Vasu Srividya, McClenaghan Neville H, McCluskey Jane T, Flatt Peter R
SAAD Centre for Pharmacy and Diabetes, University of Ulster, Cromore Road, Coleraine BT52 1SA, Northern Ireland, UK.
Biochim Biophys Acta. 2014 Jan;1840(1):136-45. doi: 10.1016/j.bbagen.2013.08.022. Epub 2013 Sep 1.
Molecular mechanisms of toxicity and cell damage were investigated in the novel human beta cell line, 1.1B4, after exposure to proinflammatory cytokines - IL-1β, IFN-γ, TNF-α.
MTT assay, insulin radioimmunoassay, glucokinase assay, real time reverse transcription PCR, western blotting, nitrite assay, caspase assay and comet assay were used to investigate mechanisms of cytokine toxicity.
Viability of 1.1B4 cells decreased after 18h cytokine exposure. Cytokines significantly reduced cellular insulin content and impaired insulin secretion induced by glucose, alanine, KCl, elevated Ca(2+), GLP-1 or forskolin. Glucokinase enzyme activity, regulation of intracellular Ca(2+) and PDX1 protein expression were significantly reduced by cytokines. mRNA expression of genes involved in secretory function - INS, GCK, PCSK2 and GJA1 was downregulated in cytokine treated 1.1B4 cells. Upregulation of transcription of genes involved in antioxidant defence - SOD2 and GPX1 was observed, suggesting involvement of oxidative stress. Cytokines also upregulated transcriptions of NFKB1 and STAT1, which was accompanied by a significant increase in NOS2 transcription and accumulation of nitrite in culture medium, implicating nitrosative stress. Oxidative and nitrosative stresses induced apoptosis was evident from increased % tail DNA, DNA fragmentation, caspase 3/7 activity, apoptotic cells and lower BCL2 protein expression.
This study delineates molecular mechanisms of cytokine toxicity in 1.1B4 cells, which agree with earlier observations using human islets and rodent beta cells.
This study emphasizes the potential usefulness of this cell line as a human beta cell model for research investigating autoimmune destruction of pancreatic beta cells.
在新型人β细胞系1.1B4暴露于促炎细胞因子白细胞介素-1β（IL-1β）、干扰素-γ（IFN-γ）、肿瘤坏死因子-α（TNF-α）后，对其毒性和细胞损伤的分子机制进行了研究。
采用MTT法、胰岛素放射免疫分析法、葡萄糖激酶分析法、实时逆转录聚合酶链反应、蛋白质免疫印迹法、亚硝酸盐分析法、半胱天冬酶分析法和彗星试验来研究细胞因子毒性机制。
细胞因子暴露18小时后，1.1B4细胞的活力下降。细胞因子显著降低了细胞胰岛素含量，并损害了由葡萄糖、丙氨酸、氯化钾、钙离子浓度升高、胰高血糖素样肽-1（GLP-1）或福斯高林诱导的胰岛素分泌。细胞因子使葡萄糖激酶酶活性、细胞内钙离子调节和胰腺十二指肠同源盒-1（PDX1）蛋白表达显著降低。在细胞因子处理的1.1B4细胞中，参与分泌功能的基因胰岛素（INS）、葡萄糖激酶（GCK）、前蛋白转化酶枯草溶菌素2（PCSK2）和缝隙连接蛋白α1（GJA1）的mRNA表达下调。观察到参与抗氧化防御的基因超氧化物歧化酶2（SOD2）和谷胱甘肽过氧化物酶1（GPX1）转录上调，提示氧化应激参与其中。细胞因子还上调了核因子κB亚基1（NFKB1）和信号转导子与转录激活子1（STAT1）的转录，这伴随着一氧化氮合酶2（NOS2）转录显著增加和培养基中亚硝酸盐积累，提示存在亚硝化应激。氧化应激和亚硝化应激诱导的细胞凋亡从尾部DNA百分比增加、DNA片段化、半胱天冬酶3/7活性、凋亡细胞增加以及B细胞淋巴瘤-2（BCL2）蛋白表达降低中明显可见。
本研究阐述了1.1B4细胞中细胞因子毒性的分子机制,这与早期使用人类胰岛和啮齿动物β细胞的观察结果一致。
本研究强调了该细胞系作为人类β细胞模型在研究胰腺β细胞自身免疫性破坏方面的潜在用途。
