敲除SIRT1对急性髓细胞白血病小鼠白血病细胞凋亡及细胞周期的影响

Influences of SIRT1 knockdown on cell apoptosis and cell cycle in mice with acute myeloid leukemia

目的探讨急性髓细胞白血病小鼠SIRT1表达及对白血病细胞凋亡及细胞周期的影响。方法 C57BL/6J小鼠10只,随机分为观察组和对照组各5只。2组小鼠应用半致死剂量X射线(4.75 Gy)照射7 min后,经尾静脉移植2.5×105个病毒感染的白血病细胞制备急性髓细胞白血病模型。移植第14天,观察组腹腔注射pIpC溶液5 mg/kg,隔天注射1次,共3次,诱导SIRT1敲除;对照组注射等量生理盐水。移植第24天处死2组小鼠,取股骨骨髓提取白血病细胞,采用实时荧光定量PCR法检测SIRT1 mRNA、细胞周期蛋白依赖性激酶1 mRNA、p19 mRNA、p21 mRNA、p27 mRNA相对表达量,采用流式细胞术检测细胞凋亡和细胞周期。结果观察组SIRT1 mRNA相对表达量(0.11±0.01)低于对照组(1.01±0.01)(P<0.05),p19 mRNA、p21 mRNA、p27 mRNA相对表达量(3.76±0.22、1.53±0.06、2.23±0.02)高于对照组(1.01±0.01、1.02±0.01、1.01±0.01)(P<0.05),细胞周期蛋白依赖性激酶1 mRNA相对表达量(1.05±0.02)与对照组(1.01±0.02)比较差异无统计学意义(P>0.05)。观察组细胞凋亡率[(30.3±0.3)%]与对照组[(27.3±0.4)%]比较差异无统计学意义(P>0.05)。观察组G0期细胞比率[(55.22±0.60)%]高于对照组[(19.20±0.30)%](P<0.05),G1期、S/G2/M期细胞比率[(24.20±0.50)%、(20.10±0.30)%]与对照组[(56.11±0.60)%、(24.38±0.40)%]比较差异无统计学意义(P>0.05)。结论敲除SIRT1不影响急性髓细胞白血病小鼠白血病细胞凋亡,可使白血病细胞停滞于G0期,其机制可能与上调细胞周期抑制基因p19、p21、p27表达有关。

Objective To investigate the expression of SIRT1 in acute myeloid leukemia mice and its influences on the leukemia cell apoptosis and cell cycle. Methods Ten C57 BL/6 J mice were randomly divided into observation group and control group, with 5 mice in each group. Two groups were irradiated with semi-lethal dose X-ray(4.75 Gy) for 7 min, and were prepared AML mice models via tail vein injection with 2.5×10~5 of virus-infected leukemia cells. On the 14 th day after transplantation, observation group was intraperitoneally injected with 5 mg/kg pIpC solution once every other day, totally 3 times, while control group was intraperitoneally injected with equivalent volume of normal saline. The mice were sacrificed on the 24 th day after transplantation to obtain the myeloid leukemia cells. Real-time fluorescence quantitative PCR technique was used to detect the relative expressions of SIRT1, cyclin dependent kinases 1, p19, p21 and p27 mRNAs. Flow cytometry was used to detect the cell apoptosis and cell cycle. Results The relative expression of SIRT1 mRNA was lower in observation group(0.11±0.01) than that in control group(1.01±0.01)(P<0.05), the relative expressions of p19, p21 and p27 mRNAs were higher in observation group(3.76±0.22, 1.53±0.06, 2.23±0.02) than those in control group(1.01±0.01, 1.02±0.01, 1.01±0.01)(P<0.05), and the relative expression of cyclin dependent kinases 1 mRNA showed no significant difference between observation group(1.05±0.02)and control group(1.01±0.02)(P>0.05).There was no significant difference in the apoptotic rate between observation group((30.3±0.3)%)and control group((27.3±0.4)%)(P>0.05).The cell ratio of G0 phase was higher in observation group((55.22±0.60)%)than that in control group((19.20±0.30)%)(P<0.05),and the cell ratios of G1 and S/G2/M phases showed no significant differences between observation group((24.20±0.50)%,(20.10±0.30)%)and control group((56.11±0.60)%,(24.38±0.40)%)(P>0.05).Conclusion SIRT1 knockdown does not affect leukemia cell apoptosis,and it can keep leukemia cells in G0 phase,probably by uprelating the expressions of cell cycle suppressor gene p19,p21 and p27.