应用基因表达谱芯片筛选高能X射线诱导HFL⁃1细胞胶原相关基因

摘要

目的 探讨高能X射线对人胚肺成纤维细胞(HFL?1)细胞胶原表达产生影响,并筛选胶原相关基因,为临床放射性肺纤维化的治疗提供理论依据.方法 将体外培养的HFL?1细胞随机分为对照组和放射组,放射组细胞予6 MV X射线5 Gy单次照射.照射后24 h以消化法检测两组细胞羟脯氨酸(HYP)表达情况,RT?PCR及蛋白印迹法检测Ⅰ、Ⅲ型胶原基因及蛋白表达情况,采用基因芯片技术分析基因表达谱改变情况.结果 照射后24 h放射组细胞HYP表达显著高于对照组(1834μg/ml:4912μg/ml,P=0000).放射组照射后24 hⅠ、Ⅲ型胶原基因及蛋白表达均明显高于对照组(18535:186779,P=0000;4337:24425,P=0000).两组细胞差异表达的基因为1879个,其中放射组上调基因771个、下调基因为1108个.参与纤维化的基因上调5倍以上的有TGF?β1、TGF?β3、MMP28、MMP26、MMP27和SMAD6.结论 高能X射线能诱导HFL?1细胞产生胶原,表达谱基因芯片技术可有效筛选高能X射线诱导胶原相关基因.放射性肺纤维化的发生发展由多个类型基因参与调控.%Objective To investigate the effects of high?energy X?ray irradiation on collagen expression in human fetal lung fibroblasts ( HFL?1) and identify the genes related to collagen production induced by X?ray, and to provide a theoretical basis for the clinical treatment of radiation?induced pulmonary fibrosis. Methods HFL?1 cells cultured in vitro were randomly divided into control group and irradiation group. The irradiation group was irradiated with 6 MV X?ray at a single dose of 5 Gy. At 24 hours after irradiation, the expression of hydroxyproline ( HYP ) in the two groups was determined by the digestion method. The expression of collagen types Ⅰ and Ⅲ at mRNA and protein levels was measured by RT?PCR and Western blot. And gene chip was used to identify the differentially expressed genes between the two groups. Results At 24 hours after irradiation, the irradiation group had significantly higher expression of HYP than the control group (1834 μg/ml vs.4912 μg/ml, P=0000), and the irradiation group had significantly higher mRNA and protein expression of collagen types Ⅰ and Ⅲ than the control group ( 18535 : vs. 86779, P=0000;4337 : vs. 4425, P=0000) . A total of 1879 genes were differentially expressed between the two groups, including 771 up?regulated genes and 1108 down?regulated genes in the irradiation group. The genes involved in fibrosis which were up?regulated more than 5?fold were transforming growth factor ( TGF)?beta 1, TGF?beta 3, matrix metalloproteinase ( MMP )?28, MMP?26, MMP?27, and SMAD6. Conclusions High?energy X?ray can induce collagen production in HFL?1 cells. Gene chip can effectively identify the genes related to collagen production induced by X?ray. Many different genes are involved in the development and progression of radiation?induced pulmonary fibrosis.