De-differentiation Response of Cultured Astrocytes to Injury Induced by Scratch or Conditioned Culture Medium of Scratch-Insulted Astrocytes |
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Authors: | Hao Yang Xi-Ping Cheng Jing-Wen Li Qin Yao Gong Ju |
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Institution: | (1) Institute of Neurosciences, The Fourth Military Medical University, Xi’an, Shaanxi, 710032, People’s Republic of China |
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Abstract: | Our previous reports indicated that astrocytes (ASTs) in injured adult rat spinal cord underwent a process of de-differentiation,
and may acquire the potential of neural stem cells (NSCs). However, the AST de-differentiation and transitional rejuvenation
process following injury is still largely unclear. The aim of the present study was to determine whether injured in vitro
ASTs can re-enter the multipotential-like stem cell pool and regain NSC characteristics, and to further understand the mechanism
of AST de-differentiation. We used an in vitro scratch-wound model to evoke astrocytic response to mechanical injury. GFAP
and nestin double-labeled indirect immunofluorescence were carried out to characterize these scratched cells at various periods.
Western-blot analysis was used to determine the changes of GFAP and nestin expression following injury. Furthermore, the rate
of proliferation was determined by immunocytochemical detection of BrdU incorporating cells. These scratch-wound ASTs were
cultured with stem cells medium to explore their ability to generate neurospheres and examine the self-renewal and multi-potency
of such neurospheres. Moreover, scratched AST culture supernatant as conditioned cultured medium (ACM) was used to investigate
if some diffusible factors derived from injured ASTs could induce de-differentiation of AST. The results showed: (1) the nestin
positivity first appeared in GFAP-positive cells at the edge of the scratch, subsequently, disseminated into un-insulted zone.
The expression of nestin in AST was increased with longer culture, while that of GFAP was decreased. Furthermore, these nestin-immunoreactive
ASTs could generate neurospheres, which showed self-renewal and could be differentiated into neurons, ASTs and oligodendrocytes.
(2) Scratched ASTs culture supernatant can induce astrocytic proliferation and de-differentiation. These results reveal that
the in vitro injured ASTs can de-differentiate into nestin-positive stem/precursor cells, the process of de-differentiation
may arise from direct injury or some diffusible factors released from injured ASTs. |
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Keywords: | Astrocyte De-differentiation Rat Nestin GFAP |
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