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Objective:To explore the therapeutic effects of intravenous injection of small extracellular vesicles (sEVs) derived from human umbilical cord mesenchymal stem cells (MSCs) on experimental autoimmune uveitis (EAU) in mice.Methods:MSCs from human umbilical cord were cultured and the supernatant was collected.The sEVs were isolated by ultracentrifugation method and a NanoSight instrument was used to analyze the particle size.The expression of surface markers sEVs, CD9, CD81 and CD63 was determined via Western blot.The morphology of sEVs was observed with a transmission electron microscope.Forty-eight 7-week-old female C57BL/6 mice were seclected to establish the EAU model through immunization with interphotoreceptor retinoid-binding protein peptide 651-670 (IRBP 651-670). The mice were divided into sEVs treatment group and phosphate buffer solution (PBS) control group using a random number table, with 24 mice in each group.The mice in the sEVs treatment group were injected with 50 μg of MSCs-derived sEVs via tail vein on the 11th day after modeling.In the PBS control group, the mice were injected with the same volume of PBS.Six mice in each group were randomly selected to observe the inflammation of the retina after mydriasis with an ophthalmoscope every other day from 8th day following modeling and the inflammation scores were evaluated.Six mice were randomly selected and sacrificed on the 14th day and 6 on the 18th day following modeling in each group, and both eyeballs of the mice were enucleated.Retinal tissue sections of the 6 mice sacrificed on the 18th day were stained with hematoxylin-eosin and the pathological scores were evaluated.The infiltration of helper T 1 (Th1) cells and Th17 cells in the eyeballs of the 6 mice sacrificed on the 18th day following modeling was detected by flow cytometry.T cells were isolated from spleen and lymph nodes of the 6 mice sacrificed on the 14th day, and the proliferation of T cells under different concentrations of IRBP 651-670 (0, 1, 10 and 20 μg/ml) was detected using a 5-bromodeoxyuridine (BrdU) method.To further study the effects of MSCs-derived sEVs on Th1/Th17 cells differentiation, naive T cells of spleen from another 3 normal mice were isolated by magnetic bead negative sorting and incubated with 10 μg/ml MSCs-derived sEVs or 10 μg/ml PBS, and then were cultured under Th1/Th17 cell differentiation conditions, respectively.Flow cytometry was used to measure the differentiation of naive T cells into Th1/Th17 cells.This study protocol complied with the regulations of the care and use of laboratory animals in China and was approved by an Ethics Committee of Tianjin Medical University (No.TJYY2019103022). Results:The isolated human MSCs-derived sEVs was with an average diameter of (102.4±33.6) nm and showed a double-layer membrane vesicle structure under the transmission electron microscope.The CD9, CD63 and CD81 proteins were highly expressed in sEVs.The inflammation scores of the sEVs treatment group were significantly lower than those in the control group on day 14, 16, 18, 20 and 22 after modeling (all at P<0.05). The pathological score of mice in the sEVs treatment group was significantly lower than that of PBS control group on the 18th day following modeling ( P<0.05). The flow cytometry results showed that on day 18 after modeling, the proportions of Th1 and Th17 cells in eyeballs in the sEVs treatment group were (15.55±2.03)% and (15.67±2.15)%, respectively, which were significantly lower than (21.35±0.72)% and (20.90±1.10)% in the PBS control group ( t=6.58, 5.31; both at P<0.01). BrdU results showed that when the IRBP 651-670 concentration was 20 μg/ml, the T cell proliferation ability in the sEVs treatment group was inhibited obviously compared with the control group ( P<0.05). The proportions of naive T cells differentiated into Th1 cells and Th17 cells in the sEVs treatment group were (28.15±1.32)% and (11.60±2.23)% respectively, which were significantly lower than (31.58±1.75)% and (23.52±1.76)% of the PBS control group, and the differences were statistically significant ( t=3.93, 10.26; both at P<0.05). Conclusions:Intravenous injection of human umbilical cord MSCs-derived sEVs can reduce the inflammation in EAU mice.The mechanism may be related to inhibiting the differentiation of naive T cells to Th1 and Th17 cells, and reducing the infiltration of Th1 and Th17 cells in the eyeballs.
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Objective To explore the expression and significance of vitamin D (VitD) in patients with rheumatoid arthritis (RA),and analyze the relationship between its expression and clinical indicators.Methods Clin-ical parameters and laboratory examinations of RA cases (n=250) were collected.Clinical parameters included were gender,age,disease course,swollen joints number,tenderness joints number,visual analog pain score (VAS),disease activity score (DAS)28 score.Laboratory examinations included erythrocyte sedimentation rate (ESR),C-reactive protein (CRP),rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) antibody,antinuclear (ANA) antibody,antikeratin (AKA) antibody,anti-perinuclear factor (APF),anti-mutated citrullinated vimentin (MCV),antibody and anti-6-glucose phosphate isomerase (GPI) antibody,lymphocyte subsets in the peripheral blood and lymphocyte subsets of CD4+T cells.The level of 25-(OH)-Vit-D and clinical parameters,laboratory examinations were analyzed retrospectively.One-way ANOVA and KruskalWallis test were used for comparison among the groups;and the correlation analysis was performed by Pearson and Spearman rank correlation analysis.Results ① The level of 25-(OH) D in RA patients was significantly lower than that in healthy controls (t=11.676,P＜0.01).② According to 25-(OH)D level,RA patients were divided into the deficiency group,insufficient group and normal group,the tender joints count (x2=17.793,P＜0.001),the number of swollen joints (x2=12.635,P=0.002),ESR (F=6.330,P=0.002),VAS score (F=5.095,P=0.007,DAS28 (F=4.990,P=0.008) were different significantly amorg the three groups.③RF (x2=6.742,P=0.034) and anti-CCP antibody (x2=6.836,P=0.033) were different significantly among the three groups and the level of 25-(OH) D was negatively correlated with RF (r=-0.202,P=0.001),anti-CCP antibody (r=-0.220,P＜0.01),anti-MCV antibody (r=-0.109,P=0.002) and AKA (r=-0.215,P=0.001).④ The level of 25-(OH) D in the RF (t=-2.715,P=0.007),anti-CCP antibody (t=-2.03,P=0.044),AKA (t=-2.108,P=0.036) negative group was significantly higher than that in patients with antibody positive group.⑤ The level of Th1 (IFN-γ) cells (F=3.259,P=0.043) and Treg (CD4+CD25+Foxp3+) cells (F=4.342,P=0.031) were significantly different among the three groups and the level of 25-(OH) D was positively correlated with Treg (CD4+CD25+Foxp3+) cells (r=0.146,P=0.025).Conclusion Vitamin D is generally deficient in RA patients,which is significantly correlated with disease activity,RF,anti-CCP antibody,anti-MCV antibody,AKA and Th1,Treg cells.It is suggested that vitamin D may play an important role in the immunological pathogenesis and disease progression of RA.
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Objective To determine the CD4+CD25+Foxp3+T regulatory (Treg) cell levels in peripheral blood (PB) of patients with autoimmune diseases (AID) and age-and sex-matched healthy controls. Methods Clinical data and laboratory examinations of AID cases (n=1561) and healthy controls (n=196) were enrolled. The levels of PB Treg cells, other T lymphocyte subsets [total T, CD4+T, CD8+T, T helper1 (Th1), T helper 2 (Th2), and T helper17(Th17) cells] and clinical indicators, laboratory test results were analyzed retro-spectively. Data were analyzed by t test, Mann-Whitney U test,χ2 test and Spearman correlation analysis. Results ①The absolute counts of Treg [22.9(18.31, 36.47) vs 30.24(21.85, 41.34), Z=-3.974, P<0.01], total T (Z=-4.234, P<0.01), CD8+T (Z=-3.801, P<0.01), Th17 (Z=-3.740, P<0.01) cells in PB of patients with AID were significantly lower than those of healthy controls, the levels of CD4+T/Treg (Z=-3.366, P=0.001), Th1/Treg (Z=-3.213, P=0.001) and Th2/Treg (Z=-2.490, P=0.013) in PB of AID patients were higher than those of healthy controls.② The levels of inflammatory indicators were associated with numbers of T lymphocyte subsets. ③ The levels of Treg (Z=-3.624, P<0.01), total T (Z=-2.954, P=0.009), CD4+T (Z=-3.005, P=0.003), Th2 (Z=-1.896, P=0.049) cells in PB of the patients who had been treated with hormones and/or biological or non-biological disease-modifying anti-rheumatic drugs (DMARDs) were significantly lower while the levels of total T/Treg (Z=-2.460, P=0.014), CD8+T/Treg (Z=-3.197, P=0.001) in PB were higher than those of the primary treatment patients. ④ The levels of Treg (Z=-7.105, P<0.01), total T (Z=-2.954, P<0.01), CD4+T (Z=-6.909, P<0.01), Th1 (Z=-4.875, P<0.01), Th2 (Z=-5.751, P<0.01), Th17 (Z=-5.121, P<0.01) cells in PB of the patients with important organs involvement were lower while the ratios of total T/Treg (Z=-4.500, P<0.01), CD8+T/Treg (Z=-5.925, P<0.01) were higher than those non-organ involvement patients. ⑤ The absolute counts levels of T lymphocyte subsets in the AID patients were not significantly correlated with whether there was a single AID and/or multiple AID overlaps. Conclusion The absolute number of peripheral Treg cells decreases significantly in AID, and is correlated with inflammatory indicators. Patients with retreated and organ involve-ment have fewer Treg cells. Our results suggest that Treg cells may play an important role in the pathogenesis of AID.
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Objective To investigate the effect of CDH1 3'-UTR + 54C/T single nucleotide polymorphism(SNP) on expression of luciferase reporter gene and its association with susceptibility to cervical cancer. Methods The luciferase gene expression vectors containing CDH1 3'-UTR +54C/T SNP C or T allelotype were constructed. The effect of CDH1 3'-UTR + 54C/T SNP on expression of luciferase reporter gene in 293 T cells were tested by daul lucfferase reporter assay system. The CDH1 3'-UTR + 54C/ T SNP was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis in 280 cervical cancer patients and 330 healthy controls. Results The lucfferase activity analysis showed that the relative luciferase activity (RLA) of 293T cells with C allelotype was 1.46, which was significantly lower than that of the 293 T cells with T allelotype (3.01; t=2. 94, P =0. 042). There was no significant difference in age distribution between the cervical cancer patients and the healthy controls. The genotype frequency distribution of CDH1 3 '-UTR + 54C/T SNP in healthy controls did not significantly differ from that expected by Hardy-Weinberg equilibrium (P>0.05). The C allelotype frequency of CDH1 in cervical cancer patients was 80. 7%, which was significantly higher than that in healthy controls (74. 5%;χ2 =6.59, P=0.010). The T/T, T/C and C/C genotype frequencies of cervical cancer patients and healthy controls were 4. 3%, 30. 0%, 65. 7% and 5. 8%, 39. 4%, 54. 8%, respectively, which were significandy different (χ2=7.45, P =0.024). Compared with individuals with T/T or T/C genotypa, individuals with C/C genotype had significantly higher risks of developing cervical cancer (OR = 1. 578,95%CI=1.136 -2.191). Conclusion The C allelotypa of CDH1 3'-UTR + 54C/T SNP might decrease the expression of lucfferase reporter gene and the C/C genotypa might be a potential risk for cervical cancer development.