Dissertation
Dissertation > Medicine, health > Internal Medicine > Respiratory system and chest diseases > Trachea and bronchial disease > Bronchial disease > Bronchial asthma

TIM-3对16HBE airway epithelial cells of NF-κB and expression levels of inflammation-related factor regulation of

Author LiuZuo
Tutor HuLiHua
School Huazhong University of Science and Technology
Course Clinical Laboratory Science
Keywords 16HBE A549 GLC-82 PBMC hTim-3 PCR Clone Transfection Filter 16HBE cells House dust mite Dexamethasone Tim-3 NF-κB IFN-γ TNF-α IL-8 RANTES ICAM-1
CLC R562.25
Type PhD thesis
Year 2010
Downloads 194
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Allergic asthma is a common complication associated with immune inflammatory airways disease characterized primarily airway hyperresponsiveness and response to environmental allergen-specific IgE or elevated total IgE, generally influenced by genetic, environmental, individual immune status and other multiple factors and disease. It is reported that human Tims Tim-3 is an important member of the gene family, it is located was highly linked with asthma chromosome 5q31-33 region. Currently, many of the Tim-3 gene as a candidate gene for asthma susceptibility is important for Tim-3 gene polymorphisms and asthma association studies. In the part of the population to carry out the study results show, Tim-3 gene polymorphisms and asthma have some relevance. Recent studies have shown that the human body under normal circumstances, TH1 and TH2 cells in these two cell subtypes regulated by various transcription factors, secrete different cytokines and coordination and antagonistic to maintain homeostasis. TH1/TH2 cells and their cytokine secretion imbalance factors affecting the incidence of asthma. Asthma occurs when the polarization offset TH2 cells, TH2 cells and their relative increase in the secretion of cytokines. Tim-3 was originally found TH1 cell proliferative response after antigen expressed in terminally differentiated cells of the TH1 and the Tim-3 ligand binding galectin-9 play after termination of TH1-mediated immune response capabilities. Tim-3 and its ligand galectin-9 binding, causing an inhibitory signal, resulting in a TH1 cell apoptosis, the negative tone of the TH1 type immune response. However, Tim-3 expression is not limited to T cells. Recent studies by reverse transcription - polymerase chain reaction confirmed Tim-3 in a variety of normal and malignant human epithelial tissue, microglia, monocytes, dendritic cells, M2 macrophages, mast cells both T cells and a variety of expression, suggesting that Tim-3 may be associated with a plurality of tissues and organs of the body and the immune system function-related. So whether Tim-3 expression in the respiratory system somewhat, it is through the respiratory system and the immune system is associated with asthma needs further study. In this study, we first of all in the human respiratory tract-related cell lines screened for expression of Tim-3 and establish hTim-3 plasmid stably transfected 16HBE cell lines, the application of house dust mite and dexamethasone treatment increases hTim-3 and did not raise hTim -3 16HBE cells were detected among treatments NF-κB and expression levels of inflammation-related factors, analyze hTim-3 对 16HBE cells NF-κB and inflammatory cytokine expression levels. Objective: Screening of Tim-3 expression was related to human respiratory cells, in order to further clarify the Tim-3 may be associated with respiratory-related basis; construct pEGFP-C2-hTim-3 eukaryotic expression vector for subsequent further research hTim- 3 functions. The recombinant plasmid was sequenced completely correct pEGFP-C2-hTim-3 transfected into human bronchial epithelial cell line 16HBE, a screened-positive cell clones to provide a platform for subsequent experiments. Methods: with 10% fetal bovine serum high glucose DMEM culture based on 37 ℃ 5% CO2 humidified incubator in cultured human airway-related cell lines 16HBE, A549, GLC-82, to adjust the cell to the best state, logarithmic growth period when the cells were collected, extracted total RNA, and reverse transcription of cDNA. Reference to human Tim-3 gene in GenBank, full-length sequence, using its Primer5.0 primers were designed and synthesized by PCR detection of these three cells of Tim-3 mRNA expression. Human peripheral blood of healthy full-length cDNA Tim-3 gene fragment was cloned into the eukaryotic expression vector pEGFP-C2, by colony PCR, restriction enzyme digestion and sequencing, gene construct Tim-3 contains recombinant plasmid pEGFP-C2-hTim- 3. The use of liposome-mediated pEGFP-C2-hTim-3 plasmid and pEGFP-C2 plasmids were transfected 16HBE cells, fluorescence microscopy 16HBE cells transiently transfected efficiency, and using a medium containing G418 selection for the integration of pEGFP- C2-hTim-3 and pEGFP-C2-positive cell clones, using fluorescence microscopy pEGFP-C2-hTim-3 and pEGFP-C2 position in 16HBE cells expressing with Real-time PCR and Western Blot technique to detect the transfection pEGFP-C2-hTim-3 and transfected with pEGFP-C2 of 16HBE cells hTim-3 expression. Results: 1.PCR revealed that human respiratory-related cell lines 16HBE, A549, GLC-82 were expressed Tim-3mRNA. 2 constructed with filter characteristics and stable expression of pEGFP-C2-hTim-3 plasmid vector and sequenced fully consistent with the desired design. 3 after transient transfection by fluorescence microscopy examination showed positive expression of pEGFP-C2-hTim-3 green fluorescent protein 16HBE cells less than 8%, while the positive expression of pEGFP-C2 green fluorescent protein 16HBE cells less than 15%. Obtained after G418 selection using the expression of wild-type hTim-3 and exogenous pEGFP-C2-hTim-3 of 16HBE cells expressing hTim3 levels than transfected with empty vector pEGFP-C2 16HBE cells was significantly higher (P lt; 0.01), The fluorescence microscope positioned on the membrane of the former, while the latter is positioned in the cytoplasm. CONCLUSION: Human respiratory-related cell lines 16HBE, A549, GLC-82 were expressed Tim-3 mRNA, Tim-3 may be associated with the occurrence and development of the human respiratory related. Successfully constructed the eukaryotic expression vector pEGFP-C2-hTim-3. Successfully screened to obtain a high expression of pEGFP-C2-hTim-3 clones of 16HBE cells for subsequent research use. Objective: To study T cell immunoglobulin and mucin domain protein 3 (abbreviated TIM-3) on airway epithelial cells 16HBE by house dust mite and / or after dexamethasone treatment NF-κB and related regulation of cytokine expression, and to explore appropriate mechanisms. Methods: The experiment divided into two groups, namely transfected with pEGFP-C2 and transfected with pEGFP-C2-hTim-3 of 16HBE cell lines. Two cell lines were used to a certain concentration of house dust mite and / or dexamethasone treatment. Extraction cell mRNA, and reverse transcribed into cDNA, real-time PCR was used to detect the difference after transfection cells were treated with Tim-3, NF-κB, IFN-γ, galectin-9, T-bet, IL-4 and GATA -3, etc.] mRNA expression levels. After extraction of cellular proteins, Western blot was used to detect the difference after transfection cells were treated with NF-κB protein expression levels. Analysis hTim-3 对 by house dust mite and / or dexamethasone treated 16HBE cells NF-κB and related cytokines expression regulation, and to explore appropriate mechanisms. Results: 1 Real-time PCR results show that when 16HBE cells were treated with house dust mite and / or dexamethasone treatment, transfected PEGFP-C2-hTim3 plasmid 16HBE cell lines Tim-3 (p lt; 0.01) , NF-κB (p lt; 0.01), IFN-γ (p lt; 0.01), T-bet (p lt; 0.01), galectin-9 (p lt; 0.01), IL-4 (p lt; 0.01) expression and appropriate treatment of transfected plasmid pEGFP-C2 16HBE cells expressing the above indicators have significant differences, and GATA-3 and no significant change (p gt; 0.05). Moreover, transfection of pEGFP-C2-hTim3 plasmid 16HBE cell lines, Tim-3 expression and IFN-γ, T-bet and galectin-9 expression was positively correlated with NF-κB and IL-4 in expression was negatively correlated; however, transfected with pEGFP-C2 plasmid 16HBE cells, not the existence of such a strong correlation. Also, when using a certain concentration of house dust mite and / or dexamethasone transfected pEGFP-C2-hTim3 plasmid 16HBE cells, Tim-3 对 NF-κB by upregulating the expression of IFN-γ, T- bet, galectin-9 and reduced the expression of IL-4 implementation. 2.Western Blot results show that, when using the house dust mite and / or dexamethasone treated 16HBE cells transfected with pEGFP-C2-hTim3 plasmid 16HBE cells of Tim-3 expression and NF-κB protein expression was negatively Related; However, transfection of pEGFP-C2 such 16HBE cells does not appear clear trend. This indicates that transfection of pEGFP-C2-hTim3 plasmid 16HBE cells upregulation of Tim-3 inhibits NF-κB protein expression. Conclusion: In 16HBE cells, Tim-3 pathway upregulation by upregulating IFN-γ, T-bet, galectin-9 and down the expression of IL-4 attenuated the expression of NF-κB, and thus play a suppression of inflammation effect. Objective: To study T cell immunoglobulin and mucin domain protein 3 (abbreviated TIM-3) on airway epithelial cells 16HBE by house dust mite and / or after dexamethasone treatment of inflammation-related factor expression regulation, and to explore appropriate mechanisms . Methods: The experiment divided into two groups, namely transfected with pEGFP-C2 and transfected with pEGFP-C2-hTim-3 of 16HBE cell lines. Two cell lines were used to a certain concentration of house dust mite and / or dexamethasone treatment. Extraction cell mRNA, and reverse transcribed into cDNA, real-time PCR was used to detect the difference after transfection cells were treated with Tim-3, NF-κB, TNF-α, IL-8, RANTES, ICAM-1 mRNA expression, etc. level. After separation of the supernatant, ELISA was used to detect the difference after transfection, cells were treated with NF-κB, TNF-α, IL-8 protein levels. Analysis hTim-3 对 by house dust mite and / or dexamethasone treated 16HBE cells NF-κB and expression of inflammatory cytokines related regulation, and to explore appropriate mechanisms. Results: 1 Real-time PCR results show that when 16HBE cells were treated with house dust mite and / or dexamethasone treatment, transfected with pEGFP-C2-hTim3 plasmid 16HBE cell lines Tim-3 (p lt; 0.01) , NF-κB (p lt; 0.01), TNF-α (p lt; 0.01), IL-8 (p lt; 0.01), RANTES (p lt; 0.01), ICAM-1 (p lt; 0.01) Expression and appropriate treatment of transfected with pEGFP-C2 plasmid 16HBE cells above indicators expression significantly different; Moreover, transfection of pEGFP-C2-hTim3 plasmid 16HBE cell lines, Tim-3 expression and NF-κB , TNF-α, IL-8, RANTES and ICAM-1 expression was negatively correlated; however, transfected with pEGFP-C2 plasmid 16HBE cells, not the existence of such a strong correlation. When a certain concentration of house dust mite and / or dexamethasone transfected with pEGFP-C2-hTim3 plasmid 16HBE cells, the level of TNF-α mRNA levels may be involved in the reduction of the expression of NF-κB down, Tim -3 for IL-8, RANTES and ICAM-1 expression possibly through reduced NF-κB-dependent way to achieve this. 2.ELISA results show that, when using the house dust mite and / or dexamethasone treated 16HBE cells transfected with pEGFP-C2-hTim3 plasmid 16HBE cells TNF-α, IL-8 expression and NF-κB protein expression was positively correlated; However, transfection of pEGFP-C2 16HBE cells does not appear this apparent trend. This indicates that the transfection of pEGFP-C2-hTim3 plasmid 16HBE cells at the protein level, reduced levels of TNF-α may be involved in NF-κB downregulation, Tim-3 对 IL-8 expression by decreasing NF may -κB-dependent manner to produce colors. Conclusion: In 16HBE cells, Tim-3 pathway possibly through upregulation of NF-κB-dependent manner reduced IL-8, RANTES and ICAM-1 expression, TNF-α levels also may be involved in the reduction of NF-κB expression downward, and thus play an anti-inflammation effect.

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