Expression of specific proteins of neural cells in rat's cultured amniotic epithelial cells

Xiaoqian LOU; Xiaoting MENG; Dawei WANG; Dong CHEN

ABSTRACT

BACKGROUND:

It has been suggested that amniotic epithelial cells (AECs) express almost all of the markers of neural cell and secret a lot of neurotrophic factors and neurotransmitters. If AECs could substitute neural cells, its neurotrophic effect will bring promising prospect in treating neuron injuries and degenerative neural disease.

OBJECTIVE:

To detect specific proteins of neural cells in rat's cultured AECs.

DESIGN:

Repeated measurement design.

SETTING:

Second Clinical Medical College , Jilin University; Department of Histology & Embryology, School of Basic Medical Science, Jilin University.MATERIALS This experiment was conducted at the Department of Histology & Embryology, School of Basic Medical Science, Jilin University from October 2004 to October 2005. The rat amniotic epithelial tissue was mechanically peeled from an embryonic 12 to 14days Wistar rats. Mouse anti Nestin was purchased from Chemicon Co.,and anti-ChAT rabbit anti-NSE and anti-NT-3 antibodies from Wuhan Boshide Company. Mouse anti-Musashi antibody was donated by Pro.Okano.

METHODS:

AECs were dissociated and purified from the amnion of pregnancy 12-14 day rats. AECs were treated with trypsin for 5 minutes,then cultured in DMEM/F12 medium at a humidified atmosphere of 0.05 volume fraction of CO2 in air at 37 ℃. Cells were inoculated at a concentration of 5×109 cells/L in culture flask. After 3 days, cells were inoculated onto poly-lysine-treated 35 mm culture Petri dish at a density of 1 × 108 cells/L for immunocytochemically staining. The cells were fixed with 40 g/L paraformaldehyde for 20 minutes. Immunocytochemical staining method was used to detect the expression of microtubule-associated protein-2 (MAP-2),neuron specific enolase(NSE), glial fibrillary acidic protein (GFAP) and choline acetyl transferase(ChAT).MAIN OUTCOME

MEASURES:

① Morphological observation of rat'AECs at different culture time. ② Expression of specific protein of neural cells in rat' cultured AECs.

RESULTS:

① After cultured for 24 hours, the AECs were flat and presented fibroblast-like morphology. 3 to 5 days later, cell bodies were well stacked; AECs had a big and round nucleus and were connected with each other by flourishing dendrites. ② Immunocytochemical staining results after culture for 4 days showed that AECs expressed Nestin, ChAT,NSE, Musashi, MAP-2, GFAP.

CONCLUSION:

AECs are homologous to neural cells in morphology, and it may be a new cell source to treat nervous system disease.