放射致小鼠骨髓造血细胞损伤与衰老模型的建立

Establishment of mouse model of irradiation-induced bone marrow hematopoietic cell injury and senescence

目的建立放射致小鼠骨髓细胞损伤与衰老模型。方法取50只小鼠,随机分为对照组和4.0、6.5、8.5和10.5 Gy不同剂量照射实验组,观察小鼠30 d存活率。另取20只小鼠,分别于6.5 Gy照射后第1、3、7和30天检测外周血血常规及骨髓单个核细胞(Mononuclear cells,MNCs)数的变化,观察骨髓造血细胞大小及形态并计数集落数量,流式细胞仪检测骨髓MNCs凋亡情况,采用衰老相关的β-半乳糖苷酶(Senescence-associatedβ-galactosidase,SA-β-gal)染色法检测SA-β-gal活性,细胞免疫荧光法分析p16Ink4a蛋白的表达水平。结果小鼠30 d存活率对照组和4.0 Gy实验组均为100%,6.5 Gy实验组为80%,8.5及10.5 Gy实验组均为0。6.5 Gy一次性全身照射后第1、3、7、30 d,小鼠外周血白细胞、血小板、骨髓MNCs数、CFU数量均下降,以照射后第3天最明显,第7天开始恢复正常;照射后骨髓MNCs凋亡率与对照组[(7.32±1.87)%]相比,均明显升高(P<0.05),第3天最高[(35.71±0.30)%],随时间的延长凋亡率逐渐下降;SA-β-gal染色和细胞免疫荧光染色结果显示,照射后阳性细胞率与对照组比较,均明显升高(P<0.05),以第3天最高,随时间延长逐渐降低。结论以6.5 Gy照射,可建立小鼠的骨髓造血细胞损伤与衰老模型,为进一步筛选抗骨髓抑制的药物奠定了基础。

Objective To establish the mouse model of irradiation-induced bone marrow hematopoietic cell injury and senescence. Methods Fifty mice were randomly divided into one control and four test groups. The mice in test groups 1, 2, 3 and g were irradiated with γ-ray at dosages of 4, 6. 5, 8. 5 and 10. 5 Gy respectively, while those in control group were not irradiated. All the mice were observed for survivals for 30 d. Another 20 mice were irradiated with γ-ray at a dosage of 6. 5 Gy, of which the peripheral blood was subjected to routine blood test on days 1, 3, 7 and 30 after irradiation, while the bone marrow mononuclear cells were counted, observed for morphology and counted for colonies, then determined for apoptosis by flow cytometry, for senescence-associated β-galactosidase （SA-β-gal） activity by SA-β-gal staining, and for expression level of p16^kkda protein by cell IFA. Results The survival rates of mice 30 d after irradiation were both 100% in control group and test group 4, 80% in test group 2 and 0% in both test groups 3 and 4. However, on days 1, 3, 7 and 30 after a single systemic irradiation with γ-ray at a dosage of 6. 5 Gy, the leukocyte and platelet counts in peripheral blood as well as bone marrow mononuclear cell counts and CFU of mice decreased, which decreased remarkably on day 3 and begun to recover to normal on day 7 after irradiation. The apoptosis rate of bone marrow mononuclear cells after irradiation increased significantly as compared with those received no irradiation [ （7. 32 ± 1.87）% ] （P 〈 0. 05）, which reached a peak value on day 3 [（35.71 ± 0. 30）%] then decreased gradually as time went on. Senescence- associated SA-β-gal staining and cell IFA showed that both the percentages of positive cells increased significantly as compared with those received no irradiation （P 〈 0. 05）, which reached a peak value on day 3 then decreased gradually as time went on. Conclusion Mouse model of bone marrow hematopoietic cell injury and senescence was established by irradiation with γ-ray at a dosage of 6. 5 Gy, which laid a foundation of further screening anti-irradiation drugs.