Selection of insulinoma cell lines with resistance to interleukin-1β- and γ-interferon-induced cytotoxicity
Selection of insulinoma cell lines with resistance to interleukin-1β- and γ-interferon-induced cytotoxicity
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DOI：
10.2337/diabetes.49.4.562
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发表时间：
2000-04-01
期刊：
影响因子：
7.7
通讯作者：
Newgard, CB
中科院分区：
文献类型：
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作者：
Chen, GX;Hohmeier, HE;Newgard, CB
工程化胰岛素瘤细胞系可能是分离胰岛移植治疗1型糖尿病的替代方案。这种方法的成功可能需要开发能够耐受精氨酸介导的损伤的细胞系。为此，我们在递增浓度的白细胞介素-1 β（IL-1 β）+ γ-干扰素（IFN-γ）中培养INS-1胰岛素瘤细胞，在8周的时间内大约每周迭代一次。基于C，N二苯基-N ′-[4，5-二甲基噻唑-2-基]-2，5-二苯基溴化四唑鎓（MTT）活力测定，所选细胞（称为INS-1（res））在用10 ng/ml IL-1 β处理5天后100%存活。在暴露于10 ng/ml IL-1 β和100 U/ml IFN-γ的组合5天后，这些细胞的存活率也为78 +/-1.2%，而以相同方式处理的亲本INS-1细胞的存活率仅为0.3 +/-0.03%。INS-1（res）细胞也对活化的大鼠外周血单核细胞上清液的处理具有抗性，而只有20%的亲本INS-1细胞在这种处理中存活。通过该程序赋予的对IL-1 β的抗性是稳定的，而对IFN-γ的部分抗性是短暂的，但在细胞因子存在下通过培养可再诱导。用含有人胰岛素cDNA的质粒稳定转染INS-1（res）细胞，并在不存在细胞因子的情况下扩增转染的集落，产生的细胞系平均对IL-1 β + IFN-γ的抗性（53 +/-11%）高于来自亲本INS-1细胞的类似转染的克隆（15 +/-7%）。重要的是，几个INS-1（res）衍生的克隆保留了响应于超过正常生理范围的葡萄糖浓度而分泌胰岛素的能力。关于赋予选择的机制，我们发现与亲本INS-1细胞相比，INS-1（res）细胞中IFN-γ受体mRNA的水平正常，但IL-1受体I型（IL-1 R1）的表达降低60%。发现通过p38 MAP激酶的IL-1 β信号传导在INS-1（res）细胞中是正常的，表明它们的IL-1 R1表达足以维持细胞因子作用。然而，在INS-1（res）细胞系中，正常的IL-1 β介导的NF-κ B易位和诱导型一氧化氮合酶表达和一氧化氮产生的诱导严重受损，提示IL-1 β抗性的机制。总之，本研究确定了一种分离抗马槟榔碱β细胞系的策略，并提供了一种新的系统，用于研究可以实现这种抗性的机制。
Engineered insulinoma cell lines may represent an alternative to isolated islets for transplantation therapy of type 1 diabetes. Success of this approach may require development of cell lines that can withstand cytokine-mediated damage. To this end, we have cultured INS-1 insulinoma cells in increasing concentrations of interleukin-1 beta (IL-1 beta) + gamma-interferon (IFN-gamma), with approximate weekly iterations over an 8-week period. Based on the C,N diphenyl-N'-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) viability assay the selected cells, termed INS-1(res), were 100%, viable after 5 days of treatment with 10 ng/ml of IL-1 beta. These cells were also 78 +/- 1.2%, viable after 5 days of exposure to the combination of 10 ng/ml IL-1 beta and 100 U/ml IFN-gamma, whereas parental INS-1 cells treated in the same manner were only 0.3 +/- 0.03% viable. INS-1(res) cells were also resistant to treatment with supernatants from activated rat peripheral blood mononuclear cells, whereas only 20% of parental INS-1 cells survived such treatment. The resistance to IL-1 beta conferred by this procedure was stable, whereas the partial resistance to IFN-gamma was transient but reinducible by culture in the presence of cytokines. Stable transfection of INS-1(res) cells with a plasmid containing the human insulin cDNA and expansion of the transfected colonies in the absence of cytokines produced cell lines that were on average more resistant to IL-1 beta + IFN-gamma (53 +/- 11%) than similarly transfected clones derived from parental INS-1 cells (15 +/- 7%). Importantly, several INS-1(res)-derived clones retained the capacity to secrete insulin in response to glucose concentrations over the normal physiological range, With regard to the mechanism by which selection was conferred, we found normal levels of IFN-gamma receptor mRNA, but a 60% reduction in expression of the IL-1 receptor type I (IL-1R1) in INS-1(res) cells compared with parental INS-1 cells. IL-1 beta signaling through p38 MAP kinase was found to be normal in INS-1(res) cells, suggesting that their expression of IL-1R1 is sufficient to maintain cytokine action, However, normal IL-1 beta-mediated translocation of NF-kB and induction of inducible nitric oxide synthase expression and nitric oxide production was severely impaired in the INS-1(res) cell lines, suggesting a mechanism for the IL-1 beta resistance. In sum, this study defines a strategy for isolation of cytokine-resistant beta-cell lines and provides a new system for studying the mechanisms by which such resistance can be achieved.