Notch信号途径对放疗后骨髓谱系造血重建的影响及其机制

Effect of Notch signaling pathway on bone marrow pedigree hematopoietic reconstruction after radiotherapy

目的探讨Notch信号途径对放疗后骨髓谱系造血重建的影响及其机制。方法通过交配获得骨髓造血细胞特异性敲除Notch信号MxCre^+RBP-J^(flox/flox)的双转基因小鼠(敲除组)和未敲除MxCre^+RBP-J^(flox/+)对照小鼠(未敲除组),给予全身一次性^(60)Co-γ射线电离辐照,观察辐照后第7天两组生存状态、造血重建情况。C57/B6小鼠接受全身一次性^(60)Co-γ射线电离辐照,通过腹腔注射Notch配体重组蛋白D1R或PBS分别建立Notch信号激活小鼠(激活组)和未激活小鼠(未激活组),观察给药第7天重组蛋白D1R的体内活性、两组造血重建情况以及髓系前体细胞周期、增殖信号通路变化。另取C57/B6小鼠随机分为阴性对照组、D1R组、阳性对照组,D1R组和阳性对照组同时接受等剂量^(60)Co-γ射线电离辐照后腹腔注射D1R和PBS,阴性对照组不予处理,停药3个月,观察重组蛋白D1R长期毒性。结果与未敲除组比较,敲除组生存状态更差,骨髓造血干/祖细胞、骨髓和外周血有核细胞及髓系细胞形态正常,但数量明显减少(P均<0.05)。与未激活组比较,激活组骨髓造血细胞Notch信号活化片段ICN1的核表达增多,Notch信号激活,骨髓造血干/祖细胞、骨髓和外周血有核细胞及髓系细胞数均明显高于对照组(P均<0.05),骨髓髓系前体细胞G_0/G_1期比例减少,S期比例增多,PI3K蛋白表达增加(P均<0.01)。停药3个月,D1R组细胞形态、组织构架及造血谱系与阴性对照组无明显差别,但骨髓造血干/祖细胞、骨髓和外周血髓系细胞数明显高于阳性对照组(P均<0.05)。结论 Notch信号途径可正向调控放疗后造血重建,尤其髓系重塑,其机制可能与Notch信号激活髓系前体细胞PI3K增殖信号通路、加快细胞周期进程有关。

Objective To investigate the effect of Notch signaling pathway on hematopoietic reconstruction of bone marrow pedigree after radiotherapy,and to explore its possible mechanism. Methods The double transgenic mice MxCre~＋RBP-J^（flox/flox）（ knockout group） and control mice MxCre~＋RBP-J^（flox/＋）（ non-knockout group） were generated to conditionally disrupt Notch signaling in the bone marrow. Mice were subjected to total body irradiation with γ-ray from a ^（60）Co irradiator. We observed the hematopoietic reconstitution on the 7 th day after irradiation. The C57/B6 mice received disposable total body irradiation for 7 days and were injected intraperitoneally with recombinant protein D1R or PBS to establish Notch signaling-activated mice（ active group） and control mice（ inactive group）. We evaluated the intracorporal activity of D1R,the hematopoietic reconstruction,the cell cycle,and proliferation signal pathway of myeloid progenitor between the two groups. C57/B6 mice were randomly divided into the negative control group,D1R group,and positive control group.Mice in the D1R group and positive control group were given D1R and PBS intraperitoneally after equal dose of ionizing radiation,while mice in the negative control group were not treated. We detected the long-term toxicity after 3-month withdrawal of D1R. Results Compared with the non-knockout group,the survival status aggravated,the morphology of bone marrow hematopoietic stem/progenitor cells,bone marrow and peripheral blood nucleated cells,and myeloid cells was normal,but the number became less in the knockout group（ all P〈0. 05）. Correspondingly,D1R efficiently increased nuclear activation fragment NICD,suggesting the activation of Notch signaling in bone marrow hematopoietic cells. Compared with the inactive group,the number of bone marrow hematopoietic stem/progenitor cells,bone marrow and peripheral blood nucleated cells,and myeloid cells was higher（ all P〈0. 05）,the proportion of myeloid precursor cells in the G_0/G_1 phase decreased,and increased in the S phase,respectively,and the expression of PI3K protein increased in the active group（ all P〈0. 01）. After 3-month withdrawal,there was no significant difference in cell morphology and hematopoietic lineage between the D1R group and negative control group,but the number of bone marrow hematopoietic stem/progenitor cells as well as bone marrow and peripheral blood myeloid cells was significantly higher than that of the positive control group（ all P〈0. 05）. Conclusion Notch signaling pathway can positively regulate the hematopoietic reconstitution after radiotherapy,especially myeloid remodeling. The mechanism may be that Notch signaling pathway activates PI3K proliferation signaling pathway in myeloid precursor cells and accelerates cell cycle progression.