神经干细胞的自发永生化

Spontaneous immortalization of human neural stem cell isolated from fetal cortex

目的 观察培养达24个月的神经干细胞（NSC）是否会发生永生化和恶变.方法 培养流产胎儿皮层来源的NSC.对培养超过24个月的NSC,分析其核型,检测其自我更新、增殖、衰老以及移植后的致瘤性等.检测其Bmi-1-p16INK4a/p14ARF基因通路的改变.结果 体外培养超过24个月的NSC自我更新旺盛,克隆形成率（14.89±4.75）%,显著高于正常培养8周NSC的（8.04±2.07）%;生长速度快,表现为传代1周后的新生神经球的直径为（85.98±30.24）μm,显著高于体外培养8周NSC的（15.97±9.97）μm.此外,培养超过24个月的NSC衰老细胞为（5.96±2.81）%,远低于正常培养8周NSC的（78.14±8.99）%;而且染色体数目异常,这些结果可以确定其发生了永生化.裸鼠移植未发现致瘤性,所以该细胞未发生恶变.此2株细胞中Bmi-1、p16INK4a、p14ARF基因均缺失.结论 体外长期培养的人类皮层神经干细胞能够自发永生化,表现为不进入衰老状态并不断增殖.其增殖不依赖于Bmi-1基因,而其不衰老状态可能和p16INK4a、p14ARF基因缺失有关.

Objective To identify whether human fetal cortex-derived neural stem cells （NSCs） can be spontaneously immortalized or transformed after more than 24 months in vitro culture. Methods Human NSCs were cultured. NSC cultured for more than 24 months in vitrowere examined for self-renewal,proliferation, senescence, karyotype, teratoma formation after nude mice transplantation, and genetic changes of Bmi-1-p16INK4a/p14ARF pathway. Results In terms of both self-renewal and proliferation ability of NSC, compared with those cultured for 8-12 weeks, which are （8.04 ± 2. 07） % and （ 15.97 ± 9.97 ）μm respectively, NSC cultured more than 24 months in vitro increased significantly to （ 14. 89 ± 4. 75 ） %and （ 85.98 ± 30. 24） μm. Together with loss of senescence[the percentage of 3-gal positive cells,（5.96 ±2. 81）% ,is significantly lower than that of 8-10 week NSC, （78. 14 ± 8. 99 ）%]and abnormal chromosome phenotype, these cells can be identified as immortalized. After being transplanted into nude mice, these cells did not form tumor in 3 months,so they are not transformed. By reverse transcription-polymerase chain reaction （RT-PCR） ,we find the transcription of Bmi-1 ,p16INK4a and p14ARF are all absent in the neural stem cells cultured more than 24 months in vitro, meanwhile, all present in the cells cultured for 8-12 weeks. Conclusion Long term in vitro culture of human cortex derived neural stem cell may lead to spontaneous immortalization, characterized by continuous self-renewal, proliferation, and loss of senescence. Their self renewal and proliferation have nothing to do with Bmi-1, and senescence loss may be the result of p16INK4a and or p14ARF absence.