摘要： 目的 探讨DNA损伤修复通路因子53BP1在骨髓干细胞自我更新及定向分化过程中发挥的作用。方法 构建53BP1全身敲除C57/6小鼠模型，免疫荧光染色检测53BP1在细胞中的定位，Western blotting对53BP1蛋白表达水平进行检测及鉴定；流式细胞术进行细胞分选和计数、细胞周期检测、以及细胞表面分子marker分析，对比WT组和Mut组细胞生长速率以及定向分化过程，Co-Ip检测分析表观遗传学磷酸化修饰蛋白，SPSS 22.0对数据进行统计学分析。结果 体内和体外实验证实伴随KSL细胞分化发育进行，53BP1表达含量显著增加，53BP1基因敲除可影响KSL细胞的自我更新和定向分化以及表面分子标记的表达；53BP1基因敲除与否并不显著影响处于细胞周期G₁/G₀的KSL细胞百分含量；53BP1可影响磷酸化修饰蛋白的表达水平，53BP1基因敲除后磷酸化修饰蛋白p-H4（k20）和p-H3（k79）表达水平明显增加，但p-ATM和p-53-Ser-20含量显著减少。结论 53BP1参与并影响骨髓干细胞的自我更新及定向分化过程，53BP1基因敲除可显著影响磷酸化蛋白p-H4（k20）、p-H3（k79）、p-ATM和p-53-Ser-20的表达水平。

DNA损伤修复通路因子53BP1在骨髓干细胞自我更新和分化发育中的作用

尤放, 王美莲

中国医科大学基础医学院病原生物学教研室, 沈阳 110122

收稿日期:2020-04-14出版日期:2021-01-30发布日期:2021-01-06
通讯作者:王美莲E-mail:bigwangfei@qq.com
作者简介:尤放(1993-),女,硕士研究生.
基金资助:国家自然科学基金（81770001）


关键词: DNA损伤修复应答, 53BP1, 骨髓干细胞, 自我更新, 分化
Abstract: Objective To determine the role of the DNA damage repair pathway factor,53BP1,in the self-renewal and differentiation of bone marrow stem cells. Methods A whole-body knockout 53BP1 C57/6 mouse model was constructed. In addition,53BP1 was mutated in KSL mouse hematopoietic stem cells. Immunofluorescence staining was used to detect the localization of 53BP1 in cells. The expression level of the 53BP1 protein was detected and identified by western blotting. The cell growth rate and directional differentiation process were compared between wild-type and 53BP1-mutated cells and between wild-type and 53BP1 knockout mice. Phosphorylated proteins were detected by co-immunoprecipitation,and the experimental data were analyzed statistically using SPSS 22.0. Results In vivo and in vitro experiments confirmed that the 53BP1 content significantly increased with the differentiation of KSL cells,and the mutation of 53BP1 affected the regeneration and directional differentiation of these cells as well as the expression of surface molecular markers. The mutation of 53BP1 did not significantly affect the percentage of KSL cells in the G₁/G₀ cell cycle phase. 53BP1 affected the expression levels of phosphorylated proteins. Specifically,53BP1 knockout led to a significant increase in the phosphorylated (p) -H4 (k20) and p-H3 (k79) proteins but a significant decrease in p-ATM and p-53-Ser-20. Conclusion 53BP1 was involved in and could affect the processes of self-renewal and directional differentiation of bone marrow stem cells. Knockout of 53BP1 affected the expression levels of p-H4 (k20),p-H3 (k79),p-ATM,and p-53-Ser-20.
Key words: DNA damage response, 53BP1, bone marrow stem cell, self-renewal, differentiation
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