Role Of DNA Damage Repair Pathway Factor 53BP1 In Bone Marrow Stem Cells And Differentiation

Objective:To explore the internal relationship between DNA damage repair pathway factor 53BP1 and the directional differentiation and development of bone marrow stem cells.We aimed to study the role of 53BP1 in the above process and analyze the relevant potential regulatory mechanisms.Methods:In order to study the effects of 53BP1 on the self-renewal and differentiation of bone marrow stem cells,we observed the expression of 53BP1 in the process of cell differentiation,as well as the changes of cell growth rate,colony forming ability and corresponding surface molecular markers after the loss of 53BP1(-/-)in KSL cells.1.Bioinformatics analysis of 53BP1 was performed by searching and comparing the database.2.Construct a C57/6 mouse model with whole-body knockout of 53BP1(-/-),and form negative control and positive control with wild-type 53BP1,namely 53BP1(WT/Mut).3.Western blot(WB)was used to detect and identify the expression level of 53BP1 protein.4.DAPI fluorescence staining was used to observe the expression of 53BP1(WT/Mut)in Embryogenesis,ES cells,C2C12 cells and KSL cells differentiation.5.The BD ProCount Progenitor Cell Enumeration Kit and BD Trucount tube were used for cell sorting,cell counting,cell cycle detection and surface molecular marker analysis by flow cytometry.6.Epigenetic phosphorylation proteins p-H4(k20)?p-H3(k79)?p-ATM and p-53-ser20 were detected and analyzed by Co-Immunoprecipitation.7.Graphpad Prism 8.0 and statistical software SPSS 22.0 were used to analyze the directional differentiation of peripheral blood lymphocytes.ANOVA and Post Hoc were used for data analysis.Enumeration data was tested by ?2 test and statistical significance was declared at p<0.05 level.Results:1.The expression of 53BP1 gradually decreased with the development of mouse embryogenesis(Day12,16,18);however,with the differentiation of embryonic stem cells(24h,48h,72h),adult stem cells C2C12 cells(Dayl and Day4)and KSL cells(Day5 and Day 10),the content of 53BP1 increased significantly.2.The loss of 53BP1(-/-)did not affect the counting of KSL cells in the resting period of cell cycle(G0 phase)(P>0.05,No significant differences),while it did significantly increased the counting of KSL cells in the intercellular phase(G1 phase)compared with that in the wild-type 53BP1(+/+)(P<0.05,Statistically Significant).In addition,the loss of 53BP1(-/-)significantly increased the common myelid precursor compared with that of wild-type 53BP1(+/+),on the other hand,it significantly reduced the common lymphoid precursors compared with that of wild-type 53BP1(+/+)(P<0.05,Statistically Significant).3.In vitro,the loss of 53BP1(-/-)slowed the growth rate of cultured KSL cells compared to that of wild-type 53BP1(+/+)(Day 1,Day 5,Day 10,Day 15),but the loss resulted in significantly higher colony number of cultured KSL cells than that of wild-type 53BP1(+/+).At the same time,the loss of 53BP1(-/-)significantly increased the percentage of Mar/Gr-1+ markers on the surface of KSL cells,while it significantly decreased the percentage of CD45+ markers on the surface of KSL cells compared to the wild-type 53BP1(+/+)(P<0.05,Statistically Significant),the bone marrow analysing results were consistent with the above.In addition,the loss of 53BP1(-/-)resulted in a significant increase in the contents of monocytes,eosinophil and basophils in the peripheral blood compared with that of wild-type 53BP1(+/+)(P<0.05,Statistically Significant).4.Epigenetic phosphorylation analysis showed that the loss of 53BP1(-/-)resulted in a significant increase in phosphorylated histone 4 lysine 20(p-H4(k20)and histone 3 lysine 79(p-H3(k79)compared to wild-type 53BP1(+/+),but it resulted in a significant decrease in phosphorylated p-ATM and p-53 Serine 20(p-53-ser20)compared to wild-type 53BP1(+/+).Conclusion:53BP1 was involved in and could affect the self-renewal and directional differentiation of bone marrow stem cells,and it mainly played a role in the early process of embryogenesis.The loss of 53BP1(-/-)could affect the expression levels of phosphorylated proteins p-H4(k20)?p-H3(k79)?p-ATM and p-53-Ser20.