探讨造血生长因子GM-CSF/IL-3融合蛋白、GM-CSF及IL-3抗γ射线辐射诱导Tf-1细胞凋亡的分子机制.采用荧光强度比色法检测辐射后Tf-1细胞内Caspase-3活性;RT-PCR半定量及免疫组化实验方法分别观察Bcl-2、Caspase-3 mRNA及蛋白表达水平的变化.GM-CSF/IL-3融合蛋白、GM-CSF、IL-3以及GM-CSF与IL-3合用均可使辐射后Tf-1细胞内Caspase-3活性明显降低,其活性是不加入任何细胞因子对照组的15.71%-43.65%.在Tf-1细胞辐射后48 h时,各细胞因子组Bcl-2 mRNA及蛋白的表达水平较对照组明显提高;其中GM-CSF/IL-3融合蛋白10μg/L组在辐射后24h时观察到Bcl-2 mRNA表达增强.细胞辐射后48h时,GM-CSF/IL-3融合蛋白10μg/L、100μg/L及IL-3 10μg/L组Caspase-3mRNA的表达明显增强;辐射后48h时,各细胞因子组均观察到Caspase-3蛋白表达增多.三种细胞因子可通过明显抑制Caspase-3活性、促进Bcl-2、Caspase-3 mRNA、蛋白质的表达以及抑制Caspase-3酶原激活而发挥抗辐射诱导的Tf-1细胞凋亡.%To investigate the molecular mechanisms of the anti-apoptosis effects of hematopoietic growth factors GM-CSF/IL-3 fusion protein, GM-CSF and IL-3 in Tf-1 cells induced by γ-irradiation. Caspase-3 activity was detected using fluorescence chromatometry. RT-PCR semiquantitative analysis was used to identify the expression changes of Bcl-2 and Caspase-3 mRNA, and an immunohistochemical technique was used to examine the protein expression in irradiated Tf-1 cells. The Caspase-3 activity in irradiated Tf-1 cells was markedly reduced to 15.71 %-43.65 % in GM-CSF/IL-3 fusion protein, GM-CSF, IL-3, and the combination of GM-CSF and IL-3-treated groups compared to those in the control groups without any growth factor. Bcl-2 mRNA and its protein expression were significantly increased by the above cytokines after incubation with the cells for 48 h after irradiation, while the enhancement of the Bcl-2 mRNA level by GM-CSF / IL-3 fusion protein (10 μg / L) began from 24 h. After 48h of incubation after irradiation, the expression of Caspase-3 mRNA was dramatically augmented by GM-CSF / IL-3 fusion protein at 10 μg / L, 100 μg / L and IL-3 10 μg / L, as was the corresponding Caspase-3 protein expression by the addition of each cytokine. The cytokines exerted the anti-apoptosis effects in irradiated Tf-1 cells through the up-regulation of Bcl-2 and Caspase-3 expression, and the inhibition of Caspase-3 activity.
展开▼
