Objective To optimize and establish the methodology for culturing and inducing differentiation of mouse preadipocytes 3T3-L1. Methods The mouse cells 3T3-L1 were incubated in DMEM medium contained with 10%FBS, during which the incubation medium was refreshed every 2 to 3 days. Two methods were used to introduce differentiation, including three-drug combination group and four-drug combination group. The protocol of mediumⅠin three-drug combination group including insulin 10 mg/L, IBMX 0.5 mmol/L and DEX 1.0μmol/L. The protocol of mediumⅠin four-drug combination group including indometacin 0.1 mmol/L based on those of three-drug combination group. Both of them were incubated for 2 days and continuous for 2 times. And medium Ⅱ included insulin 10 mg/L for 2-day culturing and continuous for 2 times. Oil red O staining was used to observe the morphological changes of two groups of cells before and after treatment under inverted microscope. Results Mouse preadipocytes 3T3-L1 appeared in good conditions and grew in a paving stone fashion. These cells covered homogeneously the bottom of incubators, the culture medium refreshed every 2 days. The results of four-drug combination group were better than those of three-drug combination group. After three-drug combination induced differentiation, there was no significant change in cell morphology. Comparing with three-drug combination induced differentiation, four-drug combination was successfully achieved in over 90% of the cell inducing, which were round-shaped, with jacinth ester droplets by oil-red O staining. Conclusion We have optimized the method for culturing and inducing differentiation of mouse preadipocytes 3T3-L1 by adding indometacin on the basis of the three-drug combination induced differentiation.

OBJECTIVE：To study the effects of Plantago asiatica polysaccharide on the proliferation ，migration and invasion of breast cancer cells ，and to investigate its mechanism preliminarily. METHODS ：Using human breast cancer cell MDA-MB- 231 as subjects ，MTT method was adopted to detect the effects of different concentrations of P. asiatica polysaccharide（8，16，32，64 mg/L）on the cell proliferation ability ，and survival rate of the cells was calculated. Scratch test and Transwell invasion test were used to detect the effects of different concentrations of P. asiatica polysaccharide（8，16 mg/L）on cell migration ability and invasion ability. Western blot assay was used to detect the expression of epithelial-mesenchymal transition （EMT）-related proteins [matrix metalloproteinase- 2（MMP-2），MMP-9，E-cadherin，N-cadherin，vimentin]. RESULTS ：Results of MTT assay showed that survival rate of the cells in 32，64 mg/L P. asiatica polysaccharide groups were significantly lower than control group （P＜0.05 or P＜0.01），so that 8，16 mg/L，which did not affect the cell survival rate ，were used as the follow-up drug concentrations. Compared with control group ，relative mobility （12，24 h），relative invasion rate and relative expression of MMP- 2，MMP-9， N-cadherin and vimentin protein were decreased significantly in 8，16 mg/L P. asiatica polysaccharide groups （P＜0.05 or P＜ 0.01），while relative expression of E-cadherin protein was increased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS ：P. asiatica polysaccharide can inhibit the proliferation of breast cancer cells MDA-MB- 231，and inhibit the migration and invasion of the cells by regulating the expression of metastasis and EMT-related proteins.