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试验旨在研究蓖麻蛋白对人外周血B淋巴细胞(IM-9)毒性作用及对相关基因表达的影响。将提取的蓖麻蛋白按不同浓度添加到培养基培养6、12h,研究剂量-时间效应,确定半数抑制浓度,然后以半数抑制浓度培养细胞2、6、10、12h,在每个时间段检测免疫相关基因CD40、IL-1β、TNF-α和凋亡相关基因Bcl-2、Bax、Caspase-3的表达情况。结果表明,蓖麻蛋白对IM-9细胞的毒性作用随浓度和时间的增加而增强。取接近半数抑制浓度20ng/mL作用IM-9细胞2、6、10、12h,免疫相关基因中,TNF-α和CD40基因在6h试验组表达量与对照组相比显著升高(P<0.05),10、12h达到极显著水平(P<0.01);IL-1β基因表达量试验组、对照组间差异不显著(P>0.05)。凋亡基因中,与对照比相比,试验组Bax基因表达量10h极显著降低(P<0.01),12h显著降低(P<0.05);Bcl-2基因表达量4个时间段均达到显著水平(P<0.05),2、12h达到极显著水平(P<0.01);Caspase-3基因除6h表达量降低外,其他时间段表达量均显著升高(P<0.05),2h为极显著水平(P<0.01)。表明蓖麻蛋白能显著影响IM-9细胞的基因表达,TNF-α和CD40基因是两个潜在用IM-9细胞评价蛋白毒性的检测基因。
The aim of this study was to investigate the effects of ricin on the cytotoxicity of human peripheral blood B lymphocytes (IM-9) and the related gene expression. The extracted ricin protein was added to the medium at different concentrations for 6 and 12 h, and the dose-time effect was studied to determine the half inhibitory concentration. Cells were cultured at half inhibitory concentration for 2, 6, 10 and 12 h, and detected at each time period The expression of CD40, IL-1β, TNF-α and Bcl-2, Bax and Caspase-3 were detected by immunohistochemistry. The results showed that the toxicity of ricin to IM-9 cells increased with the increase of concentration and time. The expression levels of TNF-αand CD40 in TNF-α and 6h groups were significantly higher than those in control group at 2, 6, 10, and 12 h (P <0.05) (P <0.01). The expression of IL-1β was not significantly different between the experimental group and the control group (P> 0.05). Compared with the control group, the expression of Bcl-2 gene in the experimental group was significantly decreased at 10h (P <0.01) and significantly at 12h (P <0.05) (P <0.05), and reached the extremely significant level at 2 and 12 h (P <0.01). The expression of Caspase-3 gene was significantly increased at other time points (P <0.05) (P <0.01). These results indicate that ricin can significantly affect the gene expression of IM-9 cells. TNF-α and CD40 genes are two potential detection genes for evaluating protein toxicity using IM-9 cells.