Study On The Telomerase Activity Of Neural Stem Cells

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.