| As the only edible sea cucumber in north of China, Stichopus japonicus (selenka) mainly distributed over Bohai Sea estuary and sea area of Lianyungang City. There are many reports about the pharmacological effects of Stichopus japonicus polysaccharide and glycoconjugate, such as anticoagulant, immunity enhance, anticancer activity, etc. But there is few report about the effect of Stichopus japonicus glycoconjugate on neural precursor cells, especially about the dedifferentiation. Besides, raw polysaccharide or preliminary depurated polysaccharide extracted from dried Stichopus japonicus rather than homogeneous glycoconjugate extracted from fresh Stichopus japonicus was used in previously research.In this paper, we obtained two homogeneous Stichopus japonicus glycoconjugates. We studied the structure and their pharmacological effects on neural precursor cells preliminarily. The main contents of the papers are as follows:1. The extraction, separation and purification of raw Stichopus japonicus extract. Hydrolyzed by double-enzyme, the raw extract was precipitated by ethanol. After decolored by macroporous adsorptive resins, the polysaccharides was first separated by DEAE-Sepharose column. Monitored by 210nm, 280nm ultraviolet light and phenol-sulphuric acid colorimetric method, four main components named A, B, C, D were obtained. Only C and D have the dedifferentiation effects on neural precursor cells. So C and D were then separated by Superdex 200 column separately. We collected every main components, and they were desalted by Sephadex G-25 column. After screening according to their dedifferentiation effects on neural precursor cells, we got two fine purification Stichopus japonicus glycoconjugates named HS-1 and HS-2.2. The study of the structure of HS-1 and HS-2. UV spectrum and Superdex 200 column chromatography showed that the purified Stichopus japonicus glycoconjugates (HS-1 and HS-2) were homogeneous; the form of Barium Sulfate showed that HS-1 and HS-2 both were sulfated; saccharides contents of HS-1 and HS-2 determined by phenol-sulphuric acid colorimetric method were 63.22% and 58.90% separately; uronic acides contents of HS-1 and HS-2 determined by sulphutic acid carbazole reaction were 38.55% and 32.29% separately; protein contents of HS-1 and HS-2 determined by Bradford method were 1.19% and 1.69% separately: the molecular weight of HS-1 and HS-2 determined by Gel Permeation Chromatography were 631 000Dal and 707 900Dal separately; the results of TLC and HPLC showed that the polysaccharide part of HS-1 was mainly composed of fucose and some other monosaccharide, and the mol ratio of fucose and galactose was 14.29:1; the results of IR and NMR showed that the glucosidicbond configuration of HS-1 was mainlyβ-configuration and HS-1 was a sulfated glycoconjugate.3. The study of pharmacological effects of the purified Stichopus japonicus glycoconjugate. We found that HS-1 and HS-2 can aggregate adherenced neural precursor cells. And the higher the Stichopus japonicus glycoconjugate concentrations are, the less the time needed for aggregating. The results of immunofluorescence staining showed that cells aggregated together were Nestin positive and NSE lepto-positive while normal cells were Nestin negative and NSE positive. The result of Trypan blue method showed that most aggregated cells were still alive. According to aforesaid results, we presumed that HS-1 and HS-2 can induce the dedifferentiation of adherenced neural precursor cells. Besides, MTT method results showed that cells in low concentration Stichopus japonicus glycoconjugate experimental group had similar survival rate with that of cells in low concentration NGF experimental group.
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