| [Objective]:This study aimed to investigate the effects of Radix Notoginseng and Rhizoma Gastrodiae on differentiation of neuronal stem cells (NSCs) and to establish the proteomic profiles using two-dimensional electrophoresis (2-DE) together with mass spectrometry (MS) technology. These would explore the mechanism and connection among of the changes of differential expressional proteins and differentiation of NSCs.[Materials and Methods]:Tissues from hippocampus of ICR embryo rats (E10-12d) were cultured for 7 days, and adopted the immunohistochemistry (IHC) staining of anti-Nestin to indentify the NSCs. Then the neurospheres were performed for the secondary culture in vitro. They were divided into six groups randomly, viz. serum-free group, serum group, Radix Notoginseng (RN) group (NSCs of serum-free group in addition of RN only), Rhizoma Gastrodiae (RG) group (NSCs of serum-free group in addition of RG only), Radix Notoginseng and serum group (NSCs of serum group in addition of RN) and Rhizoma Gastrodiaeand and serum group (NSCs of serum group in addition of RG). To detecte the differentiation of NSCs, They were harvested in 7 days of secondary culture for IHC staining for anti-Tuj 1 and anti-GFAP. To investigate the effects on the survival, proliferation and differentiation of NSCs, the numbers of the neurospheres and the positive cells were counted and the area and the axonal length, measured by ImageJ sofeware. The proteomics changes of serum-free group, serum group, RN and serum group and RG and serum group were further investigated to understand the mechanism of differentiation of NSCs. [Results]:1. NSCs were cultured in vitro and indetified successfully by ant-Nestin IHC staining.2. Comapring to the serum-free group, the numbers of neurosphere in the RN group and the RG group were significantly descreased, the areas of neurosphere and the length of axons, significantly increased; The area of anti-Tuj 1 positive cells and the length of axons, significantly increased; the area of anti-GFAP positive cells, significantly increased.3. In the serum group, comapring to the serum-free group, the numbers of neurosphere were significantly descreased; the areas of neurosphere and the length of axon, significantly increased; The numbers of anti-Tuj 1 positive cells, significantly decreased; the area of positive cells and the length of axons, significantly increased; the numbers of anti-GFAP positive cells, significantly increased, the area of positive cells, increased. The percentages of Tuj1 immuno-reactive (IR) and GFAP-IR were 23.6% and 45.6%, while those in the serum-free group were 24.6% and 33.4%.4. Comapring to the serum group, the numbers and the area of neurosphere in RN and serum group and the RG and serum group were significantly descreased. The Tuj1 IHC staining revealed the numbers and the area of neurosphere in RN and serum group and the RG and serum group were significantly increased. There was not statistically significant in GFAP IHC staining among the three groups.5. The number of protein spots in the serum-free group, the serum group, the RN and serum group and the RG and serum group were 320,230,287 and 181, respectively. The protein spots were mainly distributed between pH4 to pH9 of pI, with relative molecular weight between 10 to 90 KDa.14 interesting differential expressional ones were subjected to MS analysis. (1) Comparing to the serum-free group, five differential spots were detected in the serum group. NO.1 spot was the new emergent one; NO.2 spot was the absent one; NO.3 spot was expressional upregulated and NO.4 and NO.5 were downregulated. (2) Comparing to the serum group, seven differential spots were detected in the RN and serum group. NO.2 and NO.6 spots were the new emergent ones; NO.7 spot was the absent one; NO.8, NO.9, NO.10 and NO.11 spots were expressional upregulated. (3) Comparing to the serum group, five differential spots were detected in the the RG and serum group. NO.1, NO.12 and NO.13 spots were the absent ones; NO.14 was expressional upregulated and NO.8, downregulated.6.14 differential spots were analyzed by MS and 13 spots were indentified successfully. They were serum albumin precursor (SAP), Tumor rejection antigen gp96 (TRA) or Heat shock protein 90kda beta (HSP90B1), Serum albumin precursor (SAP), Nonmuscle tropomyosin (NMTPM), Nucleophosmin 1 (NPM1), Dihydropyrimidinase-like 2 (DPYSL2), Lactate dehydrogenase 2, B chain (LDH-B), 78 kda glucose-regulated protein precursor) (Grp78), Dihydropyrimidinase-like 2 (DPYSL2), Collapsin response mediator protein 1 (CRMP1), Fscnl protein (Fscnl), Chaperonin subunit 5 (Cct5) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).[Conclusion]:1. NSCs was extracted, cultured and indentified successfully. The serum could promote the differentiation NSCs to the neuron-like cells; the RN and the RG in the condition of serum-free would enhance the growth of the neuron-like and the astrocytes-like cells. But in the condition of serum, they could promote the neun-like cells emerging from the NSCs.2. Proteomics map of differentiation of NSCs in addition of the serum were investigated and five differential expressional proteins were found and indentified successfully. They were SAP, TRA or HSP90B1, SAP, NMTPM and NPM1. They are associated to metabolic and antioxidant activity, stress response, metabolic and antioxidant activity, cellular component movement and signal transduction and anti-apoptosis.3. Proteomics map of the RN and serum group and the RG and serum group were detected. Seven differential expressional proteins were indentified in the RN and serum group. They were TRA or HSP90B1, DPYSL2, LDH-B, Grp78, DPYSL2, CRMP1 and Fscnl. They are involved in stress response, signal transduction, glycolysis, protein catabolic process and anti-apoptosis and cell junction and motility; four differential expressional proteins were indentified in the RGand serum group. They were SAP, Grp78, Cct5, and GAPDH. They are associated to metabolic and antioxidant activity, protein catabolic process and anti-apoptosis, protein folding and apoptosis and glycolysis. |
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