Small molecule inhibitors of RORγt for Th17 regulation in inflammatory and autoimmune diseases / 药物分析学报

As a ligand-dependent transcription factor,retinoid-associated orphan receptor γt(RORyt)that controls T helper(Th)17 cell differentiation and interleukin(IL)-17 expression plays a critical role in the pro-gression of several inflammatory and autoimmune conditions.An emerging novel approach to the therapy of these diseases thus involves controlling the transcriptional capacity of RORyt to decrease Th17 cell development and IL-17 production.Several RORyt inhibitors including both antagonists and inverse agonists have been discovered to regulate the transcriptional activity of RORyt by binding to orthosteric-or allosteric-binding sites in the ligand-binding domain.Some of small-molecule inhibitors have entered clinical evaluations.Therefore,in current review,the role of RORyt in Th17 regulation and Th17-related inflammatory and autoimmune diseases was highlighted.Notably,the recently developed RORyt inhibitors were summarized,with an emphasis on their optimization from lead compounds,ef-ficacy,toxicity,mechanisms of action,and clinical trials.The limitations of current development in this area were also discussed to facilitate future research.

Study on the mechanism of Wenyang huazhuo tongluo formula contained serum inhibiting the proliferation of Th17 cells in scleroderma patients / 中国药房

OBJECTIVE To study the effects of Wenyang huazhuo tongluo formula conta ined serum on the expression and methylation of retinoic acid related nuclear orphan receptor （RORγt），and to explore the mechanism of its regulation of Th 17 cell proliferation in the treatment of scleroderma. METHODS Wistar rats were given 15，30，60 g/（kg·d）Wenyang huazhuo tongluo formula intragastrically to prepare different doses of drug-contained serum ，and peripheral blood of scleroderma patients were collected to sort Th 17 cells. Using blank serum as blank control ，methyltransferase inhibitor decitabine （DCA）as positive control ， Th17 cells were treated with different concentrations of drug-contained serum ，and then the proliferation of Th 17 cells was detected by CCK- 8；mRNA expressions of RORγt and IL-17 were detected by qRT-PCR ，and the protein expression of RORγt was detected by Western blot assay ；the protein expression of IL- 17 in the cell supernatant was detected by ELISA ，and the methylation level of RORγt gene promoter was detected by methylation-specific PCR. The transcriptional activity of RORγt gene promoter was detected by dual-luciferase assay. RESULTS Compared with blank control ，Wenyang huazhuo tongluo formula contained serum and DCA could inhibit the proliferation of Th 17 cells（P＜0.05），reduced the mRNA and protein expressions of RORγt and IL-17，enhanced the methylation level of RORγt gene promoter and attenuated ， the transcription activity of RORγt gene promoter. Except for mRNA expression of Th 17 and the methylation level of RORγt gene promoter in drug-contained serum low-dose group ，there were statistical significance in above indexes of other groups（P＜0.05）. CONCLUSIONS Wenyang huazhuo tongluo formula can promote the methylation lev el of RORγt gene，and inhibit the expression of RORγt and the secretion of IL-17，so as to inhibit the proliferation of Th 17 cells.

Effect of Warming and Heat-clearing Method on Expressions of FoxP3 and ROR-γt in Colon Tissue of Rats with Intestinal Endotoxemia Caused by Acute-on-chronic Liver Failure / 中国实验方剂学杂志

Objective::To study the effect of warming and heat-clearing method (Wenyang Jiedu Huayu decoction) on the expressions of Forkhead box P3 (FoxP3), Retinoic acid-related orphan receptor gamma t (ROR-γt) in colon tissue of mice with acute-on-chronic liver failure (ACLF), in order to explore the possible regulatory mechanism on intestinal endotoxemia (IETM) in liver failure mice. Method::The 130 SD rats were randomly divided into normal group (10 rats) and model group (120 rats). The ACLF mice model was established through the subcutaneous injection with bovine serum albumin and the intraperitoneal injection with D-galactosamine(D-Gal) and lipopolysaccharide (LPS). The model mice were randomly divided into model group, heat-clearing group (Yinchenhao decoction, 6.68 g·kg-1), warming group (Yinchen Zhufu decoction, 7.09 g·kg-1) and warming and heat-clearing group (Wenyang Jiedu Huayu decoction, 19.53 g·kg-1). The normal group and the model group were given distilled water by gastric lavage, while the other groups were given equal volume of corresponding Chinese herbal medicines for a week. The value of each index at 1, 12 and 24 h was measured. The ratio of Treg/Th17 cell in peripheral blood were detected and calculated by flow cytometry. Real-time fluorescence quantitative PCR (Real-time PCR) was used to detect the expressions of FoxP3 and ROR-γt in colon tissues of mice at different time points. In situ hybridization and immunohistochemistry were used to observe the expressions of FoxP3 and ROR-γt genes and proteins. Result::Compared with normal group, the ratio of Treg/Th17 in the model group decreased significantly at each time point (P<0.01). Compared with the model group, the Treg/Th17 ratio increased only in the warming and heat-clearing method group (P<0.05). Compared with normal group, the expression of ROR-γt in the model group was significantly higher (P<0.01), and the expression of ROR-γt in the model group was higher than FoxP3.Compared with the model group, the expressions of FoxP3 and ROR-γt mRNA in the heat-clearing group and the warming group decreased at each time point (P<0.05), and the expressions of FoxP3 and ROR-γt in the warming and heat-clearing method group decreased significantly (P<0.01). The expressions of FoxP3 and ROR-γt mRNA in warming and heat-clearing group decreased compared with those in the warming group and heat-clearing group (P<0.05). Conclusion::The mechanism of the warming and heat-clearing method on IETM in liver failure may be related to the regulation of FoxP3 and ROR-γt expressions.

Effect of Qi-Boosting Toxin-Resolving Granules and cisplatin on transplanted tumor of nasopharyngeal carcinoma and balance of Foxp3/ROR-γt / 中草药

Objective To investigate the immune tolerance of Qi-Boosting Toxin-Resolving Granules (QBTRF) to Treg in the micro- environment of nasopharyngeal carcinoma (NPC) transplanted tumor, and reveal the possible anti-tumor mechanism of traditional Chinese medicine. Methods Using tumor cells CNE2 to build grafted tumor models, the anti-tumor effect of the QBTRF and cisplatin was observed, and the protein expression levels of Foxp3, ROR-γt and the content of IFN-γ, TGF-β and IL-17 in serum were detected. Results It was found that the content of TGF-β and IL-17 in serum of the tumor granule group and the combination therapy group were significantly lower than the model group, the IFN-γ content was obviously higher than that of the model group and cisplatin group, the apoptotic rate was significantly higher than that of the model group, the protein expression of Foxp3 was significantly increased, and the expression of ROR-γt was obviously decreased, in which the combination group was obviously better than that of single-use groups (P < 0.01). Conclusion QBTRF can effectively regulate the balance of Treg/Th17 in mice, restore the function of Treg, enhance the immune function of T cells during chemotherapy in mice, and exert anticancer effect. Combined with cisplatin, it can reduce the toxicity of chemotherapy.

Effect of complete lung cancer resection on the balance of Th17/Treg in the peripheral blood of patients / 实用肿瘤学杂志

Objective The aim of this study was to investigate the effect of complete lung cancer resection on the balance of Th17/Treg cells in the peripheral blood of lung cancer patients. Methods Flow cytometry was used to detect the percentage of Th17 and Treg cells in the peripheral blood of 21 patients with lung cancer before and after operation and 21 healthy controls. RT-PCR was used to detect the expression of fork-head/winged helix transcription factor( Foxp3) and retinoic acid-related orphan receptor γt ( RORγt)in PBMC. The plasma levels of interleukin(IL) -17 and transforming growth factor(TGF) -β1 were detected by enzyme-linked immunosorbent assay(ELISA). Results After surgery,the percentage of Th17 cells in the peripheral blood decreased and the percentage of Treg cells increased in patients when compared to the pre -operation ( P <0. 01). After surgery,the expression of RORγt was decreased and the expression of Foxp3 was increased in the CD4 +T cells of patients in comparison with the pre-opera-tion(P<0. 01). After surgery,the expression of IL-17 was decreased and the expression of TGF-β1 was increased in the plasma of patients in comparison with the pre -operation( P <0. 01). In addition,the percentage of Th17 and Treg cells,the expression of RORγt and Foxp3,and the expression of IL-17 and TGF-β in the peripheral blood were increased in preoperative lung cancer pa-tients when compared to healthy controls. Conclusion There is a Th17/Treg imbalance in the peripheral blood of lung cancer pa-tients after complete resection of lung cancer.

Relationship between serum mannose-binding lectin and Th17/Treg cells in patients with silicosis / 中国职业医学

OBJECTIVE: To explore the relationship between serum mannose-binding lectin( MBL) and T helper cell 17( Th17)/regulatory T cells( Treg) balance in patients with silicosis. METHODS: A total of 101 male patients with silicosis were selected in silicosis group and 62 health individuals in control group using the cross-sectional study. The level of serum MBL was measured by enzyme linked immunosorbent assay. The ratio of Th17/Treg was recorded by flow cytometry.The relative expression of retinoid-related orphan nuclear receptor γt( RORγt) and forkhead box 3( Foxp3) mRNA in peripheral blood mononuclear cell were tested by real-time polymerase chain reaction method. RESULTS: The level of serum MBL in silicosis group was higher than that of control group( P < 0. 01). The ratio of Th17 cells and the relative expression of RORγt mRNA increased in silicosis group( P < 0. 05),while the ratio of Treg cells and the relative expression of Foxp3 mRNA decreased in silicosis group( P < 0. 05) compared to the control group. The level of serum MBL had negative correlation with forced expiratory volume in the first second,forced vital capacity and forced expiratory flow( P < 0. 05) in patients with stage Ⅰ and Ⅱ silicosis. Meanwhile,the level of serum MBL had negative correlation with Th17 ratio and RORγt mRNA relative expression( P < 0. 05),and positive correlation with Treg ratio and Foxp3 mRNA relative expression( P < 0. 05). CONCLUSION: MBL might participate in the development of silicosis through regulating the balance of Th17/Treg cells.

Balance of Treg/Th17 cells and the expression of related transcription factors in peripheral blood of patients with condyloma acuminatum / 中国感染控制杂志

Objective To evaluate the role and significance of Treg/Th17 cells imbalance in pathogenesis and recurrence mechanism of condyloma acuminatum (CA).Methods 52 patients with CA were selected as study group (CA group,27 with initial occurrence of CA,25 with recurrence of CA),30 healthy persons were as control group,flow cytometry was used to detect the proportion of Treg cells and Th17 cells in the peripheral blood,the expression level of Foxp3 mRNA and RORyt mRNA in peripheral blood mononuclear cells was determined by real-time quantitative polymerase chain reaction.Results The proportion of Treg cells and the expression level of Foxp3 mRNA in peripheral blood in CA group was higher than that in control group,recurrence CA group was higher than initial occurrence CA group,difference was significant(both P＜0.05);the proportion of Th17 cells and expression level of RORyt mRNA in CA group was significantly lower than that in control group,proportion of Th17 cells in recurrence CA group was lower than initial occurrence CA group,there was significant difference (both P＜0.05).The proporation of Treg/Th17 in CA group was higher than that in healthy controls(4.60[3.20,8.68] vs 1.39[1.05,2.05],P＜0.05),recurrence CA group was higher than initial occurrence CA group (8.19[4.21,10.81] vs 3.52 [2.47,4.85],P＜0.05).Conclusion There is an imbalance between Treg cells and Th17 cells in patients with CA,especially in patients with reccurrence of CA,the imbalance of Treg/Th17 cells may play an important role in the pathogenesis and recurrence mechanism of CA.

The changes of ROR-γt,IL-17A and Foxp3 mRNA expression and promoter methylation in TDI-induced mouse asthma / 中国职业医学

OBJECTIVE: To explore the changes of retinoic acid related orphan receptor-γt( ROR-γt),interleukin( IL)-17 A and forkhead / winged helix protein 3( Foxp3) mRNA expression and promoter methylation in the process of asthma induced by toluene-diisocyanate( TDI). METHODS: Specific pathogens free grade healthy male BALB / c mice were randomly assigned into asthma group and control group with 18 animals in each group. In the asthma group,the mice were sensitized with 0. 30% TDI( mass-volume concentration) dropped on the dorsum of both ears( 20 μL / ear) on day 1 and day 8. On day 15,the mice were challenged with 20 μL 0. 01% TDI( mass-volume concentration) by the trachea. The control group mice were sensitized and challenged by the same procedures with the same amount of solvent( acetone / olive oil). The mice were challenged 24 hours,the pathological changes of trachea and lung tissues were observed. The bronchoalveolar lavage fluid( BALF) from each group was collected,and the inflammatory cells in BALF were counted and classified. IL-4and Interferon-γ( IFN-γ) levels in BALF supernatant were measured by enzyme-linked immunosorbent assay. ROR-γt,IL-17 A and Foxp3 mRNA expression in the lung tissue were measured by real-time fluorescent quantitative polymease chain reaction. The degree of ROR-γt,IL-17 A and Foxp3 promoter methylation in lung issue were detected by Mass Array system.RESULTS: The asthmatic group demonstrated the symptoms of acute asthma,such as breathing deeply and fastly,bowing the back,lifting the forelimbs,et al. But the control group had no such symptoms in mice. Hematoxylin-Eosin staining showed obvious inflammatory lesions in the trachea and lung tissue of asthmatic mice. Compared with the control group,the white blood cell count,the neutrophil and eosinophil percentages in BALF,the IL-4,IFN-γ levels in BALF supernatant in asthma group were all significantly increased( P < 0. 01),meanwhile the lymphocyte and monocyte percentages in BALF were reduced( P < 0. 01); ROR-γt mRNA expression was significantly increased( P < 0. 01),and the degree of promoter methylation from sites 3,4,5,6,8,11 and 12 was significantly reduced( P < 0. 05); IL-17 A mRNA expression was significantly increased( P < 0. 01),and the degree of promoter methylation from sites 6 and 7 was significantly reduced( P < 0. 01); Foxp3 mRNA expression was significantly reduced( P < 0. 01),and the degree of promoter methylation from sites 1 and 10 was significantly increased( P < 0. 01). CONCLUSION: Th17 / Treg cell immune imbalance occurs in asthma induced by TDI. ROR-γt,IL-17 A and Foxp3 gene promoter methylation abnormalities may be involved in Th17 / Treg cell immune imbalance.

Clonal Expansion of Allergen-specific CD4⁺ T Cell in the Lung in the Absence of Lymph Nodes

The expansion of allergen-specific CD4⁺ T cells is a critical step in inducing airway inflammation during allergic asthma. Such clonal expansion of T cells is initiated through the interaction between allergen-bearing dendritic cells and allergen-specific naïve T cells in the draining lymph nodes. Whether such T cell clonal expansion also occurs in the lung, the primary organ encountering inhaled allergens, remains unclear. Compared with wild-type mice, we found similar frequencies of CD4⁺ T cells in the lung of lymph node-deficient Rorgt(gfp/gfp) mice after repeated exposure to inhaled allergens. In addition, we observed an evident population of CD4⁺ T cells that underwent clonal expansion in the lung of allergen-challenged mice treated with an S1P antagonist FTY720 in an in vivo proliferation study with CFSE-labeled OT-II T cells. Moreover, the expansion of allergen-specific CD4⁺ T cells was significantly enhanced in the lungs of Rorgt(gfp/gfp) mice in comparison to that of wild-type mice. These results together demonstrate that the clonal expansion of allergen-specific CD4⁺ T cells occurs in the absence of the lymph nodes, indicating that the lung can act as a primary site of the clonal expansion of CD4⁺ T cells in response to inhaled allergens.

Regulation of T cell lineage commitment by SMAR1 during inflammatory & autoimmune diseases.

Background & objectives: CD4+ T cells are involved in abnormal inflammatory responses causing adverse effects to the body. Th17 cells play a major role in immune disorders and the exact mechanism by which CD4+ T cells regulate its effector Th1 and Th17 phenotype at chromatin level is not clearly understood. This study was aimed to understand the role of matrix associated region (MAR) binding protein SMAR1 (scaffold/matrix attachment region binding protein 1) in T cell differentiation during inflammatory and autoimmune condition using SMAR1 transgenic mice as model. Methods: Wild type (C57BL/6J) and SMAR1 transgenic mice were used for isolation of T cells and further identification of different T cell lineages, along with histological analysis. Further, we studied autoimmune and inflammatory diseases using chemically induced and T cell transfer model of colitis and rheumatoid arthritis to better understand the role of SMAR1 in immune responses. Results: SMAR1 transgenic mice were resistant to dextran sodium sulphate (DSS) induced colitis with decreased expression of Th1 and Th17 specific cytokines. Overexpression of SMAR1 repressed Th17 response by negatively regulating RORγt and IL-17 expression. Downregulation of SMAR1 upregulated signal transducer and activator of transcription 3 (pSTAT3) and IL-17 expression that caused generation of more proinflammatory Th1 and Th17 cells leading to inflammation and disease. Interpretation & conclusions: Our results show an important role of SMAR1 in regulating CD4+ T cell differentiation during inflammatory disorders via regulation of both Th1 and Th17 signaling pathways. This study reveals a critical role of SMAR1 in maintaining the proinflammatory immune responses by repressing Th1 and Th17 cell function and it gives the novel insight into immune regulatory mechanisms.

Research progress of IL-17+Foxp3+T cells / 天津医药

Recently, it is reported that regulatory T cells (Tregs) can be reprogrammed into a novel population [interleu?kin (IL)-17+Foxp3+T cells] phenotypically and functionally resembling Th17 cells under complicated cytokine circumstanc?es. IL-17+Foxp3+T cells are characterized by production of IL-17 and expression of retinoic acid receptor related orphan re?ceptor (ROR)γt, demonstrating dual functions in immune response and providing novel insight into the interconnection be?tween Tregs and Th17 cells. In this review, we lay emphasis on the phenotype features, origination, differentiation and the pleiotropic functions of IL-17+Foxp3+T cells. Furthermore, we summarized the functions of IL-17+Foxp3+T cells in inflam?matory disease and tumor microenvironment.

Expression of and significance of RORγt and IL-17 in newborn mice with cyto-megalovirus infection / 中国免疫学杂志

Objective:By comparing the expression levels of retinoic acid-related orphan nuclear receptorγt( RORγt) and in-terleukin-17 ( IL-17 ) in newborn rats′spleen tissue with cytomegalovirus infection with normal newborn to provide experimental evidence for the pathogenesis of CMV infection.Methods:Forty-eight newborn BALB/c mice were randomly divided into control group and MCMV group.Mice in virus group was given intraperitoneal injection of MCMV virus suspension,while the control group were given the same dose of normal saline as controls.Eight mice in the two groups were killed at day 3,7 and 14.The animal were sacrificed at day 3,7 and 14( n=8 for each interval) and spleens were obtained from the two groups.MCMV DNA,RORγt mRNA,IL-17 mRNA and protein of RORγwere detected by using RT-PCR and Western blot between the two groups.Results:Expression of MCMV DNA was in-creased in the MCMV group but absent in the control group.RORγt mRNA, IL-17 mRNA and protein expression of RORγt were significantly higher than the normal control group,with the extension of the infection time gradually increased( P<0.05 ) .Conclusion:The IL-17 and RORγt in spleen tissue may take part in the inflammatory response induced by MCMV, and may be involved in the pathogeneses of MCMV injury.

Effects of leptin on Th17 cells and the possible mechanism / 中华微生物学和免疫学杂志

Objective To investigate the effects of leptin on Th17 cells and the possible mechanism. Methods The leptin-deficient ( ob/ob) mice and their homologous wild-type mice were used in the study.The percentages of Th17 cells in peripheral blood samples, spleen tissues and lymph nodes were measured by flow cytometry ( FCM) analysis.The splenic CD4+T cells, separated from the ob/ob mice and the wild-type mice by using magnetic beads,were respectively cultured with leptin at various concentrations and with anti-leptin neu-tralization antibody to evaluate the effects of leptin on Th17 cells.The quantitative real-time PCR was performed to analyze Th17 cell-related cytokines at transcriptional levels.The levels of IL-6 and IL-17A in the supernatants of CD4+T cell culture were measured with Luminex technology.Results Compared with the wild-type mice, the ob/ob mice showed lower percentages of Th17 cells in both peripheral blood samples and spleen tissues (0.49%±0.03%vs 1.29%±0.1%, 1.56%±0.22%vs 2.47%±0.11%).There was a decrease in the percentages of Th17 cells upon the in vitro treatment of CD4+T cells from wild-type mice with anti-leptin antibody.The per-centages of Th17 cells were increased in a dose-dependent manner upon the in vitro treatment of CD4+T cells from ob/ob mice with leptin.Moreover, the levels of IL-17A and IL-6 and the transcriptional levels of RORγt, IL-17A and IL-6 in leptin deficiency group were lower than those of wild-type group, but were increased upon the treatment with leptin.No significant difference with the transcriptional levels of TGF-βand IL-23 was ob-served between the two groups with and without intervention.Conclusion Leptin deficiency seriously hampered the generation of Th17 cells in mice and resulted in a decreased expression of RORγt, IL-17A and IL-6 at mRNA level.The treatment of CD4 T cells with leptin might promote the generation of Th17 cells through up-regulating the transcription of RORγt and IL-6.

Imbalance of Treg/Th17 cells in peripheral blood of elderly patients with laryngeal cancer and its significance / 国际肿瘤学杂志

Objective To investigate the relationship of the expression characteristics of CD4 + CD25 + Foxp3 + Treg cells and CD4 + IL-17 + Th17 cells and the progress of the elderly patients with laryngeal cancer.Methods Sixty elderly patients diagnosed as laryngeal cancer in our hospital from January 2013 to June 2014 were chosen.Among them,28 cases had lymph node metastasis and 32 without.Thirty cases without laryngeal diseases were chosen as control.The peripheral blood of elderly patients with laryngeal cancer and normal controls was extracted.The expression levels of Treg and Th17 cells in all groups were detected with flow cytometry,and the expression changes of Foxp3,RORγt in peripheral blood mononuclear cell (PBMC) were detected with real time quantitative PCR.Results Compared with the control group,CD4 + CD25 + Foxp3 + Treg and CD4 + IL-17 + Th17 cell percentage,Treg and Th17 cells specific transcription factor Foxp3,RORγt expression levels in elderly laryngeal cancer patients without lymph node metastasis were significantly higher (t=9.817,P=0.018; t=12.120,P=0.009; t=6.090,P=0.023; t=7.130,P=0.028).Compared with elderly laryngeal cancer patients without lymph node metastasis,Treg and Th17 cell percentage and Foxp3,RORγt expression levels were significantly increased (t =9.070,P =0.014; t =12.140,P =0.009; t =10.130,P =0.009 ; t =13.070,P =0.008),and the ratios of Th17/Treg and Foxp3/RORγt were significantly lower (2.401 ± 0.614 vs 3.763 ± 0.959 ; 0.401 ± 0.075 vs 0.563 ± 0.091 ; t =9.070,P =0.014 ; t =11.140,P =0.007) in elderly laryngeal cancer patients with lymph node metastasis.Conclusion In elderly patients with laryngeal cancer,Treg cells and Th17 cells imbalance is positively correlated to disease progression.Therefore,monitoring and correcting the expression levels of Treg and Th17 cells may be important for the diagnosis,treatment and prognosis of the elderly patients with laryngeal cancer.

The level of miR-206 in per ipheral blood mononuclear cells and their signicance in systemic lupus erythematosus patients / 中华风湿病学杂志

Objective To explore the role of miR-206 in peripheral blood mononuclear cells (PBMCs) of systemic lupus erythematosus (SLE) patients.Methods Human PBMCs were isolated by standard densitygradient centrifugation over Ficoll-Hypaque solution in SLE and healthy controls.Total RNAs were extracted from PBMCs which were stimulated by PMA and ionomycin,thenthe RNA was transcribed reversely into cDNA.The expression of miR-206 and Kruppel-like factor 4 (KLF4) and the orphan nuclear receptor RORγt mRNA was detected by real-time quantitative polymerase chain reaction (qRT-PCR) method.T test and Spearman's correlation test were used for statistical analysis.Results The expression of miR-206 in the PBMCs of SLE patients was significantly decreased compared with that of healthy controls (0.066±0.021 vs 0.143±0.059,t=3.136,P＜0.01).The levels of KLF4 and RORγt mRNAin the PBMCs of SLE patients were increased significantly than those of healthy controls (0.637 ±0.186 vs 0.104 ± 0.028,t=6.673,P＜0.01),(0.575±0.263 vs 0.065±0.014,t=7.386,P＜0.01).Furthermore,there was a negative correlation between the expression of miR-206 and KLF4 or RORγt mRNA in SLE patients (r=-0.627,P＜ 0.01),(r=-0.853,P＜0.01).Conclusion These results indicate that the augmented expression of KLF4 mRNA may be caused by the attenuated expression of miR-206,and the high level of KLF4 mRNA evokes the proportion of Thl7 cells in SLE patients.

Level of MicroRNA-2 0 6 in Peripheral Blood Mononuclear Cells from Rheumatoid Arthritis and Their Significance / 现代检验医学杂志

Objective To explore the role of microRNA-206 (miR-206)in peripheral blood mononuclear cells (PBMC)from rheumatoid arthritis (RA)patients.Methods 27 patients with RA and 25 healthy controls were enrolled into the current study.Human peripheral blood mononuclear cells (PBMCs)were isolated by standard density-gradient centrifugation over Ficoll-Hypaque solution in rheumatoid arthritis and healthy control volunteers.Total RNAs were extracted from PBMCs which were stimulated by PMA and ionomycin,then the RNA was transcribed reversely into cDNA.The expression of mi-croRNA-206 and Kruppel-like factor 4 (KLF4)and the orphan nuclear receptor RORγt mRNA was detected by real-time quantitative PCR (qRT-PCR)method.Student’s unpaired t-test and spearman correlation were used for statistica1 analysis. Results The expression of miR-206 in the PBMCs of RA patients was significantly decreased compared with that of the healthy controls (0.056±0.019 vs 0.138±0.057,t=3.103,P<0.01),The levels of KLF4 and RORγt mRNAin the PB-MCs of RA patients were increased significantly verves those of the healthy controls(0.604±0.183 vs 0.098±0.027,t=6.651,P<0.01;0.583±0.271 vs 0.069±0.018,t=7.438,P<0.01),Furthermore,a negative correlated between the ex-pression of miR-206 and KLF4 or RORγt mRNA in RA patients (r=-0.639,P<0.01;r=-0.842,P<0.01).Conclusion These results indicated that the augmented expression of KLF4 mRNA may be caused by the attenuated expression of miR-206,and the high level of KLF4 mRNA evokes the proportion of Th17 cells in RA patients.

Molecular biological characteristics of the transcription factor RORγt and the expression in immune-related diseases / 医学研究生学报

RORγt, an immune cell-specific isoform of RORγ( retinoic acid receptor-related orphan nuclear receptor gam-ma) , is a key transcription factor for the development of Th 17 cells both in human and mouse .RORγt is required for the induction of IL-17 transcription and for the manifestation of Th 17-dependent autoimmune diseases in mice .RORγt natural ligands are retinoic acid . RORγt is closely implicated in the pathology of numerous autoimmune diseases , infectious diseases and cancer .With the further re-searches about the role of RORγt, we will clarify the mechanism of RORγt in autoimmune diseases , which will provide new ideas to di-agnose and treat autoimmune diseases .

The significance of peripheral blood CD4 + T cell-derived leptin in asthmatic children / 临床儿科杂志

Objectives To explore the role of CD4+T cell-derived leptin in peripheral blood mononuclear cells (PBMC) in asthmatic children. Methods Peripheral blood mononuclear cells were isolated from peripheral blood of both healthy subjects and asthmatic children in attack and remission stages. CD4+T cells were purified from PBMCs by mag-netic beads and were cultured in vitro. Supernatants were used to detect the levels of leptin by ELISA. The expression of the orphan nuclear receptor (ROR)γt was detected by real-time quantitative PCR (qRT-PCR) method. Results There was significant difference in CD4+T cell-derived leptin levels of asthmatic children in attack stage (68.46±13.08 pg/ml), remis-sion stage (36.73±6.13 pg/ml) and normal controls (32.82±5.79 pg/ml) (P0.05). The plasma leptin of children in attack stage and remission stage, as well as in normal subjects were 16.64 ± 3.53, 14.91 ± 3.24 and 13.72 ± 5.79 ng/ml respectively with no significant differences (P>0.05). The levels of RORγt mRNA were 0.341 ± 0.175, 0.089±0.028 and 0.068±0.018 in children with asthma during attack stage, remission stage and in normal children respec-tively (P0.05). Furthermore, the result of this study showed CD4+T cell-derived leptin positively correlated to RORγt in asthmatic children in attack stage (r=0.681, P<0.01). Conclusions CD4+T cell-derived leptin is elevated in asthmatic children in attack stage and its level is closely related to the pathological process of asthma.

Study of the level and function of Th17 cells in the peripheral blood of asthmatic / 重庆医学

Objective To explore the role of Th17 cells in the pathogenesis of asthma .Methods Peripheral blood was obtained from 10 patients with asthma(asthma group) and 10 healthy volunteers(control group) .RORγt mRNA expression of lymphocytes was measured by RT-PCR ,percentage of Th17 cell was detected by FCM ,IL-17 in plasma was examined by ELISA .Results RORγt mRNA level in asthma group(0 .46 ± 0 .07) was significantly higher than that in control group (0 .15 ± 0 .02) ,proportion of Th17 cell in asthma group(28 .53 ± 7 .20)% was significantly higher than that in control group(14 .72 ± 2 .33)% ,P<0 .01 .Com-pared with IL-17 level in control group(59 .68 ± 8 .85)pg/mL ,there was significant increase in asthma group(102 .31 ± 11 .45)pg/mL(P<0 .01) .Conclusion Th17 cell is associated with asthma ,and probably aggravating asthma condition through increasing in-flammation secreting IL-17 .

The expression and significance of interleukin-9, interleukin-17 and interferon-γ in CD4+ T cells from patients with rheumatoid arthritis / 中华风湿病学杂志

Objective To detect the levels of IL-9,IL-17,and IFN-γ in CD4+T cells from patients with rheumatoid arthritis (RA).Methods The peripheral blood mononuclear cells (PBMCs) from patients with RA and healthy controls were obtained,then the CD4+ T lymphocytes were tested by immunomagnetic beads.The protein levels of IL-9,IFN-γ and IL-17 were measured by flow cytometry (FCM).The mRNA levels of IL-9,IL-17,RORγt and IFN-γwere also detected by qRT-PCR.Data were analyzed by comparison between groups using variance analysis,and Pearson's correlation analysis was used for linear correlation analysis.Results The isolation of untouched human CD4+ T cells from PBMC was effective and its purity was over 90％.The protein levels of IL-9,IL-17,IFN-γwere higher in patients with active RA as compared with patients with inactive RA (P＜0.01) which were (1.62±0.23)％ vs (1.15±0.24)％(P＜0.01),(1.47±0.20)％ vs (1.04±0.26)％(P＜0.01) and (8.1±0.6)％ vs (6.9±1.0)％(P＜0.01) respectively,so did the patients with RA when compared with healthy controls (P＜0.01).The mRNA levels of IL-9,IL-17,RORγt and IFN-γ were higher in patients with active RA as compared with inactive RA patients (P＜0.01),which were (3.0±0.6) vs (1.8±0.4) (p＜0.01) (4.2±0.9)vs (2.3±0.7) (P＜0.01),(4.1±0.7)vs (2.9±0.3) (P＜0.01)and (4.0±0.8)vs (2.3±0.6) (P＜0.01) respectively,so did the patients with RA when compared with healthy controls (P＜0.01).Intracelluar IL-9 levels were positively correlated with IL-17 (r=0.632,P=0.001),IFN-γ (r=0.515,P=0.008),DAS28 (r=0.519,P=0.009) and ESR (r=0.857,P=0.038) but had no correlation with CRP (r=0.38,P=0.61).Conclusion The levels of IL-17,IL-9,IFN-γare higher in the PBMCs of RA patients,and these cytokines may participate in the pathogenesis of RA.