Specific surface markers of Th1/Th2 subsets and their identification

CD4+Tcells can be divided into Th1/Th2 subsets.Th1/Th2 imbalance participates many disease processes.A stable surface marker distinguishing Th1 and Th2 will greatly facilitate the investigation of Th1/Th2 interaction.Several surface molecules have been reported to be differentialy expressed between Th1 and Th2 cells.LAG-3, active ligands for P-and E-selectin, IL-18R, IL-12Rβ2, CC chemokine receptor(CCR5)were shown to be dominantly expressed on Th1 cells, whereas expression of CD30, ST2L, CRTH2, CCR3, CCR4 was reported to be preferential to Th2 cells.In this review, several surface molecules were mainly discussed.     

Role of B cells in CD45RB antibody-induced transplantation immune tolerance

AIM: To investigate the role of B cells in CD45RB antibody-induced transplantation immune tolerance. METHODS: Single cell suspension was made from the spleen of BALB/c nude mice disposed by CD45RB antibody, then mixed cultured with T cells of BALB/c mice and spleen cells of C57BL/6 mice. The Th1, Th2, Treg and Tm cells were monitored by flow cytometry during the culture process. The skin graft model was set up with B6.μMT^-/- mice as receptors and BALB/c mice as donors. CD45RB antibody was intraperitoneally injected into the receptors after transplantation and then CD3⁺CD45RB^hi cells were detected by flow cytometry. In another mixed lymphocyte culture, CD45RB antibody was added, and then B cells were isolated and injected into B6.μMT^-/- mice through the tail vein. The heart transplantation model was established with B6.μMT^-/- mice as receptors and BALB/c mice as donors, and then the survival and the migration of B cells to the thymus were observed. RESULTS: When T lymphocytes were co-cultured with B lymphocytes treated with anti-CD45RB monoclonal antibody(mAb) in vivo, the percentages of Th2 and Treg cells were up-regulated and Th1 cells were down-regulated, but Tm cells were not altered as compared with the control. In vivo without B lymphocytes, anti-CD45RB mAb also down-regulated the expression of CD45RB in T lymphocytes. The reduction was faster and the percentage of CD3⁺CD45RB^hi T cells was not altered as compared with the control. The B lymphocytes treated with anti-CD45RB mAb in vitro prolonged the lifetime of receptor in heart transplantation model but failed to induce complete tolerance. After recieving B cells treated with anti-CD45RB mAb and allogeneic heart transplantation, B cells migrated to the thymus in B6.μMT^-/- mice. CONCLUSION: B lymphocytes play a definite role in the transplantation immune tolerance induced by anti-CD45RB mAb through their affection on T-cell subgroups and also in the central tolerance. However, the induction of immune tolerance can not only rely on B cells.     

Effect of mesalazine on Th1, Th17 and Treg cells in mice with DSS-induced ulcerative colitis

AIM: To investigate the effect of mesalazine treatment on regulation of Th1, Th17 and Treg cells in mice with dextran sulfate sodium (DSS)-induced ulcerative colitis (UC). METHODS: The expression of IL-17, IFN-gamma and Foxp3 in the peripheral blood mononuclear cells (PBMC) and intestinal mucosa lamina propria mononuclear cells (LPMC) of DSS-induced UC mice was detected by flow cytometry analysis. The effect of mesalazine treatment on regulaiton of Th1, Th17 and Treg cells in the mice with DSS-induced ulcerative colitis was examined.RESULTS: The expression of IL-17, IFN-γ and Foxp3 on CD4+T cells were significantly higher in the PBMC of DSS-induced mice than those in control group. CD4+ IFN-γ+T cells and CD4+ Foxp3+T cells were higher in LPMC than those in control group, except CD4+IL-17+T cells. Moreover, the Th1, Th17 and Treg cells were higher in DSS group than those in control group in LPMC. However, only Tregs was higher in PBMC. Pre-treatment with mesalazine significantly decreased the number of Th17, Th1 and Treg cells of UC model mice both in PBMC and LPMC.CONCLUSION: The Th1, Th17 and Tregs cells in DSS-induced mice were significantly higher than those in control mice, suggesting that CD4+T cell subsets play an important role in the pathogenesis of UC. Mesalazine may play a role in the treatment of UC by regulating the Th1, Th17 and Tregs cells.     

Expression of cytokines and their receptors in leukemia cell lines and normal blood cells

AIM: To study the expression of cytokines and their receptors in leukemia cell lines and normal blood cells. METHODS: RT-PCR was used to detect expression of mRNA for cytokines in leukemia cell lines(HL-60,U937,K562,HEL,DAMI,MEG-01,HUT78 and CA) and normal blood cells, including CD34⁺ cells, megakaryocytes,platelets, peripheral mononucleates cells and granulocytes. RESULTS: ①CD34⁺ cells simultaneously expressed mRNA for IL-1(α,β),IL-3, IL-6 , G-CSF, GM-CSF and their receptors and SCFR,MPL as well. The granulocytes only expressed IL-6,IL-6R,G-CSFR,GM-CSF. Megakaryocytes and platelets only expressed IL-3R,IL-6,IL-6R,MPL.Interestingly, TGFβ₁ ,TNFα and their receptors sustained to express in normal cells.②Most leukemia cell lines were found to simultaneously express at least two or more stimulating cytokines and receptors ,while TGFβ 1 , TNFα and their receptors were expressed in all the leukemia cell lines we observed. CONCLUSIONS: ①Multi-autocrine loops exist in leukemia cells;②Imbalance of autocrine loops of positive and negative cytokines may be related to leukemia.     

TMT-tagged quantitative proteomics analysis of differentially expressed proteins and enrichment of signaling pathways in nasopharyngeal carcinoma cells with or without SATB1 over-expression

AIM: To analyze the effects of special AT-rich sequence binding protein 1 (SATB1) expression on the protein expression profiles in human nasopharyngeal carcinoma (NPC) cells, and to enrich the differential signaling pathways through bioinformatics analysis. METHODS: SATB1 over-expressing lentivirus and negative control lentivirus were used to infect the CNE1 cells, and then the cell lines were obtained by puromycin stressed method. The total proteins of the 2 cells were extracted, and the differentially expressed proteins were screened by TMT-labeled protein quantification technique and tandem mass spectrometry. The mRNA levels of the differential protein-coding genes were verified by RT-qPCR. GO analysis was used to annotate and enrich the differentially expressed proteins, and the KEGG database was used to enrich and analyze the signaling pathways of differential proteins. RESULTS: SATB1 over-expressing CNE1 cells were established through infected with associated lentivirus. Compared with the control group, 278 differentially expressed proteins were identified in SATB1 over-expressing CNE1 cells, in which 115 were up-regulated and 163 were down-regulated. 10 representative differential protein-coding genes were verified by RT-qPCR, which showed the consistence with the proteomic results. GO analysis indicated differentially expressed proteins were mainly involved in cellular processes, single-organism processes, biological regulation, metabolic processes, protein binding and catalysis. Cell components of differentially expressed proteins mainly existed in cell part, cells and organelles. KEGG analysis showed that differentially expressed proteins were involved in signaling pathways closely related to tumors, includeing MAPK, PI3K-Akt, AMPK, JAK-STAT, p53, PPAR, Hippo and HIF-1 signaling pathways. CONCLUSION: Over-expression of SATB1 significantly alters the protein expression profiles in the NPC cells and affects multiple signaling pathways closely related to tumors. Proteomics also provides a possible macro approach to the screening of molecular mechanisms, therapeutic and prognostic targets for NPC.     

Suppression of allergic airway inflammation in a mouse model by Der p2 recombined BCG-induced adaptive CD4+CD25+ Treg

AIM: The bacillus calmette-guerin (BCG) vaccine is the most widely used Th1-inducing vaccine. In recent years, some studies argued that mycobacterium vaccae can be used as adjuvant to induce regulatory T cells (Treg) and then suppress asthmatic airway inflammation. We previously have engineered recombined BCG that expressed Der p2 of house dust mites (Der p2 rBCG) on the cell wall. The aim of this study is to investigate the immune regulatory mechanisms of Der p2 rBCG. METHODS: Mice were vaccined with PS, BCG or rBCG. The relative proportion and the absolute numbers of related Tregs in spleen cells were analyzed. The suppressive activity of Der p2 rBCG-induced CD4+CD25+ T cells was detected both in vitro and in vivo. RESULTS: (1) Der p2 rBCG induced a CD4+CD25+Foxp3+ T cell subtype. (2) Der p2 rBCG-induced CD4+CD25+ T cells suppressed the proliferation of Th2 effector cells in vitro in an antigen-specific way. (3) Der p2 rBCG-induced CD4+CD25+ T cells mediated Der p2 specific suppression of airway allergy in vivo. CONCLUSION: Der p2 rBCG induces a CD4+CD25+Foxp3+ T cell subtype, which suppresses inflammation in allergic airway in a mouse model.     

Influence of ovariectomy and estrogen replacement on gene expression profile of myocardium in rats

AIM:To investigate the influence of ovariectomy and estrogen replacement treatment on profile of gene expression in myocardium by cDNA microarray,and to characterize the targeting genes of estrogen.METHODS:cDNA microarray containing 1 400 rat cDNAs was used to study the genes differentially expressed in myocardium between sham (Ⅰ),ovariectomy (Ⅱ,OVX) and estrogen replacement treatment (Ⅲ,OVX+E2) group.Then down-regulated genes in myocardium of OVX rats were further confirmed by RT-PCR.RESULTS:177 genes were differentially expressed in myocardium between sham and OVX rats,with 91 genes up-regulated and 86 genes down-regulated in OVX rats.164 genes were differentially expressed in myocardium between OVX and OVX+E2 rats,with 113 genes up-regulated and 54 genes down-regulated in OVX rats.There were 54 genes differentially expressed in OVX compared to sham and OVX+E2.They are involved in membrane channels and transporters (18),cell receptors (9),intracellular transducers/effectors/modulator (7) and metabolism (6).Most of the genes (45) were down-regulated in OVX rats and up-regulated in OVX+E2 rats.RT-PCR test confirmed the results of cDNA microarray.CONCLUSIONS:Long-term estrogen replacement may influence the expression of genes involved in membrane channels and transporters,cell receptors,intracellular transducers/effectors/ modulator and metabolism.Long-term estrogen replacement has some beneficial effects on ionic concentration and cardiac function which partially comes from the results of influence of expression on Na+,K+-ATPase and Na+/H+ exchanger.Estrogen has an inhibitory effect on the expression of dopamine receptor,which partially clarify the myocardial protection of estrogen.     

Functional expression of B7H1 on pancreatic carcinoma panc-1 cells

AIM: To observe the effect of B7H1 expression in pancreatic carcinoma cells on the proliferation and activation of co-cultured T lymphocytes. METHODS: B7H1 expression in panc-1 cells before and after interferon-γ(IFN-γ) treatment or B7H1-siRNA transfection was evaluated by RT-PCR and flow cytometry. The influence of B7H1 expression on co-cultured PHA-activated T lymphocytes was determined by the methods of MTT and enzyme-linked immunosorbent assay (ELISA). RESULTS: B7H1 was highly expressed in panc-1 cells and up-regulated after IFN-γ stimulation. Such up-regulation led to the significant inhibition of T cell proliferation and secretion of cytokines such as IFN-γ and interleukin-2(IL-2). However, IL-10 production was enhanced. In contrast, knockdown of B7H1 expression in panc-1 cells by RNA interference resulted in increased T cell proliferation as well as IFN-γ and IL-2 production. Meanwhile, the IL-10 secretion decreased. CONCLUSION: B7H1-expressing panc-1 cells suppress T cell function by inhibiting T cell proliferation and production of Th1 cytokines. Suppression of B7H1 expression through siRNA restores T cell immune functions, indicating a potential strategy for immunotherapy against pancreatic cancer.     

Kinetic expressions of PD-L1 and PD-L2 on the surface of human lymphocytes and monocytes

AIM: To investigate the expression kinetics of PD-L1 and PD-L2 on the surface of the resting and activated B/T cells as well as monocytes from healthy human peripheral blood. METHODS: Fluorescent antibody staining together with flow cytometry were used to detect the percentages of the resting as well as the activated B cells and T cells that expressed PD-L1 and PD-L2. Meanwhile the percentages of the resting and activated monocytes that expressed PD-L2 were determined. RESULTS: Both resting B cells and T cells did not express PD-L1 on their surface, however PD-L1 expression was significantly up-regulated on the surface of the activated B cells after 6 h stimulation with LPS or pokeweed mitogen (PWM), and the percentages of B cells that expressed PD-L1 reached a plateau at 24 h, which were (46.26±10.71)% with LPS and (43.67±6.14)% with PWM stimulation, respectively. No markedly change of PD-L1 expression on the surface of the activated T cells after stimulation with LPS was observed, but upregulation of PD-L1 expression was observed when stimulation with PWM. The percentages of T cells that expressed PD-L1 reached a plateau at 24 h, which was (25.42±9.23)%. PD-L2 expression was not found on the resting as well as the activated B cells and T cells. In addition, the resting monocytes did not express PD-L2. Combination of INF-γ plus LPS markedly induced the PD-L2 expression, and the percentages of monocytes that expressed PD-L2 reached a peak at 48 h, which was (28.70±14.22)%. CONCLUSION: The activated lymphocytes only express PD-L1, reaching a plateau at 24 h. PD-L2 is expressed on the surface of the activated monocytes, reaching a peak at 48 h.     

Study on polarized Th1/Th2 cell in vitro from adult human peripheral blood

AIM: To investigate the details of CD4⁺ T cell polarized to Th1/Th2 in vitro. METHODS: After isolated the PBMCs and blood-plasma from adult human peripheral blood by Ficoll-Hypaque centrifugation, the PBMC culture procedure with or without the self-blood -plasma was applied to polarize T cells in vitro, these cells were polarized by PHA(20 mg/L),non-PHA respectively. The polarized rates of Th cell after 24 h,48 h,72 h were estimated respectively by flow cytometry following two-color immunofluorescent staining. RESULTS: CD4⁺T cell would polarize to Th1/Th2 two subsets after self-cytokines and PHA activation in vitro. The polarized rates of T cell after cultured for 24 h,48 h and 72 h were (13.28%±1.59%)/(12.70%±1.65%),(17.19%±1.03%)/(17.50%±1.30%),(19.49%±2.87%)/(18.58%±1.49%) respectively, but the polarized rates of T cell were very low if without self-blood-plasma. The difference between them was significant. The ratios of Th1/Th2 cells were about 1. CONCLUSION: CD4⁺T cell from adult human peripheral blood would polarize to Th1/Th2 two subsets in the presence of self-blood-plasma and PHA(20 mg/L) in vitro, and the cell number of Th1 and Th2 would be in balance.     

IL-1β promotes differentiation of naïve T cells into Th22 cells and its expression in non-small cell lung cancer

AIM:To investigate the mechanism of interleukin-1β (IL-1β) promoting the transformation of naïve T cells into Th22 cells and the correlation of its peripheral blood expression in non-small cell lung cancer patients. METHODS:CD4⁺ naïve T cell magnetic bead sorting kit was used to isolate the peripheral blood mononuclear T cells from healthy people. Transforming growth factor-β (TGF-β) and IL-2 were added to promote differentiation and proliferation. IL-1β was used to induce differentiation into Th22 cells. The proportion of CD4⁺ IL-22⁺ T cells was analyzed by flow cytometry, and the expression of IL-22 was detected by ELISA. We selected 60 cases of non-small cell lung cancer patients in our hospital, including 18 in I phase, 20 in Ⅱ phase, 13 in Ⅲ phase and 9 in IV phase, as well as 25 healthy persons. The proportion of Th22 (CD4⁺ IL-22⁺) cells in peripheral blood was detected by flow cytometry, and the serum levels of IL-1β and IL-22 were measured by ELISA. RESULTS:IL-1β induced the transformation of naïve T cells into Th22 cells and promoted the secretion of IL-22 (P<0.05). The proportion of Th22 cells and the IL-22 and IL-1β levels in peripheral blood of the patients with non-small cell lung cancer were higher than those in healthy subjects, and correlated with the clinical stage. CONCLUSION:IL-1β induces the differentiation of Th22 cells and the expression of IL-22. The levels of IL-1β and IL-22 are related to the progression of non-small cell lung cancer, which may be involved in immunosuppression and promote the occurrence of non-small cell lung cancer.     

CTLA4Ig induces immune tolerance of T cells to oxidized-low density lipoprotein in vitro

AIM: Recently,it is widely accepted that atherosclerosis (AS) is an auto-immune related disease and the oxidized-low density lipoprotein (ox-LDL) is the most important AS-related antigen.In order to prevent immune injuries in AS and find new strategies to prevent AS,the immune tolerance of T cells to ox-LDL in vitro was induced in this study.METHODS: Human monocytes were separated from peripheral blood to induce dendritic cells (DCs).DCs were treated with LPS (30 μg/L),ox-LDL (10 mg/L) and LDL (10 mg/L) for 48 h.Then DCs were mixed with allogenic T lymphocytes to carry out mixed lymphocytes reaction (MLR).CTLA4Ig in different concentrations was added in the MLR of ox-LDL group.MTT method was used to assay the proliferation of T cells and expressed in stimulation index (IS).The CD25 expression and apoptosis of T cells in MLR were tested by flow cytometry.The excretion of IL-2,IFN-γ and IL-4 was assayed by ELISpot method.RESULTS: SI in ox-LDL group was higher than that in LDL group significantly (P<0.05) and CTLA4Ig inhibited the SI in ox-LDL group with dose-dependent effect (P<0.05,P<0.01).CTLA4Ig decreased the CD25 expression (P<0.05,P<0.01) and induced apoptosis of T cells in MLR (P<0.05,P<0.01).CTLA4Ig decreased the ELISpot counts of IL-2 and IFN-γ (P<0.01),while increased that of IL-4 (P<0.05).CONCLUSION: CTLA4Ig induces T cells tolerance to ox-LDL in vitro.CTLA4Ig inhibits T cells activation,promotes T cells apoptosis and Th1/Th2 immune deviation,which is the important mechanism in it′s induction of tolerance.     

大白菜杂交种和亲本之间基因表达差异分析

利用cDNA-AFLP技术研究大白菜白引2号杂交种及其亲本在幼苗期、莲座期、结球期的基因差异表达。结果表明：在3个发育期,发现有29条差异片段,其中5个差异片段出现在幼苗期,14个差异片段出现在莲座期,10个差异片段出现在结球期|19个差异片段在杂种中表达,3个差异片段在双亲中同时表达,2个差异片段仅在母本中表达,5个差异片段仅在父本中表达。将29个差异片段的序列在白菜基因组序列数据库网站（http://brassicadb.org/brad/）进行BLAST分析,均找到了对应的染色体位置。其中17个差异片段分别有明确的基因及编码蛋白与其相对应。其中有两个基因片段分别含有AP2/EREBP结构域和PPR结构域,这两个结构域在控制植物生长发育方面具有重要作用。     

Adipose-derived stem cells mediate immunosuppression of Th17 in multiple sclerosis

AIM: To investigate how human adipose-derived stem cells (hASCs) regulates the differentiation of Th17 cells in multiple sclerosis. METHODS: hASCs were isolated from the adipose tissues. Magnetic-activated cell sorting (MACS) kit was used to isolate CD4⁺ T cells from peripheral blood mononuclear cells (PBMCs) which were isolated by density gradient centrifugation. The percentage of CD4⁺ T cells was detected by flow cytometry. The activated CD4⁺ T cells were co-cultured with hASCs for about 4 d at different ratios of hASCs to CD4⁺ T cells (1:4 and 1:10) in a Th17 polarised condition. Another group adding anti-leukemia inhibitory factor (LIF) antibody was set up. Th17 cell proportion of the CD4⁺ T cells was determined by flow cytometry. The level of LIF in the supernatant of co-cultured system was measured by ELISA. The mRNA expression of retinoid-related orphan receptor γt (RORγt), interleukin-6 receptor (IL-6R), interleukin-23 receptor (IL-23R), LIF and leukemia inhibitory factor receptor (LIFR) was detected by real-time PCR. RESULTS: The result of flow cytometry suggested there were mainly hASCs, and the percentage of CD4⁺ T cells in the PBMCs were above 90% after MACS. The Th17 cell proportion decreased in 1:4 and 1:10 co-cultured groups in a dose-dependent manner. The mRNA expression of IL-6R, IL-23R and RORγt was downregulated and the expression of LIFR and LIF was up-regulated. When the anti-LIF was added into the co-cultured system, the ratio of Th17 cells increased and reached to the control level. The protein level of LIF obviously increased after co-cultured. After anti-LIF added, the mRNA expression of RORγt and IL-6R was up-regulated. CONCLUSION: hASCs inhibits the differentiation of Th17 cells from multiple sclerosis patients through the competitive inhibition of LIF/IL-6 by secreting LIF.     

Effects of inactivated HIV-1 particles on human CD4+T cell activation and Th1/Th2 cytokine secretion in whole blood

AIM: To investigate the effects of AT-2-inactivated HIV-1 particles on human CD4+T cell activation and cytokine secretion in whole blood (WB) in vitro. METHODS: HIV-1ⅢB particles were inactivated by AT-2 chemical and the concentration of p24 antigen was determined by p24 ELISA. AT-2-inactivated HIV-1ⅢB particles were added to human WB culture system in serial concentrations to stimulate the cells. PHA was used as positive control. After 24 h, all the cultural supernatants were harvested and the concentrations of Th1 (IL-2, IFN-γ and TNF-α) and Th2 (IL-4, IL-6 and IL-10) cytokines released to the supernatants were detected by cytometric bead array (CBA). The percentage of CD69 expression on CD4+T cells from WB was detected by immuno-fluorescence staining plus flow cytometry. RESULTS: The concentration of p24 antigen in the AT-2-inactivated specimen was 85.5 μg/L. 24 h later, the percentage of CD69 expression on CD4+T cells from control group was (1.62±0.63) %, whereas it was (38.82±6.00)%, (3.83±1.07)%, (5.94±0.85)% and (9.30±1.22)% in PHA group, HIV-1 (1/500) group, HIV-1 (1/50) group and HIV-1 (1/5) group, respectively. Cytokines secreted by WB in control group were mainly TNF-α and IL-6. However, all the six cytokines tested were strikingly increased in PHA group, as well as in HIV-1ⅢB groups. CONCLUSION: AT-2-inactivated HIV-1ⅢB particles activate CD4+T cells from WB, and up-regulate both Th1 and Th2 cytokine secretion in WB. Besides the effects of viral proteins, other mechanisms may be proposed that HIV-1 particles act as antigen presenting cell (APC) because many host-derived immune molecules are incorporated into HIV-1 envelop when it is released from infected cells by budding, and exert immune modulation.     

Proteomic study on mice hepatic cell membrane proteins after exposed to hypoxia

AIM: To identify the differential expression of mice hepatic cell membrane proteins after hypoxic exposure.METHODS: The hepatic cells of C57BL/6 mice were cultured for 8 h under hypoxic or normoxic conditions and the membrane proteins were extracted. The differentially expressed membrane proteins were separated by two-dimensional electrophoresis. The technique of matrix-assisted laser desorption/ionization mass time-of-flight spectrometry(MALDI-TOF-MS) was used to analyze the proteins.RESULTS: Compared with the proteins extracted from the cells under normoxic condition with a threshold of 1.5-fold, 28 differentially expressed proteins were identified in the proteins extracted from the cells under hypoxic condition according to the database NCBInr 20071130, in which 9 were down-regulated and 7 were up-regulated, including prohibitin, cytochrome b-c1 complex subunit 1 and p22HBP.CONCLUSION: The results suggest that prohibitin, cytochrome b-c1 complex subunit 1 and p22HBP might play important roles in sensing of cellular oxygen and the related signal transduction pathways.     

The immunity effect of B7-H1 blockade on immature dendritic cells

AIM: To investigate the immune stimulation capacity of B7-H1 blockade on immature dendritic cells (DCs) in vitro. METHODS: The human monocyte-derived dendritic cells were induced in the presence of cytokine GM-CSF and IL-4. The expression of B7-H1 was detected by FCM. On blockade of B7-H1, the maturation and endocytic activity, T cells stimulatory proliferation capacity, IL-12 production, T cell differentiation effect of DCs were detected by FCM, MTT assay, ELISA and ELISPOT, respectively. RESULTS: The expression of B7-H1 was increased with the induction of DCs. On day 7, the positive expression was 54.12%, and the TNF-α induced mature DCs had the positive expression rate of 83.64%. The blockade of B7-H1 on immature DCs had sharply increased their T cells stimulatory proliferation capacity and IL-12 production, and efficiently induced the development of Th1/Tc1 cells, but had no effect on their maturation and endocytic activity. CONCLUSION: The blockade of B7-H1 on immature DCs increases its immune stimulation activity. It is valuable to investigate the antitumor immune responses of DCs vaccine with B7-H1 blockade.     

Bone marrow-derived mesenchymal stem cells regulate the function of Th17/ Treg in peripheral blood of severe asthmatic children

AIM：To investigate the regulatory function of bone marrow-derived mesenchymal stem cells (MSCs) on T helper 17 cells (Th17) and regulatory T cells (Treg) in peripheral blood of severe asthmatic children. METHODS：MSCs were isolated, cultured and identified in vitro. MSCs digested with mitomycin were cocultured with T lymphocytes (TLC) at different ratios (1∶1, 1∶2, 1∶10 and 1∶20) from severe asthmatic children for 72 h. The proliferation of TLC was measured by CCK-8 method. In the coculture system of the 1∶2 ratio and the single TLC system, the supernatant levels of interleukin-17 (IL-17) and transforming growth factor-β (TGF-β) were measured by ELISA. The mRNA expression of retinoic acid-related orphan nuclear receptor C (RORC) and forkhead box protein 3 (Foxp3) in TLC was detected by qRT-PCR. RESULTS：After cocultured with MSCs, the proliferation of TLC decreased significantly in a dose-dependent manner (P<0.05). It also showed decreases in IL-17 (3 799±441 vs 4 890 ±373, P<0.05) and RORC mRNA level (1.21±0.14 vs 3.85±0.48, P<0.05), while an increase in TGF-β level (209±32 vs 117±26, P<0.05) was observed. No influence on the mRNA expression of Foxp3 was found (P>0.05). CONCLUSION：MSCs suppresses Th17 polarization of naive peripheral blood CD4+ T cells and matures Th17 cells secreting IL-17, which may effectively revise Th17/Treg imbalance of asthma.     

Screening of differentially expressed genes in colorectal cancer based on TCGA database and verification of novel gene CCDC78

AIM To explore novel genes closely related to the prognosis of colorectal cancer through bioinformatics analysis of RNA sequencing (RNA-Seq） data of gene expression profile in large samples of colorectal cancer tissues from The Cancer Genome Atlas (TCGA) database, and to validate the new genes and study their functions. METHODS The RNA-Seq data were downloaded from TCGA database to screen differentially expressed genes, R-survival package was used to carry out survival analysis of differentially expressed genes to screened out genes related to prognosis of colorectal cancer, and the most significantly up-regulated genes that have not been reported in cancer studies were selected for further verification. RT-qPCR was employed to detect mRNA expression of these novel genes in human colon cancer cell lines and human colorectal cancer tissues. The colon cancer cell line with stable silencing of the novel gene expression was constructed by transfection of lentivirus vector, and its viability, migration and invasion were detected by CCK-8 assay and Transwell assay. RESULTS (1) A total of 4 017 differentially expressed genes were found in the gene expression profile of the colorectal cancer samples. Kaplan-Meier survival analysis showed that 69 genes were closely related to poor prognosis in patients with colorectal cancer, 36 of which were up-regulated, and 11 of these 36 genes have not been reported in cancer studies. (2) The mRNA expression of the top 3 up-regulated genes CCDC78, PGGHG and TSPEAR of the 11 genes was significantly increased in colon cancer cell lines, and the expression of CCDC78 mRNA was also up-regulated in colorectal cancer tissues. (3) CCDC78 gene silencing significantly suppressed the viability, migration and invasion of colon cancer cells (P<0.01). CONCLUSION Bioinformatics analysis based on TCGA database is helpful to discover nov?el genes related to prognosis of colorectal cancer, and CCDC78 may be a novel oncogene associated with poor prognosis of colorectal cancer.