| Objective: To explore the feasibility of Salvia Miltorrhiza injection inducing mesenchymal stem cells(MSCs) to differentiate into neuron-like cells in vitro and the dose-effective relationship between them.Methods: MSCs were isolated and purified by adhering to the culture glassware wall. After 4 passages in culture, the MSCs were implanted into 96 foveola with cell density of 0.08,0.1,0.2,0.4,0.6,0.8,1,2,4,6,8,10(x104/ml) respectively. Meanwhile, the MSCs were cultured in Dulbecco's modified Eagle's medium (DMEM) culture medium with different concentration of fetal bovine serum, the 3-4,5dimethylthiazol-2-yl-2,5-diphenyl tetrazelium bromide( MTT) assay were used to determine the proliferation index of MSCs 72 hours later, the cell cycle were determine by cytometer also. With pre-induction of lOng/ml basic flbroblast growth factor (bFGF) for 24 hours, the MSCs were induced to neural differentiation by free-FBS (fetal bovine serum) culture medium contained different concentration of Salvia Miltorrhiza injection including 1,5,10,50,100,200 and 400g/L respectively for 8 hours, the expression of Nestin, neuron specific enolase(NSE) and glial fibrillary acid protein(GFAP)were detected by immunocytochemistry. Different inducing methods were compared also.Results: The MSCs isolated with adherent assay have the capability of proliferating quickly. The G0~G1 stage cells account for 92.9%. Compared to seeded with (0.08-2)X104/ml or 10X104/ml, the proliferation ratio of MSCs were higher when seeded with cell density of (4~8)x104/ml, when planted in culture medium with 15% FBS, the/4 value were (0.112+0.0096),while (0.053+0.027)in 5% FBS group and (0.088+0.010)in 10% FBS group ,the A value of 15% FBS group were compared with that of 5% FBS group and 10% FBS group, F value were 17.25 , 12. Irrespectively P<0.05 ,on the other hand , compared with that of in 20% FBS group (0.102+0.012) and in 25% FBS group (0.120+0.0092), F value were 1.79,1.18 respectively, P >0.1. With the presence of 100g/L Salvia Miltorrhiza injection for approximately 8 hours, the MSCs differentiated into neuron-like cells , expression rate of Nestin and NSE are (52.14 + 2.16) %, (56.44 + 2.49 ) % respectively. GFAP was not detected . The Salvia Miltorrhiza injection could not induce the MSCs into neural differentiation when its concentration was less than 10g/L.The most effective induction concentration of Salvia Miltorrhiza injection were betweenlOO g/L and 200 g/L. When the inducing medium contains bFGF, the expression rate of Nestin and NSE were (68.61+2.07) %, (76.54 + 3.64) % respectively. But without bFGF in inducing medium their expression rate were (51.14+1.99) % , (57.47 + 3.06) % respectively, F =17.463, P <0.01. No bFGF both in pre-inducing medium and inducing medium , their expression rate were (47.03 + 1.86) % , (45.19 + 3.11) % respectively, F =21.574, P<0.01.Conclusion: Adherent assay is a simple practical method of isolating and culturing MSCs. The MSCs proliferated significantly when cultured with cell density of (4~8)x104/ml and 15%FBS in the medium. Concentration of Salvia Miltorrhiza injection between 100g/L and 200g/L are superiority to inducing the BMSCs to differentiate into neural-like cells. bFGF could promote not only the neural differentiation of MSCs but also the survival of differentiated neuron-like cells significantly in vitro. |
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