Effect Of Dendrobium Officinale Polysaccharides On Mouse Embryonic Stem Cells And Study On Its Antitumor And Immune Activity

In recent years, increasing attention has drawn on plant polysaccharides, Dendrobium officinale is valuable as a traditional Chinese herbal medicine, and it is significant to explore the potential pharmacological activity of Dendrobium officinale polysaccharides. This paper primarily studied the effect of Dendrobium officinale polysaccharides on mouse embryonic stem cells (mESCs). Since the21st century, cancer has been a serious threat to human life and health, so the research and development of efficient and low-toxic anticancer drugs has a great significance under this circumstance. This paper also explored the anti-cancer and immune activity of Dendrobium officinale polysaccharides.The main contents were divided into the following four parts:1. Dendrobium officinale polysaccharides were preliminarily extracted in water and precipitated in ethyl alcohol and purified by removing proteins. Then the polysaccharides content of Dendrobium officinale was measured by phenol-sulfuric acid method. The results showed that the efficiency of Dendrobium officinale polysaccharides was10.01%, which had reached a high extraction rate. The content of polysaccharides in Dendrobium officinale polysaccharides by extrated was87.35%, indicating that the. polysaccharides also had a high purity.2. Culture mESCs in vitro. The effect of Dendrobium officinale polysaccharides on the proliferation of mESCs was analyzed through clonogenic, MTT method and growth curves assay. QRT-PCR method was used to detect the relative expression of mES-marker genes, such as Nanog, Oct4, Sox2, Klf4, Lin28, Rexl and Aire, which were used to analyse the effect of Dendrobium officinale polysaccharides on maintaining the pluripotency of mESCs. The results confirmed Dendrobium officinale polysaccharides could improve the cologenic efficiency of mouse embryonic stem cells, and promote their proliferation, while not affect the maintenance of their pluripotency at the same time.3. Embryonic bodies (Ebs) of mESCs were cultured in suspension, which were used to simulate the process of early embryonic differentiation to the three germ layers. Real-time quantitative PCR assay was used to detect the ectodermal marker genes Nestin and Fgf5, mesoderm marker gene Flkl and SM22a and primitive endoderm marker genes AFP and Sox17, in order to study the effect of Dendrobium officinale polysaccharides on EBs differentiation. The results preliminary indicated that the Dendrobium officinale polysaccharides in low concentration (25-200μg.ml-1) were not conducive to the differentiation of EBs, while the high concentration (200～400μg.ml-1) had no significant effect on EB differentiation to three layers.4. Culture human hepatoma cells HepG2, human lung cancer cells A549, murine teratoma stem cells F9and human teratoma stem cells NCCIT in vitro, morphological analysis of these cells showed that Dendrobium officinale polysaccharides could inhibit the growth HepG2, A549, NCCIT and F9, indicating its anticancer activity. Mouse spleen cells and peritoneal macrophages were isolated and cultured in vitro and were used to research the effect of Dendrobium officinale polysaccharides on their proliferation. Experimental results showed Dendrobium officinale polysaccharides could promote the proliferation of mouse spleen cells, indicating its immunological activity (mainly related to humoral immune associated with spleen cells).