedback regulation of MSCs.
RESUMEN
Objective To explore the relationship between the clinical features,serological markers and European League Against Rheumatism SS Disease Activity Index (ESSDAI) scores of primary Sj(o)gren's syndrome (SS).Methods We enrolled 106 patients,who fulfilled the 2002 classification criteria for primary SS from December 2008 to January 2015,to evaluate the relationship among the clinical characteristics,laboratory features,serological variables and ESSDAI scores.According to serological variables,the prognosis was subdivided into three distinct groups:favourable (no serological markers),intermediate (one serological marker) and poor (two or more serological markers).These data were analyzed by Chi-square test and variance analysis.Results The mean ESSDAI score of 106 pSS patients was (11±7).ESSDAI score was categorized according to the EULAR-SS recommendations as low activity,moderate activity and high activity (scores of 0-4,5-13 and ≥14,respectively),and the positive rate of antinuclear antibody (ANA) 1:100 (6 cases,37.5％;37 cases,66.1％;32 cases,94.1％) in three different ESSDAI levels was statistically different (x2＝18.110,P＜0.01).Those with positive ANA 1:100[positive (13±7) and negative (7±4)],anti-SSA antibody postive (12±7) and negative (9±7),anti-RNP antibody (positive 16±9 and negative 10±6) had higher ESSDAI scores than those with negative ones (F=8.812,P=0.0001;F=3.862,P=0.024;F=5.786,P=0.004).No statistical difference in ESSDAI means were found between patients with positive anti-SSB antibody,rheumatoid factor (RF),FS level,dry mouth,Raynoud's phenomenon and psychosomatic diseases.The ESSDAI scores of favourable group,intermediate group and poor group were significantly different (8±5,10±7,14±7,F=8.715,P=0.000 1).In comparison with the other two groups,the poor pSS patients had a higher frequency of positive ANA 1:100 (15 cases,55.6％;20 cases,57.1％;40 cases,90.9％),anti-SSA antibody(11 cases,0.7％;23 cases,41.1％;36 cases,81.8％),anti-SSB antibody (6 cases,2 2.2％;13 cases,37.1％;23 cases,52.3％),anti-RNP antibody (0 case,0;2 cases,5.7％;9 cases,20.5％) (x2=17.408,P=0.002;x2=14.306,P=0.006;x2=12.330,P=0.015;x2=1 1.482,P=0.022).Conclusion Patients with two or more serological markers may have higher ESSDAI score,and which in turn may associate with poor prognosis.
RESUMEN
Objective To investigate how human umbilical cord mesenchymal stem cells (MSCs) in vitro regulate the miRNA profile of activated peripheral blood CD4+T cells from patient with primary Sj?gren's syndrome (pSS). Methods Peripheral blood CD4+T cells from patient with pSS were sorted and divided into healthy naive group, pSS naive group, pSS activated group, MSC treatment group and MSC (pre-stimulated by IFN-γ) treatment group. CD4+ T cells were counted. MiRNA microarray technology was used to detect the expression profile of CD4+T cells, and the expression of miRNA125b and miRNA155 was verified by real time quantification-polymerase chain reaction (RT-PCR). Mean in groups were compared using ANOVA, and multiple comparisons were used with LSD method. Results Both MSCs and IFN-γ-MSCs could inhibit the proliferation of activated CD4+ T cells in a MSC-dependent manner, but there was no significant difference between two groups. Microarray analysis found that the differentially enriched miRNAs in pSS na?ve (vs healthy na?ve), pSS activation (vs pSS na?ve), MSC treatment (vs pSS activation) and pre-IFN-γ MSC treatment (vs pSS activation) were 42 miRNAs, 56 miRNAs, 21 miRNAs and 24 miRNAs, respectively. Furthermore, the expressions of miRNA125b and miRNA155 were verified by RT-PCR and found that miRNA125b relative level in 5 groups was 1.02 ±0.13, 0.80 ±0.11, 0.44 ±0.17, 0.76 ±0.17 and 0.81 ±0.15 (F=18.32, P<0.01), and miRNA155 was 1.5 ±0.8, 3.9 ±1.3, 8.4 ±2.6, 10.1 ±4.2 and 11.2 ±5.0 (F=26.65, P<0.01). Conclusion MSCs can regulate miRNA profile of activated CD4+ T cells in peripheral blood of patient with pSS, and partially reverse down-regulated miR-125b in activated CD4+T cells, which may play a regulatory role in inhibiting the activation of CD4+T cells by MSCs.
RESUMEN
Objective To observe the regulatory role of microRNA-1187(miR-1187)in hepatocyte apoptosis through miR-1187 targeting regulation of caspase-8 mRNA expression.Methods The acute liver failure model was established by injection of D-galactosamine plus lipopolysaccharides(LPS)in BALB/c mice.The liver tissues were collected for LNA-miRNA array analysis and functional analysis of genes targeted by miRNA.Embryonic murine hepatocyte cell line 2(BNL-CL2)was cultivated in vitro and treated with tumor necrosis factor(TNF)-α and D-galactosamine to induce the transfection of miR-1187 in transfected group or untransfected group.The expressions of miR-1187 and caspase-8 mRNA were detected by real-time polymeramse chain reaction(PCR)and caspase-8 protein was determined by Western blot.The apoptosis rate was detected by flow cytometry.The comparison of means between groups was done by t test.Results The miR-1187 signal was deceased with the development of acute liver failure.The 3'UTR of caspase-8 mRNA had direct binding sites with miR1187.In BNL-CL2 cell experiments,miR-1187 was down-regulated in untransfected group and decreased more slowly in transfected group(t=6.371,P<0.01).The expression of caspase-8 mRNA was up-regulated in untransfected group and increased less in transfected group(t=4.539,P<0.01).The apoptosis rate in transfected group was significantly lower than untransfected group(t=3.365,P<0.05).Conclusios miR-1187 is one of inhibitors of hepatocyte apoptosis.High expression of miR-1187 could regulate the expression of caspase-8 mRNA,thus inhibit the apoptosis of hepatocytes.
RESUMEN
Objective To investigate the expressions of microRNAs (miRNA) and cytokines in the peripheral blood mononuclear cells (PBMCs) of patients with hepatitis B virus (HBV) infection and analyze their relationship with inflammation. Methods PBMCs were collected from acute hapatitis B (AHB) patients of 3 groups, including acute episode, virus clearance, recover period, and chronic hepatitis B (CHB) of mild, medium, severe type, and HBV-related liver cirrhosis. Each group included 20 patients, and 17 healthy donors were as control. Reverse transcription-polymerase chain reaction was used to measure miRNA146, miRNA155, miRNA181, interferon (IFN)-α, IFN-β,interferon induced gene 54 (ISG54) and interferon regulate factor 5 (IRF5). Comparisons among groups were done by one factor analysis of variance. Results The expression of miRNA155 was high in acute episode of AHB (2. 386± 1. 835), and higher than healthy control (1. 498± 1. 276) (F=1. 137,P-=0. 045), while reduced in acute episode, virus clearance (1. 633±2. 291), and recover period (0. 642±0. 836) (F=2. 122,P=0. 022). The expressions of IFN-α and IFN-β in AHB reduced in acute episode, virus clearance and recover period ( F = 1. 880, P = 0. 038 ; F= 1. 835, P = 0. 048).The expression of miRNA155 in AHB is closely correlated with IFN-α and IFN-β (r = 0. 483, P=0. 004; r= 0. 660, P= 0. 0002, respectively). In addition, in HBV infectious patients, the expression of miRNA155 was correlated with alanine transarninase (ALT), serum bilirubin (TBil) (r=0. 342,P=0. 006; r=0. 322, P= 0. 011, respectively), but not with HBV DNA load. Compared with healthy control (1. 307+ 0. 935), miRNA181 was higher in patients with HBV infection (F= 2. 072, P=0. 045) except AHB in recover period (1. 873±0. 998). There was no statistical difference in the miRNA146 expression between various groups. Conclusions The exprossion of miRNAs might be involved in the host anti-HBV immune response during HBV infection, and closely correlated with expression of IFN-related immune factors.
RESUMEN
Objective To study the expression profile of microRNA (miRNA) and the roles in pathogenesis of acute liver failure in mouse model. Methods Eighty-five BALB/c mice were divided into four groups: 40 in model group of acute liver failure were intraperitoneally injected with Dgalactosamine (D-GalN) and lipopolysaccharides (LPS); 20 in D-GalN group were injected with DGalN only; 20 in LPS group were injected with LPS only; 5 in control group were injected with saline.Liver histology of mouse was observed at hour 0, 5, 7 of injection, and sera and liver tissues were collected at hour 0, 1, 3, 5, 7, 9 of injection. Meanwhile, levels of inflammatory factors [tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)] in serum and liver tissue were detected by realtime polymerase chain reaction (PCR). Lock nucleic acid (LNA)-based miRNA microarray technology was used to detect the expression profile of hepatic miRNA, and the expression of miRNA was verified by real time quantification-polymerase chain reaction (RT-PCR). Mouse macrophage Raw264.7 cells were induced by LPS in vitro and the expressions of miRNA at different time points were detected.The comparison of means among groups was analyzed using one way ANOVA and the correlation were analyzed by Pearson and Spearman correlation. Results Microarray analysis found that the expression profile of miRNA during the acute liver failure changed dramatically. There were 97 miRNA in model group changed significantly compared with control group (P＜0.01), including 21 up-regulated and 27down-regulated at hour 5 and 7 of injection. Furthermore, the expressions of miR 146a and miR-155were verified by RT-PCR and found they both increased progressively over time after injection.Correlation analysis showed that miR-155 was well correlated with both TNF-α and IL-6 expressions.It was further found that miR-146a and miR-155 were both up-regulated in activated Raw264.7 cells in vitro. Conclusions The expression profile of miRNA changes during acute liver failure in mouse model. Inflammation associated-miR-146a and miR-155 are both up-regulated significantly, which indicatcs that they may play an important regulatory role in pathogenesis of acute liver failurc.
RESUMEN
Objective To explore the genetic impact of three newly discovered single nueleotide polymorphism (SNP) sites of the transforming growth factor (TGF)-β1 gene on the susceptibility of the chronic hepatitis B virus (HBV) infection. Methods Genome DNA was extracted from the peripheral blood samples of 115 cases suffering from the chronic HBV infection (74 chronic hepatitis B, 41 cirrhosis) as well as 41 healthy volunteers. Thereafter, genotyping of rs2241715, rs2241716 and rs4803455 sites of the TGF-β1 gene was performed by genotype-specific polymerase chain reaction (PCR) analysis. The data were analyzed by the ehi square test and Fisher exact test. Results There was a significant difference of rs2241715 genotypes and allele frequencies between healthy volunteers and patients with chronic hepatitis B and cirrhosis (χ2 = 11.419, P＜0.01 and χ2 = 6.218, χ2 = 5.961,P＜0.05,respectively). Interestingly, the risk relative of subjects with T/T genotype suffered from chronic hepatitis B (OR = 2. 974, 95% CI = 1.209 - 7. 314, P = 0.018) and cirrhosis (OR = 3.228, 95%CI=1.201-8.675, P=0.020) was dramatically higher than that in patients with T/G or G/G genotypes. Conclusion The TGF-β1 rs2241715 T/T genotype appears to be associated with the chronic HBV infection.