缺氧复合梭曼中毒后PC12细胞IL-6、GP130、JAK1、STAT1基因表达的变化

The expression changes in IL-6,GP130,JAK1 and STAT1 genes in PC12 cell after combined soman and hypoxia injury

目的研究缺氧复合梭曼中毒后PC12细胞IL-6、GP130、JAK1、STAT1基因mRNA和蛋白表达的变化。方法制备缺氧复合梭曼中毒细胞模型,设对照组、梭曼中毒组、缺氧复合梭曼中毒组和Genistein抑制剂组。采用半定量RT-PCR和Western blot法检测各组PC12细胞IL-6、GP130、JAK1、STAT1基因mRNA和蛋白的表达水平,并对PCR产物进行测序。结果梭曼中毒后PC12细胞IL-6、GP130、JAK1、STAT1mRNA和蛋白表达水平升高,在中毒后12h达峰值,中毒后24h的表达量低于12h,与对照组比较均有显著差异。缺氧复合梭曼中毒组PC12细胞中IL-6、GP130、JAK1、STAT1 mRNA和蛋白表达水平6h后达峰值,且明显高于对照组、梭曼中毒组和Genistein抑制剂组。RT-PCR产物直接测序证实扩增产物片段与GenBank中相应序列相同。结论梭曼中毒或(和)缺氧后PC12细胞中JAK1/STAT1基因的表达增强,在缺氧复合梭曼中毒所致脑损伤中可能发挥重要作用。

Objective To investigate the complex effect of soman and hypoxia on PC12 cell line as shown by the expression changes in IL-6/GP130, JAK1 and STAT 1. Methods A cell model of intoxication by combining effect of hypoxia and soman was reproduced, and it was divided into four groups： control, soman intoxication, hypoxia combined with soman intoxication, and C-enistein inhibition groups. PC12 cells were cultured in RPMI 1640 and were treated with NGF （50ng/ml） for seven days. The differentiated PC12 cells were then exposed to hypoxia in an incubator containing 5% CO2, 95% N2 and （or） incubated with soman （20μmol/L） for 2, 6, 12 or 24 hours. The expression levels of IL 6/GP130, JAK1, STAT 1 mRNA and protein were assessed by RT PCR and Western blot in PC12 cells. The prod ucts were sequenced by Sanger＇s double strand DNA sequence determination. Results In soman intoxicated group, the expression levels of IL-6/GP130, JAK1, STAT 1 mRNA and protein were elevated in PC12 cell, reaching the peak level at 12 hours, and then lowered at 24 hours, but remaining higher than that of control group. In combined soman intoxicated and hypoxia group, the expression levels of IL6/ GPI30, JAK1, STAT 1 mRNA and protein reached the peak value at 6 hours, being higher than that of control group, soman intoxicated group and C-enistein inhibition group. It was shown that the sequences of the products as amplified by RT-PCR were the same as that found in the Genl3ank. Conclusion Soman intoxication or （and） hypoxia up-regulate the expression of IL-6/GPI30. Both hypoxic condition and soman treatment can up-regulate the expression of JAK1/STAT 1 mRNA and protein in PC12 cells. JAK-STAT pathway may play a role in the mechanisms of brain injury resulted from hypoxia and soman poisoning.