RÉSUMÉ
Objective:To analyze the methylation characteristics of the lymphocyte-specific protein-tyrosine kinase (LCK) promoter region in the peripheral blood circulation of rheumatoid arthritis (RA) patients and its correlation with clinical indicators.Methods:Targeted methylation sequencing was used to compare the methylation levels of 7 CpG sites in the LCK promoter region in the peripheral blood of RA patients with healthy controls (HC) and osteoarthritis (OA) patients. Correlation analysis and ROC curve construction were performed with clinical information.Results:Non-parametric tests revealed that compared with HC [0.53(0.50, 0.57)] and OA patients [0.59(0.54, 0.62), H=47.17, P<0.001], RA patients [0.63(0.59, 0.68)] exhibited an overall increase in methylation levels. Simultaneously, when compared with the HC group [0.38(0.35, 0.41), 0.59(0.55, 0.63), 0.60(0.55, 0.64), 0.59(0.55, 0.63), 0.58(0.53, 0.62), 0.45(0.43, 0.49), 0.57(0.54, 0.61)], the RA group [0.46(0.42, 0.49), 0.70(0.65, 0.75), 0.70(0.66, 0.76), 0.70(0.65, 0.75), 0.69(0.64, 0.74), 0.55(0.51, 0.59), 0.68(0.63, 0.73)] showed a significant elevation in methylation levels at CpG sites cg05350315_60, cg05350315_80, cg05350315_95, cg05350315_101, cg05350315_104, cg05350315_128, and cg05350315_142, with statistically significant differences ( Z=-5.63, -5.89, -5.91, -5.89, -5.98, -5.95, -5.95, all P<0.001). Compared with the OA group [0.65(0.59, 0.69), 0.65(0.60, 0.69), 0.64(0.58, 0.68), 0.50(0.45, 0.54), 0.63(0.58, 0.67)], the RA group [0.70(0.66, 0.76), 0.70(0.65, 0.75), 0.69(0.64, 0.74), 0.55(0.51, 0.59), 0.68(0.63, 0.73)] exhibited a significant increase in methylation levels at CpG sites cg05350315_95, cg05350315_101, cg05350315_104, cg05350315_128, and cg05350315_142, with statistically significant differences ( Z=-3.56, -3.52, -3.60, -3.67, -3.62; P=0.036, 0.042, 0.031, 0.030, 0.030). Furthermore, Pearson correlation coefficient analysis revealed a positive correlation between the overall methylation level in this region and C-reactive protein (CRP) ( r=0.19, P=0.004) and erythrocyte sedimentation rate ( r=0.14, P=0.035). The overall methylation level of the LCK promoter region in the CRP (low) group [0.63 (0.58, 0.68)] was higher than that in the CRP (high) group [0.65(0.61, 0.70)], with statistically significant differences ( Z=2.60, P=0.009). Finally, by constru-cting a ROC curve, the discriminatory efficacy of peripheral blood LCK promoter region methylation levels for identifying RA patients, especially seronegative RA patients, from HC and OA groups was validated, with an AUC value of 0.78 (95% CI: 0.63, 0.93). Conclusion:This study provides insights into the methylation status and methylation haplotype patterns of the LCK promoter region in the peripheral blood of RA patients. The overall methylation level in this region is positively correlated with the level of inflammation and can be used to differentiate seronegative RA patients from the HC and OA patients.
RÉSUMÉ
ObjectiveUsing the technology of siRNA to inhibit the gene expression of no-receptor tyrosine protein kinase Lck in T cells of asthmatic mice,and to study the therapeutic effect of Lck specific siRNA in asthmatic mice.MethodsReceptor tyrosine protein kinase Lck specific siRNA fragments were taken from chemosynthesis.In vivo-jetPEITM was used to transfect the siRNA into mice body through tail vein injection.The mice were killed 48 hours later,and the levels of IL-4,IL-17 in bronchoalveolar lavage fluid (BALF) were detected with respondent ELISA kits.The change of inflammatory histopathology in lung was observed with H.E.staining.The expression of Lck in lung was detected with immunohistochemistry (IHC),and the level of Lck in lung tissue homogenate was detected with Western Blot.Results Compared with asthmatic group[ (234.68 ± 11.15 ) pg/ml,( 96.76 ± 8.28 ) pg/ml],the levels of IL-4,IL-17 [ (234.68 ± 11.15)pg/ml,(96.76 ±8.28) pg/ml] in the BALF of siRNA interference group decreased, and the inflammation in the lung relieved.IHC indicated that the expression of Lck in lung decreased and the level of Lck in lung tissue homogenate decreased ( P ＜ 0.05 ).Conclusions Lck specific siRNA could reduce the level of IL-4,IL-17 in the lung tissues of asthmatic mice,and relieve the inflammatory reaction in lung.
RÉSUMÉ
Objective Using the technology of siRNA to inhibit gene expression of T cells'nonreceptor tyrosine protein kinase Lck in asthmatic mice,and to study the effect of siRNA inhibited Lck to the function of T cells in asthmatic mice.Methods The 21 - 23 bp RNA fragments of mouse T cell Lck were made by chemosynthesis.INTERFERinTMsiRNA Transfection Reagent was used as transfection reagent to transfect the siRNA into the spleen T cells of asthmatic mice for 48 hours.Then T cells were mixed with bone marrow dendritic cells (DC) of asthmatic mice for another 48 hours.Cell culture suspension was collected and the level of IL-4,IL-13,IL-2,INF-γ were detected with respondent ELISA kits; Western Blot was used to identify if the expression of Lck was blocked.Results The expression of Lck in T cells almost could not be detected in siRNA interference group.The levels of IL-4 and IL-13 in siRNA interference group( 10.19 ± 1.66,12.34 ±0.79) were lower than no-siRNA interference(28.06 ±2.88,27.87 ± 1.61 )and control group ( 22.07 ± 2.5 1,20.47 ± 2.37 ),and the difference was statistical significant ( P ＜0.01 ).Conclusions Special siRNA could block the expression of special gene,and Lck specific siRNA could block the activation and differentiation of T cells and reduce the secretion of inflammatory cytokines in asthmatic mice.
RÉSUMÉ
Objective To investigate the Lymphocyte-specific protein-tyrosine kinase(Lck)gene ex- pression in the renal tubule epithelial cells(TECs)of lupus nephritis,and the effect of interlenkin-2(IL-2) stimulation on its expression.Methods Proximal TECs derived from 6 weeks old spontaneous systemic lupus erythematosus(SLE)BXSB mice were exposed to IL-2(100 U/ml),the expression of Lck mRNA and protein was examined by reverse transcription-polymerase chain reaction(RT-PCR)and immunoblotting respectively. The difference of Lck gene expression before and after IL-2 stimulation was investigated.The expression of Lck protein in TECs of renal tissues of BXSB mice and human with lupus nephritis was observed through im- munohistochemistry.Results The expression of Lck mRNA and protein was very low in cultured TECs of 6 weeks old BXSB mice,but increased sharply after IL-2 stimulation(P