Study Of The Effect Of Dendrobium Polysaccharides On Adipogenic Differentiation And Osteogenic Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells

In recent years, natural medicine has gradually attracted people’s attention and has been widely used in clinical practice as a kind of drug. Dendrobium is a rare and precious traditional chinese medicines, the application has a long history and the efficacy is significant, which in clinical practice has been verified for thousands of years. This paper studies the effect of polysaccharides from dendrobium officinale and dendrobium moniliforme on adipogenic differentiation and osteogenic differentiation of rat bone marrow mesenchymal stem cells, and thus makes an attempt of explain its traditional effect of "Nourishing Kidney-Yin" by modern molecular biology theory with the aim of underlying better development of dendrobium resourcesThe study includes the following four parts:1. General solvent extraction method was used to extract polysaccharides from dendrobium officinale and dendrobium moniliforme. Polysaccharides was primarily purified by ethanol precipitation and sevage method(deproteinization). The yield of crude polysaccharides from dendrobium officinale and dendrobium moniliforme was 20.4 % and 10.6% respectively, with the content of polysaccharides 76.4% and 67.5% accordingly, indicating that the polysaccharides had a higher purity.2. Whole bone marrow adherent method was used to isolate and subculture rat bone marrow mesenchymal stem cells. Cell morphology was observed under inverted microscope, and flow cytometry was used to detect the expression of specific cell surface markers. Trypan blue staining was used to detect cell viability, meanwhile the cell growth curve and the effect of dendrobium polysaccharides on its survival value by methylthiazolyl tetrazolium(MTT) assay. The results showed that BMSCs was spindle, swirling growth and cell viability was more than 95%, and cell morphology was homogeneous under inverted microscope. The expression of cell surface marker was normal, which hardly expressing CD45, but expressing CD105 by flow cytometry. The results of above indicated that the polysaccharides of dendrobium had no significant effect on the survival value of BMSCs, indicating the polysaccharides without toxicity.3. Different dendrobium polysaccharides were Added with concentration from 50 to 800 μg/m L to intervene adipogenic differentiation of BMSCs. Oil red O staining and quantitative analysis of cell differentiation were performed after 10、20 days. Quantitative RT-PCR was used to detect the m RNA expression level of peroxisome proliferator-activated receptor gamma(PPARG), lipoprotein lipase(LPL) and fatty acid binding protein 4(FABP4) during adipogenic induction. The results showed that the dendrobium polysaccharides at the concentration of 400, 800 μg/m L reduced the absorbance values after oil red O staining and turned down the m RNA expression level of PPARG, LPL, FABP4. The results of above indicated that the dendrobium polysaccharides would inhibit significantly adipogenic differentiation.4. The dendrobium polysaccharides were added with concentration from 50 to 800μg/m L to intervene osteogenic differentiation of BMSCs. Subsequently alkaline phosphatase(ALP) staining after seven days, and Alizarin Red staining after twenty eight days. Quantitative RT-PCR was used to detect the m RNA expression level of core binding factor α1(Cbfα1), collagen I(Col I) and osteopontin(OP). The results showed that osteogenic cells can be stained by Alkaline phosphatase and Alizarin Red S. Polysaccharides from dendrobium officinale had no significant effect on the expression of osteogenic differentiation marker genes,while the polysaccharides of dendrobium moniliforme enhenced significantly the expression levels of osteogenic differentiation marker genes at the concentration of 200 μg/m L, inlcuding core binding factor α1(CBFα1), collagen I(COL I), which indicating that the dendrobium moniliforme polysaccharides would partly induce osteogenic differentiation of BMSCs.