NF-κB抑制剂和IFN-γ对BAFF-R基因启动子活性的影响

目的:探讨IFN-γ和NF-κB抑制剂对人BAFF-R基因启动子活性的影响。方法:以人全血基因组DNA为模板,PCR方法扩增得到BAFF-R基因转录起始点上游5’端-1562~+261 bp的片段,与荧光素酶报告基因载体pGL3-Basic连接构建8个序列缺失质粒。将重组质粒瞬时转染人多发性骨髓瘤细胞株KM3,通过检测荧光素酶的相对活性,确定启动子活性最强的区域;分别用NF-κB抑制剂(BAY11-7082)和IFN-γ对活性最强的启动子进行干预,测定并比较荧光素酶的相对活性;同时RT-PCR检测干预前后BAFF-R基因mRNA的表达水平。结果:IFN-γ对BAFF-R基因启动子活性有促进作用并且可以上调BAFF-R mRNA的表达,NF-κB抑制剂(BAY11-7082)对BAFF-R基因启动子活性有抑制作用并且可以下调BAFF-R mRNA的表达。结论:IFN-γ和NF-κB信号通路参与了BAFF-R基因的转录调控,为进一步研究BAFF-R的调节机制提供了基本的依据。     

T细胞白血病病毒1型Tax蛋白阳性细胞中早期生长反应基因-1对NF-κB活性的影响

目的 观察人T细胞白血病病毒1型(HTLV-1)的Tax蛋白阳性细胞中早期生长反应基因-1(EGR-1)与NF-κB的关系.方法 RT-PCR扩增MT2细胞中EGR-1的cDNA全长,连接于真核表达载体pcDNA3.0;用脂质体介导的方法将pcDNA3.0-EGR-1转染TaxP/TaxN细胞,48 h后PCR检测EGR-1和p65 mRNA的表达,Western blot检测EGR-1及p65蛋白的表达;将pcDNA3.0-EGR-1和NF-κB报告基因共转染TaxP及TaxN细胞48 h后检测荧光素酶活性.结果 成功构建了重组表达质粒pcDNA3.0-EGR-1,Tax可以促进EGR-1 mRNA和蛋白的表达;EGR-1可以促进TaxP细胞p65 mRNA和蛋白的表达,上调NF-κB活性.结论 EGR-1可能通过促进NF-κB活化参与成人T淋巴细胞白血病(adult T-cell leukemia,ATL)的发病.

Abstract：

Objective To research the relation of early growth response gene-1(EGR-1) and NF-κB in human T-cell leukemia virus type 1(HTLV-1) Tax protein positive cells. Methods RT-PCR was used to amplify the aimed segments EGR-1 cDNA which was then inserted into an eukaryotic expression plasmid pcDNA3.0 to construct pcDNA3.0-EGR-1. The constructed plasmid was transfected into TaxN and TaxP cells by Tfx-50-mediated transfer method, the expression levels of EGR-1, p65 and Tax mRNA in transfected cells were assay by RT-PCR after 48 h post-transfection, the proteins of EGR-1 and p65 were detected by Western blot after 48 h post-transfection too. The constructed plasmid and pNF-κB-luc reporter gene plasmid was co-transfected into TaxN and TaxP cells by Tfx-50-mediated transfer method, and the activity of luciferase was assay after 48 h post-transfection. Results The results showed that the eukaryotic expression plasmid pcDNA3.0-EGR-1 was successfully constructed. The mRNA and protein expression of EGR-1 could be promoted significantly by Tax. EGR-1 can promote the mRNA and protein expressions of p65 in TaxP cells, the activity of NF-κB was up-regulated by EGR-1 too. Conclusion EGR-1 maybe involve in adult T-cell leukemia(ATL) by increasing the activation of NF-κB.     

乳杆菌细胞壁成分激活人阴道上皮细胞β-防御素-2表达机制研究

目的 探讨乳杆菌细胞肇成分(Lactobacillus cell wall extract,LCWE)对人阴道上皮细胞β-防御素-2的诱导作用及细胞内信号转导机制.方法 采用实时定量PCR和Western blot方法检测LCWE处理对人阴道上皮细胞(WZV-1)β-防御素-2表达的影响;Western blot方法检测LCWE处理后WZV-1细胞内NF-κB和p38MAPK信号通路活化情况;实时定量PCR和Western blot方法检测NF-κB抑制剂和p38MAPK抑制剂预处理WZV-1细胞对LCWE诱导β-防御素-2表达的影响.结果 LCWE可诱导WZV-1细胞β-防御素-2表达且存在着明显的时间效应和剂量依赖关系,50μg/mlLCWE处理细胞8 h对β-防御素-2的上调幅度最大,刺激0.5 h后可引起细胞内p38MAPK蛋白的磷酸化显著增加,1 h达到顶峰;刺激0.5 h后可引起细胞核内NF-κB含量显著增加(P<0.05),2 h达到顶峰.NF-κB抑制剂PDTC、p38MAPK抑制剂SB20380预处理WZV-1细胞,可明显抑制LCWE诱导WZV-1细胞β-防御素-2的表达.结论 LCWE具有激活NF-κB和p38MAPK信号通路诱导阴道-上皮细胞β-防御素-2的作用.

Abstract：

Objective To investigate the molecular and cell signal transduction mechanisms by Lactobacillus cell wall extract(LCWE)inducing human-β-defensin-2(hBD-2)expression in human vaginal epithelial cells.Methods The induction of hBD-2 in human vaginal epithelial cells(WZV-1)by LCWE was observed using real-time PCR and Western blot.After stimulating WZV-1.the activation of NF-κB and p38MAPK signaling pathways were determined by Western blot.The induction of hBD-2 in WZV-1 cells by LCWE was observed with signaling pathways inhibitors of NF-κB and p38MAPK using real-time PCR and Western blot.Results The results showed that LCWE significantly upregulated hBD-2 expression in the time and dose-dependent manner.The maximal stimulatory effect of LCWE on the expression of hBD-2mRNA in WZV-1 cells were observed at the concentration of 50μg/ml after treatment for 8 h.After stimulation by 50μg/ml LCWE,Western blot analysis demonstrated that the phosphorylation of p38MAPK increased at 0.5 h significantly,peaked at 1 h,moreover the concentration of NF-κB in nucleus increased at 0.5 h significantly(P<0.05),peaked at 2 h.Blocking with inhibitor of NF-κB and(or)p38MAPK pathways results in decreased levels of HBD-2 expression.Conclusion These findings suggest that p38MAPK and NF-κB pathways play the important roles in induction of hBD-2 expression by LCWE in human vagihal epithelial cells.     

Nuclear factor κB represses the expression of latent membrane protein 1 in Epstein-Barr virus transformed cells

AIM: To investigate the role of nuclear factor κB(NF-κB) in the regulation of Epstein-Barr virus(EBV) latent membrane protein 1(LMP1) in EBV transformed cells. METHODS: LMP1 expression was examined in EBV transformed human B lymphocytes with modulation of NF-κB activity. RESULTS: EBV infection is associated with several human cancers. EBV LMP1 is required for efficient transformation of adult primary B cells in vitro, and is expressed in several pathogenic stages of EBVassociated cancers. Regulation of EBV LMP1 involves both viral and cellular factors. LMP1 activates NF-κB signaling pathway that is a part of the EBV transformation program. However, the relation between NF-κB and LMP1 expression is not well established yet. In this report, we found that blocking the NF-κB activity by Inhibitor of κB stimulated LMP1 expression, while the overexpression of NF-κB repressed LMP1 expression in EBV-transformed IB4 cells. In addition, LMP1 repressed its own promoter activities in reporter assays, and the repression was associated with the activation of NF-κB. Moreover, NF-κB alone is sufficient to repress LMP1 promoter activities. CONCLUSION: Our data suggest LMP1 may repress its own expression through NF-κB in EBV transformed cells and shed a light on LMP1 regulation during EBV transformation.     

Inhibition of NF-κB Expression and Allergen-induced Airway Inflammation in a Mouse Allergic Asthma Model by Andrographolide

Andrographolide from traditional Chinese herbal medicines previously showed it possesses a strong anti- inflammatory activity. In present study, we investigated whether Andrographolide could inhibit allergen-induced airway inflammation and airways hyper-responsiveness and explored the mechanism of Andrographolide on allergen-induced airway inflammation and airways hyper-responsiveness. After sensitized and challenged by ovalbumin, the BALB/c mice were administered intraperitoneally with Andrographolide. Hyper-responsiveness was recorded. The lung tissues were assessed by histological examinations. NF-κB in lung was determined by immunofluorescence staining and Western blotting. Treatment of mice with Androqrapholide displayed lower Penh in response to asthma group mice. After treatment with Andrographolide, the extent of inflammation and cellular infiltration in the airway were reduced. Andrographolide interrupted NF-κB to express in cell nucleus. The level of NF-κB expression was inhibited by Andrographolide. The data indicate that Andrographolide from traditional Chinese herbal medicines could inhibit extensive infiltration of inflammatory cells in lung and decrease airway hyperreactivity. Andrographolide could inhibit NF-κB expression in lung and suppress NF-κB expressed in the nucleus of airway epithelial cells.     

IFN-γ和IL-6对多发性骨髓瘤B淋巴细胞刺激因子表达变化的调控研究

目的 探讨IFN-γ和IL-6对多发性骨髓瘤(multiple myeloma,MM)B淋巴细胞刺激因子(BLyS)表达变化的影响及相关机制.方法 采用流式细胞术、实时荧光定量PCR、E1.ISA及Western blot方法 分析人MM肿瘤细胞KM3在IFN-γ和IL-6以及相关信号通路特异性抑制剂作用前后BLJys表达水平的变化.结果 lFN-γ和IL-6促进KM3细胞BLyS的表达水平;NF-KB抑制剂能够抑制BLyS的表达水平;NF-KB抑制剂BAY11-7082能够完全下调IFN-γ引起的BLyS表达的上调作用;抑制促分裂原活化蛋白激酶(MAPK)的活性能够下调BLyS的表达水平.结论 MAPK与NF-KB信号通路参与了Blys的表达调节.     

MyD88/PI3-K/NF-κB pathway mediates the antagonism of lipoxin A4 on LPS-induced synthesis of interleukins in endothelial cells

In order to investigate whether lipoxin A4(LXA4) has an antagonistic effect on lipopolysaccharide (LPS)-induced synthesis of interleukin (IL)-1β, IL-6 and IL-8 in rat pulmonary microvascular endothelial cells (PMVEC) , and to explore the molecular mechanisms of signal pathway in LXA4 actions, cultured PMVEC were treated with LPS, with or without preincubation with LXA4. Proteins of IL-1β, IL-6 and IL-8 in supernatant were analyzed by enzyme-linked immunosorbent assay (ELISA) . Expressions of mRNA of IL-1β, IL-6 and IL-8 were determined by RT-PCR. Expressions of phosphorylation of phosphoinositide 3-kinase (PI3-K) and myeloid differentiation factor 88 (MyD88) were analyzed by Western blot. Activities of DNA-binding of nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1) were measured by electrophoretic mobility shift assay (EMSA) . The results showed that LPS induced production of IL-1β, IL-6 and IL-8 in rat PMVEC via MyD88/PI3-K/NF-κB and AP-1 pathway-dependent signal transduction. LPS-stimulated expression of PI3-K, activities of NFκB and AP-1, secretion of protein and expression of mRNA of IL-1β, IL-6 and IL-8 but not MyD88 expression in PMVEC were inhibited by LXA4 in a dose-dependent manner. In conclusion, LXA4 inhibits synthesis of IL-1β, IL-6 and IL-8 by down-regulation of PI3-K/NF-κB and AP-1 signal pathway in PMVEC.     

Induction of APOBEC3G in human hepatoma cells by interferon-α stimulation

To clarify the role of APOBEC3G (A3G) in cellular defense against hepatitis B virus ( HBV), the expression of A3G in normal human liver and the regulation of the A3G expression in hep-atoma cell line (HuH-7) were investigated. Expression level of APOBEC3s mRNA in human liver was determined by RT-PCR. HuH-7 and HepG2 cells were treated with various concentrations of IFN-α(0 U/ml, 100 U/ml, 500 U/ml, 1000 U/ml)for 12 h. The mRNA levels were measured by a quantitative RT-PCR, the results were normalized relative to the specimens without IFN-αstimulation. Total protein of HuH-7 cells treated with various concentrations of IFN-αfor 48 h was subjected to Western blot analysis. For reporter gene assay, HuH-7 cells were transfected with the reporter plasmids containing IRF-E sites and its mutants with different lengths. Then the cells were treated with or without 1200 U/ml IFN-a for additional 12 h (1000 U/ml) after 24 h of transfection, and the cell lysate was prepared and assayed for luciferase activity. It was found that normal human liver expressed the mRNA of A3G. A3G mRNA expression in HuH-7 and HepG2 cells were up-regulated by IFN-αstimulation in a dose-depen-dent manner. Western blot analysis indicated that A3G protein expression was also enhanced by IFN-αstimulation. Sequence analysis showed the existence of putative sites of IFN regulatory factor element (IRF-E) in 5' region of A3G gene upstream the initiation codon. IFN-αstimulation results in 6- to 8-fold increase in luciferase activity in cells transfected with the plasmid containing IRF-E sites of the 5' upstream sequences, whereas luciferase activity did not change in cells transfected with the plasmid containing mutant IRF-E sites or without IRF-E sites. As a conclusion, A3G are expressed in normal human liver. A3G expression was up-regulated by IFN-αstimulation in hepatoma cells and could be involved in host defense mechanisms against HBV. ERF-E site in 5' region of AP0BEC3G gene upstream the initiation codon plays an important role in this process.     

Effect of nuclear factor-κB p65 ASOND on I typ collagen expression and rat hepatic stellate cells proliferation

Objective Sstudy effect of nuclear factor-κB ASOND on I type collagen expression and rat hepatic stellate cells(HSC)proliferation.Methods Rat HSCs were separated by affusing and digestingof Ⅳ type collagenenzyme and density acentric method.Lipid-mediated NF-κB p65 ASOND(0.001,0.01,0.1,1μmol/L)Was transferred into rat HSCs.Toxicity of HSCs caused by NF-κB p65 ASOND and activity of LDH were determined by trypan blue staining.Proliferation affection of transferring NF-κB p65 ASOND into HSCs was determined by MTT.In different concentration NF-κB p65 ASOND.expression of Ⅰ type collagen stimulated by 1mg/L TNF-αwas determined by RT-PCR and ELISA.Results After transfection of NF-κB p65 ASODN,expression of NF-κB protein in HSCs was decreasing.Toxicity experiment indicated that NF-κB p65 ASOND of different concentration(0.001,0.01,0.1 and 1.0 μmol/L)had no effect on HSCs livability and LDH activity(P<0.05).Four different concentration NF-κ B p65 ASOND could restrain HSCs proliferation stimulated by 1 mg/L TNF-α.The expression of I type collagen and mRNA stimulated by 1mg/LTNF-αwas increased,and had a positive correlation with concentration(P>0.05).Conclusion NF-κB p65 ASOND may depress NF-κB activity to restrain HSCs proliferation and Ⅰ type collagen expression,and reduce extracellular matrix.     

Effect of nuclear factor-κB p65 ASOND on I typ collagen expression and rat hepatic stellate cells proliferation

Objective Sstudy effect of nuclear factor-κB ASOND on I type collagen expression and rat hepatic stellate cells(HSC)proliferation.Methods Rat HSCs were separated by affusing and digestingof Ⅳ type collagenenzyme and density acentric method.Lipid-mediated NF-κB p65 ASOND(0.001,0.01,0.1,1μmol/L)Was transferred into rat HSCs.Toxicity of HSCs caused by NF-κB p65 ASOND and activity of LDH were determined by trypan blue staining.Proliferation affection of transferring NF-κB p65 ASOND into HSCs was determined by MTT.In different concentration NF-κB p65 ASOND.expression of Ⅰ type collagen stimulated by 1mg/L TNF-αwas determined by RT-PCR and ELISA.Results After transfection of NF-κB p65 ASODN,expression of NF-κB protein in HSCs was decreasing.Toxicity experiment indicated that NF-κB p65 ASOND of different concentration(0.001,0.01,0.1 and 1.0 μmol/L)had no effect on HSCs livability and LDH activity(P<0.05).Four different concentration NF-κ B p65 ASOND could restrain HSCs proliferation stimulated by 1 mg/L TNF-α.The expression of I type collagen and mRNA stimulated by 1mg/LTNF-αwas increased,and had a positive correlation with concentration(P>0.05).Conclusion NF-κB p65 ASOND may depress NF-κB activity to restrain HSCs proliferation and Ⅰ type collagen expression,and reduce extracellular matrix.     

Effect of nuclear factor-κB p65 ASOND on I typ collagen expression and rat hepatic stellate cells proliferation

Objective Sstudy effect of nuclear factor-κB ASOND on I type collagen expression and rat hepatic stellate cells(HSC)proliferation.Methods Rat HSCs were separated by affusing and digestingof Ⅳ type collagenenzyme and density acentric method.Lipid-mediated NF-κB p65 ASOND(0.001,0.01,0.1,1μmol/L)Was transferred into rat HSCs.Toxicity of HSCs caused by NF-κB p65 ASOND and activity of LDH were determined by trypan blue staining.Proliferation affection of transferring NF-κB p65 ASOND into HSCs was determined by MTT.In different concentration NF-κB p65 ASOND.expression of Ⅰ type collagen stimulated by 1mg/L TNF-αwas determined by RT-PCR and ELISA.Results After transfection of NF-κB p65 ASODN,expression of NF-κB protein in HSCs was decreasing.Toxicity experiment indicated that NF-κB p65 ASOND of different concentration(0.001,0.01,0.1 and 1.0 μmol/L)had no effect on HSCs livability and LDH activity(P<0.05).Four different concentration NF-κ B p65 ASOND could restrain HSCs proliferation stimulated by 1 mg/L TNF-α.The expression of I type collagen and mRNA stimulated by 1mg/LTNF-αwas increased,and had a positive correlation with concentration(P>0.05).Conclusion NF-κB p65 ASOND may depress NF-κB activity to restrain HSCs proliferation and Ⅰ type collagen expression,and reduce extracellular matrix.     

Effect of nuclear factor-κB p65 ASOND on I typ collagen expression and rat hepatic stellate cells proliferation

Objective Sstudy effect of nuclear factor-κB ASOND on I type collagen expression and rat hepatic stellate cells(HSC)proliferation.Methods Rat HSCs were separated by affusing and digestingof Ⅳ type collagenenzyme and density acentric method.Lipid-mediated NF-κB p65 ASOND(0.001,0.01,0.1,1μmol/L)Was transferred into rat HSCs.Toxicity of HSCs caused by NF-κB p65 ASOND and activity of LDH were determined by trypan blue staining.Proliferation affection of transferring NF-κB p65 ASOND into HSCs was determined by MTT.In different concentration NF-κB p65 ASOND.expression of Ⅰ type collagen stimulated by 1mg/L TNF-αwas determined by RT-PCR and ELISA.Results After transfection of NF-κB p65 ASODN,expression of NF-κB protein in HSCs was decreasing.Toxicity experiment indicated that NF-κB p65 ASOND of different concentration(0.001,0.01,0.1 and 1.0 μmol/L)had no effect on HSCs livability and LDH activity(P<0.05).Four different concentration NF-κ B p65 ASOND could restrain HSCs proliferation stimulated by 1 mg/L TNF-α.The expression of I type collagen and mRNA stimulated by 1mg/LTNF-αwas increased,and had a positive correlation with concentration(P>0.05).Conclusion NF-κB p65 ASOND may depress NF-κB activity to restrain HSCs proliferation and Ⅰ type collagen expression,and reduce extracellular matrix.     

Effect of nuclear factor-κB p65 ASOND on I typ collagen expression and rat hepatic stellate cells proliferation

Objective Sstudy effect of nuclear factor-κB ASOND on I type collagen expression and rat hepatic stellate cells(HSC)proliferation.Methods Rat HSCs were separated by affusing and digestingof Ⅳ type collagenenzyme and density acentric method.Lipid-mediated NF-κB p65 ASOND(0.001,0.01,0.1,1μmol/L)Was transferred into rat HSCs.Toxicity of HSCs caused by NF-κB p65 ASOND and activity of LDH were determined by trypan blue staining.Proliferation affection of transferring NF-κB p65 ASOND into HSCs was determined by MTT.In different concentration NF-κB p65 ASOND.expression of Ⅰ type collagen stimulated by 1mg/L TNF-αwas determined by RT-PCR and ELISA.Results After transfection of NF-κB p65 ASODN,expression of NF-κB protein in HSCs was decreasing.Toxicity experiment indicated that NF-κB p65 ASOND of different concentration(0.001,0.01,0.1 and 1.0 μmol/L)had no effect on HSCs livability and LDH activity(P<0.05).Four different concentration NF-κ B p65 ASOND could restrain HSCs proliferation stimulated by 1 mg/L TNF-α.The expression of I type collagen and mRNA stimulated by 1mg/LTNF-αwas increased,and had a positive correlation with concentration(P>0.05).Conclusion NF-κB p65 ASOND may depress NF-κB activity to restrain HSCs proliferation and Ⅰ type collagen expression,and reduce extracellular matrix.     

Effect of nuclear factor-κB p65 ASOND on I typ collagen expression and rat hepatic stellate cells proliferation

Objective Sstudy effect of nuclear factor-κB ASOND on I type collagen expression and rat hepatic stellate cells(HSC)proliferation.Methods Rat HSCs were separated by affusing and digestingof Ⅳ type collagenenzyme and density acentric method.Lipid-mediated NF-κB p65 ASOND(0.001,0.01,0.1,1μmol/L)Was transferred into rat HSCs.Toxicity of HSCs caused by NF-κB p65 ASOND and activity of LDH were determined by trypan blue staining.Proliferation affection of transferring NF-κB p65 ASOND into HSCs was determined by MTT.In different concentration NF-κB p65 ASOND.expression of Ⅰ type collagen stimulated by 1mg/L TNF-αwas determined by RT-PCR and ELISA.Results After transfection of NF-κB p65 ASODN,expression of NF-κB protein in HSCs was decreasing.Toxicity experiment indicated that NF-κB p65 ASOND of different concentration(0.001,0.01,0.1 and 1.0 μmol/L)had no effect on HSCs livability and LDH activity(P<0.05).Four different concentration NF-κ B p65 ASOND could restrain HSCs proliferation stimulated by 1 mg/L TNF-α.The expression of I type collagen and mRNA stimulated by 1mg/LTNF-αwas increased,and had a positive correlation with concentration(P>0.05).Conclusion NF-κB p65 ASOND may depress NF-κB activity to restrain HSCs proliferation and Ⅰ type collagen expression,and reduce extracellular matrix.     

Effect of nuclear factor-κB p65 ASOND on I typ collagen expression and rat hepatic stellate cells proliferation

Objective Sstudy effect of nuclear factor-κB ASOND on I type collagen expression and rat hepatic stellate cells(HSC)proliferation.Methods Rat HSCs were separated by affusing and digestingof Ⅳ type collagenenzyme and density acentric method.Lipid-mediated NF-κB p65 ASOND(0.001,0.01,0.1,1μmol/L)Was transferred into rat HSCs.Toxicity of HSCs caused by NF-κB p65 ASOND and activity of LDH were determined by trypan blue staining.Proliferation affection of transferring NF-κB p65 ASOND into HSCs was determined by MTT.In different concentration NF-κB p65 ASOND.expression of Ⅰ type collagen stimulated by 1mg/L TNF-αwas determined by RT-PCR and ELISA.Results After transfection of NF-κB p65 ASODN,expression of NF-κB protein in HSCs was decreasing.Toxicity experiment indicated that NF-κB p65 ASOND of different concentration(0.001,0.01,0.1 and 1.0 μmol/L)had no effect on HSCs livability and LDH activity(P<0.05).Four different concentration NF-κ B p65 ASOND could restrain HSCs proliferation stimulated by 1 mg/L TNF-α.The expression of I type collagen and mRNA stimulated by 1mg/LTNF-αwas increased,and had a positive correlation with concentration(P>0.05).Conclusion NF-κB p65 ASOND may depress NF-κB activity to restrain HSCs proliferation and Ⅰ type collagen expression,and reduce extracellular matrix.     

Effect of nuclear factor-κB p65 ASOND on I typ collagen expression and rat hepatic stellate cells proliferation

Objective Sstudy effect of nuclear factor-κB ASOND on I type collagen expression and rat hepatic stellate cells(HSC)proliferation.Methods Rat HSCs were separated by affusing and digestingof Ⅳ type collagenenzyme and density acentric method.Lipid-mediated NF-κB p65 ASOND(0.001,0.01,0.1,1μmol/L)Was transferred into rat HSCs.Toxicity of HSCs caused by NF-κB p65 ASOND and activity of LDH were determined by trypan blue staining.Proliferation affection of transferring NF-κB p65 ASOND into HSCs was determined by MTT.In different concentration NF-κB p65 ASOND.expression of Ⅰ type collagen stimulated by 1mg/L TNF-αwas determined by RT-PCR and ELISA.Results After transfection of NF-κB p65 ASODN,expression of NF-κB protein in HSCs was decreasing.Toxicity experiment indicated that NF-κB p65 ASOND of different concentration(0.001,0.01,0.1 and 1.0 μmol/L)had no effect on HSCs livability and LDH activity(P<0.05).Four different concentration NF-κ B p65 ASOND could restrain HSCs proliferation stimulated by 1 mg/L TNF-α.The expression of I type collagen and mRNA stimulated by 1mg/LTNF-αwas increased,and had a positive correlation with concentration(P>0.05).Conclusion NF-κB p65 ASOND may depress NF-κB activity to restrain HSCs proliferation and Ⅰ type collagen expression,and reduce extracellular matrix.     

Effect of nuclear factor-κB p65 ASOND on I typ collagen expression and rat hepatic stellate cells proliferation

Objective Sstudy effect of nuclear factor-κB ASOND on I type collagen expression and rat hepatic stellate cells(HSC)proliferation.Methods Rat HSCs were separated by affusing and digestingof Ⅳ type collagenenzyme and density acentric method.Lipid-mediated NF-κB p65 ASOND(0.001,0.01,0.1,1μmol/L)Was transferred into rat HSCs.Toxicity of HSCs caused by NF-κB p65 ASOND and activity of LDH were determined by trypan blue staining.Proliferation affection of transferring NF-κB p65 ASOND into HSCs was determined by MTT.In different concentration NF-κB p65 ASOND.expression of Ⅰ type collagen stimulated by 1mg/L TNF-αwas determined by RT-PCR and ELISA.Results After transfection of NF-κB p65 ASODN,expression of NF-κB protein in HSCs was decreasing.Toxicity experiment indicated that NF-κB p65 ASOND of different concentration(0.001,0.01,0.1 and 1.0 μmol/L)had no effect on HSCs livability and LDH activity(P<0.05).Four different concentration NF-κ B p65 ASOND could restrain HSCs proliferation stimulated by 1 mg/L TNF-α.The expression of I type collagen and mRNA stimulated by 1mg/LTNF-αwas increased,and had a positive correlation with concentration(P>0.05).Conclusion NF-κB p65 ASOND may depress NF-κB activity to restrain HSCs proliferation and Ⅰ type collagen expression,and reduce extracellular matrix.     

黄芪多糖诱导脐血单核细胞向树突状细胞分化中的作用

目的 观察NF-κB在黄芪多糖(APS)诱导脐血单核细胞向树突状细胞(DCs)分化过程的作用,探讨APS诱生DCs过程中的信号传导通路.方法 无菌条件下采集脐血,密度梯度离心法获得脐血单核细胞分为3组.对照组:在无药物的RPMI 1640完全培养液中培养;APS组:在含有黄芪多糖100mg/L的RPMI 1640完全培养液中培养;PDTC组:用NF-κB的抑制剂吡咯烷二硫氨基甲酸酯(PDTC)10μmol/L处理脐血单核细胞30 min后,加入含有黄芪多糖100 mg/L的RPMI 1640完全培养液中培养.培养过程中用倒置光学显微镜和透射电镜观察细胞形态变化;收集培养第12天的细胞,FCM检测细胞免疫表型;免疫荧光显微镜下观察细胞内NF-κB的激活、迁移情况.结果 (1)对照组细胞无成簇生长,培养至第12天细胞呈梭形巨噬细胞形态;黄芪多糖组细胞成簇生长,形态学由圆形逐渐变为典型的树突状细胞形态;抑制剂组细胞生长缓慢,未出现聚集现象,细胞形态学未见明显变化.(2)APS组高表达DCs特异性抗原CD80、CD83和CD86,与对照组、PDTC组比较差异有统计学意义(P<0.01),对照组与PDTC组表达相似,二者差异无统计学意义(P>0.05).(3)免疫荧光显微镜下观察NF-κB可见APS组伴有大量NF-κB荧光进入细胞核,尤以72 h显著,NF-κB激活率为(75.20±7.37)%,而对照组、PDTC组NF-κB激活率分别为(13.20±3.46)%、(8.20±1.92)%,与APS组相比,差异有统计学意义(P<0.01).结论 黄芪多糖能够诱导脐血单核细胞定向分化为DCs,NF-κB是黄芪多糖诱导脐血单核细胞向DCs分化的信号传导通路中的关键元件.

Abstract：

Objective To investigatethe role of NF-κB played in the process of the cord blood monocytes differentiating into dendritic cells(DCs)induced by astragalus polysaccharide(APS)and to explore the signal transduction pathway involved in this process.Methods Umbilica]cord blood was collected in aseptic conditions.The cord blood monocytes were obtained by density gradient centrifugation and were divided into three groups afterwards.In the control group.cells were cultivated in the RPMI 1640 complete medium.In the APS group.cells were cultivated in the RPMI 1640 complete medium containing 100 mg/L APS.In the PDTC group:cells were treated with 10 μmol/L disulfide carbamate(PDTC).NF-κB inhibitor in 30 min followed by cultivalion in the RPMI 1640 complete medium containing 100 mg/L APS.,The morphological changes were observed during the process of cultivation by the optical microscope and transmission electron microscopy.Cells were collected 12 d later and the cellular immunophenotyping was assayed by FCM.,The activation and migration of NF-κB fluorescence in the cells was examined by the immunoflouresce microscopy.Results (1)Cells in the control group grown up without cluster forformation and were found fusiform and macrophage-like in 12 d.Cells in the APS group grown up in clnstem,and morphological changes were found from the circular shape to a typical dendritic cells-like shape.Cells in the inhibitor group grown up slowly and without cluster formation,and cell morphdogy had no significant change.(2)The expression of DCs-specific antigen CD80,CD83 and CD86 in the APS group was higher than that in the control group and inhibitom group(P<0.01).The expression of those antigen in the control group and PDTC group was similar and had no statistically significance(P>0.05).(3)NF-κB fluorescence in the nuclei was examined by the immunoflourescence microscopy and was much higher in the APS group than that in khe other groups,especially in 72 h with the activation rate of NF-κB (75.20±7.37)%,while(13.20±3.46)% of PDTC group and(8.20 ±1.92)%,respectively(P<0.01).Conclusion Astragalus polysaccharide can induce the differentiation of umbilical cord blood cells into DCs,and NF-κB is the key component of the signal transduction pathway involved in this process.