Differential expression profiling of proteome for systemic lupus erythematosus with quantitative proteomic technique

AIM: To identify and quantify the total proteins in peripheral blood mononuclear cells (PBMC) of systemic lupus erythematosus (SLE) patients with quantitative proteomic technique, and to establish a differential expression profile of proteome for SLE. METHODS: Four-plex isobaric tags for relative and absolute quantification coupled with multiple chromatographic fractionation and tandem mass spectrometry were used to analyze the total proteins in PBMC from healthy controls and the patients of stable SLE, active SLE and rheumatoid arthritis. The proteins were identified by database searching with peptide mass fingerprinting. The differential expression of the proteins was compared. RESULTS: More than 400 proteins were identified. Compared with healthy controls, 44 proteins were discovered to be significantly expressed by more than 2 folds in stable SLE and active SLE, among which 9 proteins were up-regulated and 35 proteins were down-regulated. Compared with rheumatoid arthritis group, 52 proteins displayed 2 or more folds of changes in stable SLE and active SLE, including 19 up-regulated proteins and 33 down-regulated ones. The up-and down-regulated proteins between active SLE and stable SLE were 17 and 13, respectively. CONCLUSION: Quantitative proteomic technique is efficiently applicable for protein identification and relative quantitation in human peripheral blood mononuclear cells. Determination of the differentially expressed proteomic profile of SLE is helpful for better understanding the pathogenesis of SLE and developing new strategies for diagnosis and treatment of SLE.     

Correlation of gene modification between histone H3 lysine 4 trimethylation and DNA methylation in IgA nephropathy patients

AIM: To investigate the correlation of gene modification between histone H3 lysine 4 (H3K4) trimethylation and DNA methylation in IgA nephropathy patients. METHODS: H3K4 trimethylation variations were analyzed in peripheral blood mononuclear cells (PBMCs) from 40 IgAN patients and 40 healthy controls by the method of chromatin immunoprecipitation linked to microarrays (ChIP-chip). ChIP real-time PCR was used to validate the microarray results. Quantitative real-time PCR (qRT-PCR) was employed to analyze mRNA expression. DNA methylation in 4 selected positive genes was analyzed by the method of methylated DNA immunoprecipitation-quantitative PCR (MeDIP-qPCR). RESULTS: IgAN patients displayed higher H3K4 trimethylation level and lower DNA methylation level than those of the healthy controls. There were significant differences in DNA methylation and H3K4 trimethylation of 4 selected genes between IgAN patients and the healthy controls (P<0.05). CONCLUSION: Our studies indicate that significant alterations of H3K4 trimethylation and DNA methylation exist in IgAN patients. There is a correlation of gene modification between DNA mathylation and histone H3 lysine 4 trimethylation in IgAN patients.     

Clinical significance of elevated serum interleukin 15 in patients with active lupus nephritis

AIM: To detect interleukin 15 (IL-15) levels in peripheral blood from patients with active lupus nephritis and investigate its clinical significance. METHODS: IL-15 level was determined by enzyme linked immunosorbent assay (ELISA). The peripheral blood mononuclear cells (PBMCs) were isolated with grads density abaxiality. The inhibitory effects of dexamethasone on production of IL-15, IgG and anti-dsDNA antibody in cultured PBMCs from LN patients were also investigated. RESULTS: (1) Serum IL-15 level in LN patients was significantly higher than that in normal controls (P<0.01). Serum IL-15 level in active LN patients was significantly higher than that in remised patients (P<0.05). (2) Serum IL-15 level was positively correlated with SLEDAI, anti-dsDNA antibody and 24 h urine protein excretion in active LN patients. (3) Serum IL-15 level was significantly reduced in LN patients treated with combination of cyclophosphamide (CTX) and steroid for 12 weeks. (4) Secretion of IL-15, IgG and dsDNA antibody in cultured PBMCs from active LN patients was significantly higher than that in normal control group, and IL-15 level in supernatant of cultured PBMCs from active LN patients was positively correlated with IgG and dsDNA antibody. Dexamethasone inhibited the secretion of IL-15, IgG and dsDNA antibody in cultured PBMCs from LN patients. CONCLUSION: Serum IL-15 level in patients with active lupus nephritis is significantly elevated, suggesting that IL-15 may be involved in the pathophysiological process of LN. IL-15 may be used as an index to assess the activity of LN.     

Chemokine receptor CXCR4 and its ligand SDF-1 in the pathogenesis of ankylosing spondylitis

AIM: To determine the role of chemokine and its receptors in the pathogenesis of ankylosing spondylitis (AS). METHODS: Gene expression profiles of peripheral blood mononuclear cells (PBMC) in 13 AS patients and 7 healthy volunteers were determined by cDNA microarray with 588 targeting gene filter. Differentiated expressed CXCR4 and its only ligand SDF-1 were confirmed by semi-quantitative RT-PCR, ELISA, immunohistochemistry and FACS analysis using PBMC, synovial fluid mononuclear cells (SFMC) and synoviocytes. RESULTS: The gene expression profile of AS patients was significantly different from those of healthy volunteers. Higher expression of CXCR4 in monocytes and CD8+ T lymphocytes from PBMC in AS patients were found with statistical significance (P＜0.05) compared to those of healthy volunteers. The expression of SDF-1 was increased in PBMC, SFMC, synovial fibroblasts and lining layer cells of synovial membrane. CONCLUSIONS: The expression of CXCR4 was significantly increased in PBMC in AS patients. Its ligand SDF-1 was also found highly expressed in synovial fibroblast cell line and synovial membrane cells of AS patients, indicating that CXCR4 and SDF-1 may play a potential key role in the development and perpetuation of joint inflammation in AS patients.     

PCR-select differential screening of a subtracted cDNA library of heat-adapted rat liver

AIM: To reveal the molecular mechanisms of heat adaptation by study the change of gene expression in rat liver when the rats exposed to hyperthermia stress and heat adaptation. METHODS: Differentially expressed genes in rat liver in association with heat adaptation were identified using the suppression subtractive hybridization (SSH) technique to prepare subtracted cDNA libraries in heat adaptation. The PCR-amplified cDNA fragments generated by SSH were then ligated into the plasmid pGEM-T (Promega). PCR-select differential screening technique was used to filtrate differentially expressed genes. Each of these differentially expressed genes was sequenced and was compared to known sequences in the GenBank database. RESULTS: eight up-regulating expressed genes segments were isolated from the up-regulating differentially expressed library, three of which were known gene such as p53 and five genes were new expressed sequence tags. CONCLUSION: Up-regulating expressed genes such as p53 maybe participate in the signal transduction pathway of heat adaptation.     

植物基因克隆技术的发展与展望

抑制性差减杂交技术和基因芯片技术是近年来发展起来的研究基因差异表达的2种非常有效的方法.SSH技术或基因芯片技术单独使用都存在不同程度的缺陷和不足,但若将2种技术结合起来使用,则能充分发挥各自的优势,并最大限度地弥补各自的不足.鉴于此,对SSH技术与基因芯片技术的原理与优缺点以及两者的结合在植物研究上的应用进行了综述.     

Identification of differentially expressed genes associated with cell adhesion and immune regulation in peripheral blood eosinophils from asthmatic patients with suppression subtractive hybridization

AIM:To determine differentially expressed genes associated with cell adhesion and immune regulation in peripheral blood eosinophils from asthmatic patients. METHODS:Peripheral blood eosinophils were isolated from the asthmatic patients at the time of exacerbation and after improvement. Total RNA was extracted. Super SMART PCR cDNA was synthesized, suppression subtractive hybridization (SSH) and PCR-select differential screening technology were used to detect expressed genes. The differentially expressed genes were sequenced. RESULTS:High efficiency subtractive cDNA library was constructed successfully. Differential screening identified 15 differentially expressed genes, which were Charcot-Leyden crystal protein (CLC protein;galectin-10), putative pre-mRNA splicing regulator female-lethal (2D), aquaporin 9 (AQP9), IL-8, slingshot 2L (SSH-2L), PP1 catalytic subunit, beta isoform, helicase with zinc finger domain (HELZ), β2-microglobin (β2-MG) and a gene associated with mitochondrion. CONCLUSION:Increased expression of these genes might be associated with eosinophil migration, adhesion and immune regulation. Intervention of these pathways may provide a theoretical base for future new targeting treatment for asthma.     

Prolactin induces interleukin-6 expression in PBMCs of rheumatoid arthritis patients

ATM: To investigate the correlation between serum prolactin (PRL) levels and disease activity in rheumatoid arthritis (RA) patients, and the regulatory role of PRL in interleukin-6 (IL-6) release from peripheral blood mononuclear cells (PBMCs), and to explore the MAPK-related mechanism of IL-6 release in PBMCs. METHODS: The clinicopathologic and hematologic parameters of 40 new-onset RA patients in the Department of Rheumatology of our hospital between March and September 2015 were collected. Chemilumineseent immunoassay (CLIA) was used to detect the serum PRL levels in the 40 RA patients and 20 healthy controls. The levels of IL-6 secretion by the PBMCs were evaluated using ELISA. Quantitative real-time PCR was employed to examine the mRNA expression of prolactin receptor (PRLR). MAPK pathway protein p-p38 levels were evaluated by Western blot. RESULTS: Serum PRL level in the RA patients was significantly higher than that in the healthy controls (P<0.01). Serum PRL level in active RA patients was significantly higher than that in inactive RA patients (P<0.01). Serum PRL level was positively correlated with DAS28, ESR and CRP (P<0.01). The expression of PRLR in the PBMCs was markedly increased in the RA patients than that in the healthy samples (P<0.01). Exposure of the PBMCs to PRL in the culture increased the release of IL-6, which was abolished by PRLR gene silencing or blocking the MAPK pathway.CONCLUSION: Serum PRL level is related to DAS28, ESR and CRP of RA patients and could be used as a predictor of disease activity. PRL/PRLR-p38 MAPK-IL-6 pathway may play a central role in the pathogenesis of RA.     

High expression level of TAL1 gene in newly diagnosed acute myeloid leukemia

AIM: To investigate the characteristic of T-cell acute lymphocytic leukemia 1 (TAL1) gene expression in acute myeloid leukemia (AML) cell lines and in primary AML cells from de novo AML patients with different subtypes. METHODS: Real-time PCR was used to determine the expression of TAL1 mRNA in acute leukemia cell lines (Jurkat, CCRF-CEM, HL-60 and NB4 cell lines) and peripheral blood mononuclear cells from 47 newly diagnosed AML patients. Twelve healthy individuals were served as healthy control group. RESULTS: A significantly increased level in TAL1 mRNA was found in AML cell lines (HL-60 and NB4), T-cell acute lymphacytic leukemia (T-ALL) cell lines (Jurkat, CCRF-CEM) and primary AML cells compared with the healthy controls. Over-expression of TAL1 was found in all detected AML subtypes, the highest level of TAL-1 mRNA was found in AML-M1 and AML-M5 subtype (P<0.05). CONCLUSION: High expression of TAL1 in AML might influence the differentiation and proliferation of myeloid cells, further investigation needs to confirm whether it would be as a biomarker for pathogenesis of AML.     

Preliminary study of IL-17 signal transduction pathway in peripheral blood mononuclear cells from lupus nephritis patients

AIM: To investigate the role of IL-17 and its signal conduction component-JNK activity in the pathogenesis of LN. METHODS: Peripheral blood mononuclear cells (PBMC) were separated and cultured from 15 cases of active lupus nephritis (LN) patients. IL-6 level was detected by ELISA, IL-6 mRNA was checked with RT-PCR, and JNK activity was measured by Western blot. RESULTS: At same IL-17 end concentrations, there was a much higher level of IL-6 in LN group than in control group (all P<0.05). IL-17 induced a significant elevation of IL-6 mRNA expression and JNK activity in PBMC from LN patients in a time- and dose-dependent manner, which could be blocked markedly by IL-17 monoclonal antibody, mIgG₂₈, and dexamethasone. Much higher IL-6 mRNA expression was observed in LN group than in control group under medium culture or IL-17-conditioned culture (all P<0.01). Under medium culture or IL-17-conditioned culture, there was much higher JNK activity of PBMC in active LN group than that in control group. There was a positive linear correlation between PBMC JNK activity and their SLEDAI or IL-6 mRNA expression level in LN patients (r1=0.638, P<0.01; r2=0.644, P<0.01). CONCLUSION: These results suggest that IL-17 may take part in the initiation and progression of LN through induction of IL-6 overexpression by PBMC,and JNK hyperactivity may be necessary in the IL-17 signal transduction.     

Expression of GFP and Luc reporter genes driven by human myocardial myosin light chain gene promoter in human cardiomyocytes

AIM:To construct the lentiviral vectors with green fluorescent protein(GFP) and luciferase(Luc) reporter genes driven by human myosine light chain 2v gene promoter(pMLC2v) and to investigate their expression in human cardiomyocyte(HCM) cell line and human lung cancer cell line A549. METHODS:Human pMLC2v-specific lentiviral vectors with GFP(pMLC2v-GFP) or Luc(pMLC2v-Luc) were constructed and transfected into HCM and A549 cell lines. The expression characteristics of the reporter genes were observed by confocal fluorescent microscopy and bioluminescence detection. Common(nonspecific) promoter-driven GFP(GFP_C) or red fluorescent protein(RFP_C) lentiviral vectors were used as controls. RESULTS:Both cell lines expressed GFP and RFP 3 days after transfected with the nonspecific vectors. HCM specifically expressed GFP and Luc 3 weeks after transfected with the pMLC2v-GFP or pMLC2v-Luc vectors. However, A549 cells didn't show the similar expression pattern. CONCLUSION:The pMLC2v-GFP and pMLC2v-Luc lentiviral vectors are specific for newly proliferative cardiomyocytes, indicating that they can be used as reliable tools for tracking the differentiation of stem cells into cardiomyocytes in vivo.     

Differential expression of genes in the rat heart during centrifuge training

AIM: Centrifuge training can improve forward acceleration (+Gz) endurance. This study was to analyzed the gene expression of rat heart affected by centrifuge, and to research the molecular mechanism of improving+Gz endurance by centrifuge training. METHODS: Differential expressed genes between high+Gz endurance (+16Gz) rats, of test group after trained12 d and control were screened using suppression subtractive hybridization (SSH) and dot blot hybridization. The obtained expressed sequence tags (ESTs)were used as probes to perform RNA slot hybridization with heart total RNA isolated from each gruop of centrifuge training and high+Gz endurance and low+Gz endurance (+12Gz) rats, respectively. The positive ESTs were sequenced and analyzed using BLAST(nr) at NCBI.RESULTS: Three down-regulated ESTs were obtained from heart samples, all of them are new, and their expression levels were decreasing during centrifuge training. CONCLUSION: Centrifuge training can significantly affect the special gene expressions of rat heart, and the expression changes of these genes may be ralated to the mechainism that+Gz endurance can be improved by centrifuge training.     

Analysis of the distribution and clonality of TCR Vβ T cells in cord blood

AIM:To investigate the distribution and clonality of TCR Vβ subfamily T cells in cord blood. METHODS:The CDR3 of TCR Vβ 24 subfamily genes were amplified in mononuclear cells from 13 cases of cord blood. To observe the usage of TCR Vβ repertoire, the PCR products were further labeled with fluorescent and analyzed by genescan technique for the CDR3 size, to evaluate clonality of the detectable TCR Vβ T cells. Peripheral bloods from 10 cases of normal individuals and T cell line Molt-4 and Jurkat served as controls. RESULTS:Only 38.78%±16.26% of 24 Vβ subfamily T cell were selectively expressed in cord blood, predominantly in Vβ 3, 5, 8, 9 and 13, whereas all 24 Vβ subfamilies could be detected in T cells from peripheral blood of normal individuals. Genescan analysis showed that all PCR products of TCR Vβ subfamilies from cord blood or normal individual peripheral blood displayed multi-peaks. CONCLUSION:Some TCR Vβ subfamily T cells were absent in cord blood. All TCR Vβ subfamily T cells in cord blood displayed polyclonality.