Effects Of Different Extracts Of Guiluerxian Glue On The Guinea Pigs’ Articular Cartilage Cells’ Cell Viability Which Is In Culture In Vitro

Objective:To explore the mechanisms of different extracts of GuiLuErXian Glue effect on Guinea pigs’articular cartilage cells from cell and molecular level and find probable effective part of GuiLuErXian Glue by observing the effects of three medicated seru m of different extracts from GuiLu ErXian Glue on the expression of Collagen-Ⅱ、pro teoglyca、Bcl-2gene and MMP-3from Guinea pigs’articular cartilage cells. Finally to provide the reference for researching and developing new traditional Chinese medici-ne.Methods:1. According to the polarity increasing of trichloromethane and butyl alcohol and water to separate the GuiLuErXian Glue in turn.It’s the method to prepare the water-extracts of GuiLuErXian Glue and butyl alcohol-extracts of GuiLuErXian Glue and tri chloromethane-extracts of GuiLuErXian Glue.2. Prepare medicated serum of three different extracts from GuiLuErXian Glue by made Wister mice taken it orally for seven days continuously.3.Establishing the vitro cultured system by Guinea pigs’ articular cartilage cells of three month.4. Using MTT assay to plot the growth curve of each generation of Guinea pigs’ articular cartilage cells,and decide the time to give medicated serum to the vitro cult ured system.5. Observing the expression of Collagen-Ⅱ from Guinea pigs’ articular cartilage cel Is by immunohistochemistry.6. Observing the expression of proteoglycan from Guinea pigs’ articular cartilage cells by Alcian blue and Periodic acid sthiff staining method.7. Observing the expression of Bcl-2gene from Guinea pigs’ articular cartilage cells by QPCR.8. Observing the expression of MMP-3from Guinea pigs’articular cartilage cells by ELISA. Results:1. The results of Toluidine blue staining and Collagen-Ⅱ with immunohistochemist ry are all positive. It means we successfully established the vitro cultured system of Guinea pigs’articular cartilage cells.2. The result of MTT assay:The effect of all groups’medicated serum on promoti ng cartilage cell proliferation best is48hours after medicated serum act on cartilage. cell culture in vitro.3. The result of immunohistochemistry: The gray value of all groups is hinger than control group (P<0.05). Butyl alcohol-extracts group> water-extracts group> trich loromethane-extracts group> control group (P<0.05)4. The result of Periodic acid sthiff staining method: The gray value of all group s is hinger than control group (P<0.05). Butyl alcohol-extracts group> water-extracts group> trichloromethane-extracts group> control group (P<0.05)5. The result of Alcian blue staining method: The gray value of all groups is hin ger than control group (P<0.05). Butyl alcohol-extracts group> water-extracts group> trichloromethane-extracts group> control group (P<0.05)6. The result of qPCR: Compared with control group, three medicated serum of d ifferent extracts from GuiLu ErXian Glue all can promote bcl-2gene expression signif icantly(P<0.01). Butyl alcohol-extracts group> water-extracts group> trichloromethane-e xtracts group> control group (P<0.05)7. The result of ELISA:Compare with control group, three medicated serum of d ifferent extracts from GuiLu ErXian Glue all can restrain MMP-3expression (P<0.05). Butyl alcohol-extracts group> water-extracts group> trichloromethane-extracts group> c ontrol group (P<0.05)Conclusions:Steroids hormone、polysaccharides and fatty acids of GuiLuErXian Glue all can pr omote the expression of Collagen-Ⅱ、proteoglycan、Bcl-2gene and restrain MMP-3e xpression from Guinea pigs’articular cartilage cells in varying degrees, then to promot ing the cell viability of Guinea pigs’ articular cartilage cells and restraining the cells apoptosis of Guinea pigs’articular cartilage cells. The results of this experiment says t he effects of alcohol-extracts from GuiLuErXian Glue is the best, and the water-extra cts from GuiLuErXian Glue is the second, that means alcohol-extracts from GuiLuErX ian Glue most possibly be its effective part.