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目的检测刚地弓形虫(Toxoplasma gondii)棒状体蛋白17(rhoptry protein 17,ROP17)对γ干扰素(IFN-γ)诱导的小鼠单核巨噬细胞(J774A.1)凋亡的调控作用。方法将重组质粒p3×Flag-CMV-14/Tg ROP17和p3×Flag-CMV-14空质粒(对照组)转染J774A.1细胞后,加入重组IFN-γ诱导细胞凋亡,通过流式细胞术和蛋白质印迹(Western blotting)检测ROP17对细胞凋亡的调控作用,检测c-Jun磷酸化水平及活化形式的半胱天冬酶3(Caspase 3)、Bcl-2、Bcl-x L和Bcl-3等凋亡相关蛋白的表达情况。J774A.1细胞共转染p3×FlagCMV-14/Tg ROP17和c-Jun sh RNA,加入重组IFN-γ诱导细胞凋亡,Western blotting检测ROP17对凋亡的调控作用及凋亡相关蛋白Bcl-3的表达。结果流式细胞术显示,过表达ROP17组细胞凋亡率为(3.73±0.51)%,明显低于对照组的(7.78±1.10)%(P<0.05)。Western blotting结果显示,过表达ROP17组磷酸化c-Jun蛋白相对表达量为0.196±0.028,对照组为0.075±0.010(P<0.05);Bcl-3相对表达量为0.461±0.063,对照组为0.108±0.013(P<0.05)。活化形式的Caspase 3相对表达量为0.015±0.004,对照组为0.174±0.026(P<0.05)。Bcl-2在过表达ROP17组和对照组中的相对表达量分别为0.119±0.013和0.117±0.018,Bcl-x L则分别为0.124±0.015和0.121±0.023,差异均无统计学意义(P>0.05)。在c-Jun sh RNA有效抑制c-Jun及其磷酸化表达的条件下,活化形式的Caspase 3在RNA干扰组和对照组的相对表达量分别为0.147±0.024和0.087±0.010(P<0.05),Bcl-3则分别为0.085±0.010和0.162±0.011(P<0.05)。结论ROP17抑制IFN-γ诱导的J774A.1细胞凋亡依赖于c-Jun的磷酸化及Bcl-3的表达。
Objective To investigate the regulatory effect of rhoptry protein 17 (ROP17) on the apoptosis of murine monocyte-macrophage (J774A.1) induced by interferon-gamma (IFN-γ) by Toxoplasma gondii. Methods The recombinant plasmid p3 × Flag-CMV-14 / Tg ROP17 and p3 × Flag-CMV-14 empty plasmid (control group) were transfected into J774A.1 cells and then induced by recombinant IFN-γ. The expression of c-Jun phosphorylation and activation of caspase 3, Bcl-2, Bcl-x L and Bcl-2 were detected by Western blotting and Western blotting respectively. -3 and other apoptosis related protein expression. J774A.1 cells were co-transfected with p3 × FlagCMV-14 / Tg ROP17 and c-Jun sh RNA, and then induced by recombinant IFN-γ. Western blotting was used to detect the effect of ROP17 on apoptosis and Bcl-3 expression. Results Flow cytometry showed that the rate of apoptosis in overexpression ROP17 group was (3.73 ± 0.51)%, which was significantly lower than that in the control group (7.78 ± 1.10)% (P <0.05). The results of Western blotting showed that the relative expression of phosphorylated c-Jun protein in ROP17 overexpression group was 0.196 ± 0.028 and that in the control group was 0.075 ± 0.010 (P <0.05); the relative expression level of Bcl-3 was 0.461 ± 0.063 and the control group was 0.108 ± 0.013 (P <0.05). The relative expression level of activated Caspase 3 was 0.015 ± 0.004 and that of the control group was 0.174 ± 0.026 (P <0.05). The relative expression levels of Bcl-2 in overexpression ROP17 group and control group were 0.119 ± 0.013 and 0.117 ± 0.018, respectively, while those of Bcl-x L were 0.124 ± 0.015 and 0.121 ± 0.023, respectively, with no significant difference (P> 0.05). The relative expression levels of activated Caspase 3 in RNA interference group and control group were 0.147 ± 0.024 and 0.087 ± 0.010 (P <0.05), respectively, under the condition that c-Jun sh RNA effectively inhibited c-Jun and its phosphorylation expression. , Bcl-3 was 0.085 ± 0.010 and 0.162 ± 0.011, respectively (P <0.05). Conclusion ROP17 can inhibit the IFN-γ-induced apoptosis of J774A.1 cells by c-Jun phosphorylation and Bcl-3 expression.