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Isloation, Structural Characterization and Effects on Rat Cardiomyocyte H9c2of One-component Polysac

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Tutor: WuMianBin
School: Zhejiang University
Course: Biochemical Engineering
Keywords: polysaccharides from Taxus Chinesis,chromatography,cardiomyocytes,MTS,flow cytom
CLC: R284.2
Type: Master's thesis
Year:  2013
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Abstract:
On the basis of the wide-ranging sources and non-toxic side effects of plant polysaccharides, much attention has been drawn on the study of the plant polysaccharides gradually in recent years, a growing number of plant polysaccharides have been separated and identified, in addition, many biological activities of these plant polysaccharides have been found, such as, immunomodulatory, hypoglycemic activities, anti-tumor, anti-viral, anti-coagulation.The crude polysaccharides were isolated by DEAE Sepharose Fast Flow chromatography at first in this thesis, the final condition for this process was optimized and related parameters were shown:pH and the salt concentration for Tris-HCl buffer solution was8.5and0.01M; Tris-HC1buffers with0.1M,0.3M、0.5M NaCl were applied for gradient elute at flowrate of11.5mL/min.Then, the different components of peaks from DEAE Sepharose Fast Flow chromatography were isolated and purified by gel chromatography with high purity and CPTC-1a、CPTC-1b and CPTC-2were obtained with the weight-average molecular weight135276Da、8862Da and73527Da accordingly. The Infrared spectrum of these polysaccharides indicates that they all have beta-type pyran ring C-H deformation vibration absorption peaks at893cm-1; both CPTC-1b and CPTC-2have the pyran ring C-H deformation vibration absorption peak at838cm-1; CPTC-2has D-glucopyranose ring vibration absorption peaks at957cm-1. CPTC-1a hasn’t corresponding absorption peaks at838cm-1and957cm-1.Subsequently, MTS tests were applied to research the effect of total sugar, CPTC-1a, CPTC-1b, CPTC-2on the cardiomyocytes H9c2, the total sugar has little effect on the proliferation in a low concentration range and the proliferation of myocardial cells were inhibited when the concentration of the total sugar is greater than125μg/mL; CPTC-2can inhibit the proliferation of cardiomyocytes when the concentration of CPTC-2is greater than8μg/mL; CPTC-1a and CPTC-1b can inhibit the proliferation of cardiomyocytes in a concentration-dependent manner and do not have a protective effect on cardiomyocytes.Finally, the results from flow cytometry indicated that all the polysaccharides don’t induce apoptosis of cardiomyocytes. The total sugar, CPTC-1b and CPTC-2can lengthen the cell cycles of cardiomyocytes H9c2while CPTC-la doesn’t, the total sugar does have a protective effect on cardiomyocytes H9c2at100ug/mL while CPTC-1b and CPTC-2have not. CPTC-1a, CPTC-1b and CPTC-2may have a synergistic mechanism on cardiomyocytes H9c2.
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