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促炎细胞因子在MIN6 β细胞中诱导NF-κB依赖性/NO非依赖性趋化因子基因表达。

Proinflammatory cytokines induce NF-kappaB-dependent/NO-independent chemokine gene expression in MIN6 beta cells.

作者信息

Baker Marshall S, Chen Xiaojuan, Rotramel Alizah, Nelson Jeffrey, Kaufman Dixon B

机构信息

Department of Surgery, Division of Transplantation, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.

出版信息

J Surg Res. 2003 Mar;110(1):295-303. doi: 10.1016/s0022-4804(03)00027-1.

Abstract

BACKGROUND

Interactions between chemokines IP-10, MCP-1, and RANTES and their receptors may mediate graft rejection following islet transplantation. The mechanisms regulating chemokine gene expression in pancreatic islet cells have not been well characterized. We examined the cytokine-induced gene expression profiles for several chemokines in a transformed pancreatic beta-cell line (MIN6) cotreated with an inhibitor of nitric oxide synthase and in a mutated clone of MIN6 made to overexpress a dominant negative inhibitor of NF-kappaB (IkappaBalphaM).

METHODS

MIN6 and MIN6-IkappaBalphaM (Bm) cells were cultured in mixtures of IL-1beta and TNF-alpha or IL-1beta, TNF-alpha, and IFN-gamma plus/minus the iNOS inhibitor L-NMMA. RT-PCR and RNase Protection Assay were used to measure mRNA expression for the following chemokines: IP-10, MIP-1alpha, MIP-1beta, MCP-1, and RANTES. Enzyme linked immunosorbant assay was used to measure IP-10 and MCP-1 protein release.

RESULTS

Cytokine-treated MIN6 and Bm demonstrated increased expression of genes for IP-10 and MCP-1. Expression in MIN6 was first detected at 2 h of incubation and peaked at 6 h. MIN6 demonstrated a more marked increase in chemokine gene expression for both IP-10 and MCP-1 and a more marked increase in IP-10 protein release than did Bm. There was no detectable gene expression for MIP-1alpha, MIP-1beta, or RANTES from MIN6 or Bm. L-NMMA completely blocked NO production from MIN6 and Bm but had no effect on chemokine gene expression in either MIN6 or Bm.

CONCLUSIONS

These results suggest that beta cells produce a complement of rejection-relevant chemokines in response to a proinflammatory stimulus and that pathways governing cytokine-induced chemokine gene expression in MIN6 are dependent on NF-kappaB but independent of NO.

摘要

背景

趋化因子IP-10、MCP-1和RANTES及其受体之间的相互作用可能介导胰岛移植后的移植物排斥反应。调节胰岛细胞中趋化因子基因表达的机制尚未完全明确。我们检测了在与一氧化氮合酶抑制剂共同处理的转化胰腺β细胞系(MIN6)以及构建的过表达NF-κB显性负性抑制剂(IkappaBalphaM)的MIN6突变克隆中,几种趋化因子的细胞因子诱导基因表达谱。

方法

MIN6和MIN6-IkappaBalphaM(Bm)细胞在白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)或IL-1β、TNF-α和干扰素-γ(IFN-γ)的混合物中培养,同时加入或不加入诱导型一氧化氮合酶(iNOS)抑制剂L-NMMA。采用逆转录聚合酶链反应(RT-PCR)和核糖核酸酶保护试验(RNase Protection Assay)检测以下趋化因子的mRNA表达:IP-10、巨噬细胞炎性蛋白-1α(MIP-1α)、巨噬细胞炎性蛋白-1β(MIP-1β)、MCP-1和RANTES。采用酶联免疫吸附测定法检测IP-10和MCP-1蛋白释放。

结果

细胞因子处理的MIN6和Bm显示IP-10和MCP-1基因表达增加。MIN6中的表达在孵育2小时时首次检测到,并在6小时达到峰值。与Bm相比,MIN6中IP-10和MCP-1的趋化因子基因表达增加更为明显,IP-10蛋白释放增加也更为明显。MIN6或Bm中未检测到MIP-1α、MIP-1β或RANTES的基因表达。L-NMMA完全阻断了MIN6和Bm中一氧化氮的产生,但对MIN6或Bm中的趋化因子基因表达均无影响。

结论

这些结果表明,β细胞在促炎刺激下产生一系列与排斥反应相关的趋化因子,并且在MIN6中,调控细胞因子诱导趋化因子基因表达的途径依赖于NF-κB,但不依赖于一氧化氮。

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