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目的研究组蛋白H2A去泛素化酶MYSM1缺陷(MYSM1-/-)小鼠骨髓造血干细胞(HSC)的静息状态、增殖与凋亡情况。方法分离MYSM1-/-小鼠和野生对照(MYSM1+/+)小鼠的骨髓细胞,利用免疫磁珠和流式细胞仪分选获得表面标志为lin-Sca-1+c-kit+的HSC。采用腹腔注射溴脱氧尿嘧啶核苷(Brd U)的方法检测HSC增殖与细胞周期、采用Hoechst 33342-派若宁(pyronin)Y双染色法检测静息态HSC的比例、采用异硫氰酸荧光素标记膜联素Ⅴ/7-氨基放线菌素D(annexinⅤ-FITC/7-AAD)双染色法检测HSC凋亡。比较MYSM1-/-小鼠和MYSM1+/+对照小鼠HSC数目、细胞周期、细胞增殖和细胞凋亡的变化情况。结果 MYSM1-/-小鼠的骨髓细胞、HSC总数与野生型小鼠相比明显降低;HSC的S期比例增加、增殖加快、静息期细胞减少但功能有缺陷的MYSM1-/-HSC凋亡率显著上升。结论 MYSM1对维持HSC的静息状态有非常重要的作用,其缺陷可引起静息态的HSC大量进入S期,异常增殖的HSC凋亡增加并最终引起HSC和骨髓细胞总数减少。
Objective To investigate the resting status, proliferation and apoptosis of bone marrow hematopoietic stem cells (HSCs) of histone H2A deubiquitinating enzyme (MYSM1 - / -) mice. Methods The myeloid cells from MYSM1 - / - mice and wild-type control (MYSM1 + / +) mice were isolated and the HSC labeled with lin-Sca-1 + c-kit + was obtained by immunomagnetic beads and flow cytometry. The proliferation and cell cycle of HSC were detected by intraperitoneal injection of bromodeoxyuridine (BrdU). Hoechst 33342-pyronin Y double staining was used to detect the proportion of resting HSC. Fluorescein isothiocyanate HSC apoptosis was detected by double staining with annexinⅤ-FITC / 7-AAD. The numbers of HSC, cell cycle, cell proliferation and apoptosis in MYSM1 - / - mice and MYSM1 + / + control mice were compared. Results The total number of myeloid cells and MNCM1 - / - mice in MYSM1 - / - mice was significantly lower than that in wild type mice. The proportion of S phase of HSCs was increased, the proliferation was accelerated, and the number of resting cells but the functionally defective MYSM1 - / - HSCs were apoptotic Rate increased significantly. Conclusions MYSM1 plays an important role in maintaining the resting status of HSC. The defects of MYSM1 can cause a large number of resting HSCs to enter the S phase, and increase the apoptosis of abnormally proliferating HSCs and eventually lead to the decrease of the total number of HSCs and myeloid cells.