【作者】 郑秀娟；

【导师】 郑志竑；

【作者基本信息】 福建医科大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 目的了解体外培养的神经干细胞端粒酶活性,观察培养至不同时间神经干细 胞端粒酶活性、端粒长度和细胞增殖速率的情况,并探讨神经干细胞分化 后端粒酶活性的变化,为神经干细胞的研究和进一步应用提供理论和实 验依据。方法1. 用无血清培养法从新生大鼠脑皮质分离培养神经干细胞,用免疫荧光细 胞染色鉴定神经干细胞。2. 用TRAP(Telomeric Repeat Amplification Protocol)-ELISA法分别测定神经 干细胞、HEK-293 细胞(阳性对照)和WI-38 细胞(阴性对照)的端粒 酶活性,并测定培养至4-12 周不同时间时神经干细胞的端粒酶活性, 用细胞计数法测定培养至4-12 周不同时间时神经干细胞的增殖速率。3. 用Southern-blot 的方法分别测定培养第1 代、第10 代时神经干细胞、 HEK-293 细胞(阳性对照)和WI-38 细胞(阴性对照)的端粒长度。4. 用含1%胎牛血清+2%B27 的DMEM/F12 诱导神经干细胞分化;用 TRAP-ELISA 法测定分化后细胞端粒酶活性;并用RT-PCR 法和 Western-blot 法进一步检测分化前后端粒酶逆转录酶(TERT)的表达情 况。结果1. 新生大鼠脑皮质分离培养出表达巢蛋白(Nestin)抗原,具有自我更新能力 和多向分化潜能的神经干细胞。2. 体外培养4-12 周内不同时间的神经干细胞均具有端粒酶活性,且其端 粒酶活性不会随培养时间而变化,细胞增殖速率也没有明显的差异。更多还原

【Abstract】 Objective:To investigate the telomerase activity of NSCs cultured in vitro;To study telomere length , telomerase activity and proliferation rate of NSCs that cultured in different time; To study the change of telomerase activity after NSCs differentiation.And to supply theory and experimental base for their further research and application.Methods:1. NSCs were cultured in serum-free media and were identified by immunofluorescence staining.2. The telomerase activity of NSCs、HEK-293(positive control) and WI-38(negative control) was respectively measured by TRAP(Telomeric Repeat Amplification Protocol)-ELISA and the telomerase activity of NSCs cultured in 4-12 weeks was also measured.The proliferation rate of NSCs cultured in 4-12 weeks was determined by cytometry.3. The telomere length of 1st and 10th passage of NSCs、HEK-293(positive control) and WI-38(negative control) was respectively determined by Southern-blot.4. NSCs were induced to differentiate with DMEM/F12 with 1%FCS+2%B27,then the telomerase activity was measured by TRAP-ELISA.The expression of telomerase reverse transcriptase (TERT) of NSCs after differentiation was examined by RT-PCR and Western-blot.Results:1. NSCs isolated and cultured from neonatal rat cerebral cortex express Nestin and have the ability to self-renew and differentiate into neurons, astrocytes and oligodendroglias.2. NSCs cultured in 4-12 weeks all express telomerase activity. It would not change during cultured and it is the same for the proliferation rate.3. NSCs contain longer telomere length. And through passage their telomere length has not decrease.4. The cells differentiated from NSCs express no telomerase activity. Data obtained from RT-PCR and Western-blot has showed that, the expression of TERT disappears after differentiation.Conclusions: NSCs isolated from neonatal rat cerebral cortex all express telomerase activity.It would not change during culture and it is the same for the proliferation rate.The telomerase of NSCs can maintain their telomere and it means that the telomerase probably plays an important role in NSCs long-term proliferation and self-renewal in vitro.The telomerase activity of cells differentiated from NSCs disappears.It may be coursed by the discontinuance of TERT expression.更多还原