pErk1/2在干扰素-γ对干扰素调节因子-1沉默后表达Akt的32D细胞生长中的作用

phosphoration Erk1/2 to the effects of IFN-γ on 32D cells of expressing Akt after IRF-1 gene silence

目的 :运用siRNA沉默干扰素调节因子-1(IRF-1)基因,观察干扰素-γ(IFN-γ)对IRF-1沉默后表达Akt的32D细胞生长增殖的作用,探讨IFN-γ由抑制造血逆转为促进造血的机制。方法 :培养表达野生型Akt与无活性型Akt的32D细胞并分别转染,针对IRF-1-siRNA质粒以沉默IRF-1基因,用实时定量聚合酶链反应检测沉默效果,Western-blotting检测IRF-1蛋白表达以验证沉默效果。将处于对数生长期的两株细胞分成3大组:siRNA干扰组、空质粒阴性对照组及空白对照组,前两组分别加入不同浓度的IFN-γ共孵育,用MTS法检测两株细胞增殖情况。用流式细胞仪检测各组细胞凋亡率;用Western-blotting检测p-Erk1/2表达。结果:(1)加IFN-γ24h后,IRF-1沉默组在IFN-γ低浓度时促进增殖,抑制凋亡。高浓度的IFN-γ仍抑制增殖,促进凋亡。在48h以后IRF-1沉默组各浓度的IFN-γ都促进增殖,抑制凋亡(P<0.05)。(2)表达野生型Akt的32D细胞在各时间点的增殖比例都大于表达无活性型Akt的细胞。且凋亡率都小于表达无活性型Akt的各组细胞,两者在48h比较差异有统计学意义(P<0.05)。(3)表达无活性型Akt的32D细胞,加入IFN-γ后各组均上调了pErk1/2的表达(P<0.05)。但IRF-1沉默前后,各组pErk1/2表达量比较差异无统计学意义(P>0.05)。表达野生型Akt的32D细胞,在IRF-1沉默后各浓度IFN-γ均下调pErk1/2的表达(P<0.05)。结论:(1)IRF-1在IFN-γ的促凋亡作用中起重要作用。(2)有活性Akt的表达可抑制Erk信号通路,其活性状态决定了IRF-1封闭后IFN-γ对pErk1/2表达的作用,pErk1/2的表达影响细胞增殖与凋亡,但并不决定IFN-γ作用的逆转。(3)Akt信号通路在IFN-γ逆转机制中起作用。

Objective： Short interfering RNA （siRNA） eukaryotic expression vector for IRF-1 to reduce the expres-sion of IRF-1 was constructed, and was transfected into Murine myeloid precursor cells（32D cells）of wild-type Akt and in-active-type Akt. To observe the effects of IFN-γon the two types cells after IRF-1 gene silence, and investigate the mech-anisms of conversion from inhibition to promotion of hematopoiesis by IFN-γ. Methods： The two type cells were cultured and IRF-1-siRNA plasmid and the blank plasmid were transfected into the logarithmic phase cells, then the silent effect of IRF-1 gene was detected by RT-PCR and applying Western-blotting the effect was validated further. The cells of logarith-mic growth phase were divided into three groups： the knock down、the empty vector negative control and the blank control group, and then the former two groups were treated with four concentrations of IFN-γ. At different times the cell viability and inhibition rate was assayed by MTS;apoptotic rate was determined by flow cytometry;and the expression of phosphor-ation Erk1/2 was investigated by applying western blot. Results：（1）After treated with IFN-γ for 24 hours, in the IRF-1 silent group in low concentration of IFN-γ promoted proliferation and apoptosis was inhibited, and in high concentration,proliferation was suppressed and apoptosis was induced. But at 48 hours, in the IRF-1 silent group in different concentra-tions proliferation was promoted and apoptosis was inhibited（P〈0.05）.（2）The proliferation’s proportion of 32D cells of wild-type Akt was higher than that of inactive-type Akt, and its apoptosis rate less than that of inactive Akt at all time pionts, especilly at 48 hours, the difference was remarkable （P〈0.05）. （3） For 32D cells of inactive-type Akt, the expression of pErk1/2 in each group was increased when adding IFN-γ （P〈0.05）. However, the expression of pErk1/2 in each group lacked statistically significant difference pre and post of the IRF-1 silence （P〉0.05）. For 32D cells of wild-type Akt, the expression of pErk1/2 in each group was decreased after IRF-1 silence （P〈0.05）. Conclusions：（1）IRF-1 plays a critical role in pro-apoptotic effect of IFN-γ on the proliferation of hematopoietic progenitor cells.（2）The expression of active Akt can inhibit Erk signaling pathway. The role of IFN-γinfluencing the expression of pErk1/2 was determined by the status of Akt when IRF-1was closed. The expression of Perk1/2 can influence the cell’s proliferation and apoptosis, but does not de-termine the reversal role of IFN-γ.（3）The Akt signal pathway may play a determined role in the IFN-γ’s reversal of pro-moting cell proliferation,and the mechanism and pathway of IFN-γ’s reversal needs further research.