Bmi-1基因RNA干扰对人胚纹状体来源神经干细胞衰老的影响

Effects of Bmi-1 RNA interference on senescence of human neural stem cells isolated from fetal striatum

目的观察原代培养3-4时期的神经干细胞（NSC）经过原癌基因Bmi-1的RNA干扰（RNAi）后是否发生衰老和衰老后的表象，以及Bmi-1下游基因p16INK4n的表达。方法培养流产胎儿皮层来源的NSC。经过Bmi-1基因RNA干扰后，检测其衰老情况和细胞增殖能力的改变，以及其下游基因p16INK4n基因通路的改变。结果体外培养3～4周的NSC增殖旺盛，经过Bmi—1基因RNA干扰，衰老细胞明显增多，表现为衰老染色阳性的细胞数目从（18．57±2．20）％增加到（33．79±7．79）％，但细胞凋亡并无明显增加。与细胞衰老相适应，NSC生长速度变慢，表现为Bmi．1基因RNA干扰1周后神经干细胞的BrdU掺入率（13．2±4．1）％，明显低于空病毒转染组的（23．1±5．5）％。此外，干扰后的NSC细胞中Bmi．1基因的转录明显下调至对照组的约40％，而其下游基因p16INK4a基因明显升高到GFP对照组的10倍以上。结论体外培养的人类纹状体神经干细胞中Bmi-1基因转录水平下降，能够诱发细胞衰老，表现为细胞增殖能力下降。Bmi-1基因下调所引发的衰老可能和p16INK4a基因转录水平升高有关。

Objective After Bmi-1 RNA interference （RNAi） ,identify whether or not human fetal striatum derived neural stem cell can be induced to become senescent, observe the profile of neural stem ceils （NSCs） and quantitatively analysis the expression of downstream gene,p16INK4n. Methods Culture human neural stem cell firstly. The characterization of the cells after RNAi includes ： senescence, apoptosis and proliferation. The transcription level of Bmi-1, p16INK4a were also measured. Results In accordance with the increase of senescence cells, as identified by the percentage of β-gal positive cells, （ 18.57 ± 2.20） % ,is significantly higher than that of control group, （ 33.79 ± 7.79 ） %. The proliferation （ Brdu incorporation rate） ability of NSC transfected by Bmi-1 RNAi virus,compared with those transfected by control GFP virus, which are （23.1 ± 5.5 ）% , decreased significantly to （13. 2 ± 4. 1 ）%. By Real-time polymerase chain reaction （PCR） , we find the transcription of Bmi-1 is significantly decreased 48 hours after the transfection, but the level of p16INK4a increased more than 10 folds than that of control group. Conclusion The decrease of Bmi-1 transcription level in NSC leads to increase of senescence, which is accompanied by decreased proliferation. Bmi-1 induced senescence and the decrease of proliferation ability, may be relat- ed to increase of p16INK4a in human fetal striatum derived NSC.