In Vitro Study On The Effects Of Wnt3a On The Proliferation And Differentiation Of Neural Stem Cells

ObjectiveTo investigate the effects of Wnt3a on the proliferation and differentiation ofneural stem cellsMethods1. NSCs were separated mechanically from the embryonic hippocampus tissues of a14-day pregnant Sprague Dawley rat and expanded by selective serum-free culturemethod. Fluorescence immunocytochemistry was used to identify the expression ofnestin antigen, transmembrane protein frizzled-3(Fzd3), and the specific antigen ofneurons (β-Tubulin-Ⅲ) and glial fibrillary acidic protein (GFAP) after the clone weredifferentiated.2. To study the effects of Wnt3a on the proliferation of NSCs, we design four groups:control group, bFGF group, Wnt3a group and bFGF+Wnt3a group. After cells werebeing cultured for7days, four groups were co-incubated with5-bromodeoxyuridine(Brdu) and immunofluorescence staining was used to detect their proliferationpotentials. Results are reported as mean±SEM of date, statistical analyses werecarried out using the Student-Newman-Keuls test with SAS9.0.3. To compare the effects of Wnt3a on the differentiation of NSCs, we design two groups:experiment group (essential medium+Wnt3a100ng/ml) and control group (essentialmedium). After cells were being cultured for14days, fluorescence immunocytochemistry was used to count the percentage of neurons (β-Tubulin-Ⅲ) andastrocytes (GFAP) in two groups. Results are reported as mean±SEM of date,statistical analyses were carried out using the Student’s t-test with SPSS16.0.Results1. The NSCs isolated from hippocampus of embryonic rats had the proliferative abilityand expression of Nestin antigen, Frizzled-3(Fzd3). The cells after differentiationcould express the specific antigens β-Tubulin-Ⅲ and glial fibrillary acidic protein(GFAP) in neuron and astrocyte.2. After7days, four groups neural stem cells were in status suspension growth and cellsgather to neurospheres. Compared the neurospheres size of four groups under theinvert microscope, we demonstrate that the control group significantly decreaseneurospheres quantity and size than others groups. However, bFGF+Wnt3a group hasa significant increase the number of than others groups, but to compare with bFGFgroup, the shape show no significant difference. Four groups were co-incubated withBrdu and immunofluorescence staining was used to observe their proliferationpotentials, the percentage of Brdu positive cells was14.8%、27.2%、38.6%、54.5%.The result of four groups show evident difference each other, and bFGF+Wnt3a grouphas significant increase the positive cells than others groups (P<0.05).3. After induced differentiation for24hours, the soma enlarged and the number of axonincreased gradually. After7days neural stem cells differentiation become stable,fluorescence immunocytochemistry was used to count the percentage of neurons(β-Tubulin-Ⅲ) and astrocytes (GFAP) in two groups. We demonstrate that GFAPpositive rate of the Wnt3a group was56.26%±4.82%and the control was68.42%±5.54%, β-tubulin Ⅲ positive rate of the Wnt3a group was11.25%±0.62%and the control was8.54%±0.48%. The percentage of GFAP positive cells andβ-tubulin Ⅲ positive cells in Wnt3a group was show significant difference incontrast with control group(P<0.05). Conclusions1. The NSCs isolated from hippocampus of embryonic rats are able to self-renew viaselective serum-free culture method. When mitogens were removed from the culturemedium, the NSCs could express the specific antigens β-tubulin Ⅲ and glialfibrillary acidic protein (GFAP) in neuron and astrocyte.2. Wnt3a has distinct effects on the proliferation of NSCs which isolated fromhippocampus of embryonic rats.3. Wnt3a is able to increase the number of NSCs differentiation into neurons and repressto differentiation into astrocytes in vitro.