【作者】 曾世明；

【导师】 张丽萍；

【作者基本信息】 东北师范大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 本论文采用水煮、醇沉的方法,得到蝙蝠蛾拟青霉粗多糖CPHP,CPHP经冻融分级、酶-Sevag法联合脱蛋白、Sepharose CL-6B柱层析和HPLC制备等方法进行纯化,得到PHP,PHP经HPLC分析,为均一组分多糖,分子量为28 kDa。GC分析表明,PHP主要由Man、Gal、Glc和Rha四种单糖组成,其摩尔比为13.36:8.18:3.59:1。PHP经部分酸水解反应发现,PHP的主链由Man和Gal两种单糖组成,糖链末端或支链主要由Glc、Gal和少量Rha组成。高碘酸氧化及Smith降解结果表明,1→、1→6键型是PHP的主要连接键型,同时还存在1→、1→2、1→2,6、1→4、1→4,6键型。PHP甲基化分析结果表明,PHP中共存在6种键型,其中Man检出1→2、1→2,6两种键型;Gal检出1→和1→6两种键型;Glc检出1→6键型;Rha检出1→2键型,除Rha外,检出的键型都能被高碘酸氧化,与高碘酸氧化结果一致。经计算,四种单糖的摩尔比为Man:Gal:Glc:Rha = 15.26:9.10:3.63:1,与气象色谱结果基本一致。CPHP对淋巴细胞的增殖及转化的影响结果表明,CPHP在100-400μg/ml浓度下,均能促进淋巴细胞的增殖,并且随着浓度的增加,促进作用越明显;在100-200μg/ml时,CPHP可以促进ConA诱导的T淋巴细胞的增殖,而对LPS诱导的B淋巴细胞的增殖无明显影响。CPHP对S180肉瘤的抑制作用结果:PHP在50、100、200 mg/kg浓度下的抑瘤率分别为0.32±0.15、0.44±0.18、0.34±0.24,而环磷酰胺阳性组的抑瘤率为0.75±0.10,该结果表明,PHP对S180肉瘤具有抑制作用,但抑制作用不如阳性药物环磷酰胺;与此同时,100 mg/kg CPHP给药组小鼠的脾指数显著高于阴性对照组(p<0.05),而CPHP给药组小鼠的胸腺指数显著低于生理盐水组;ELISA法检测血清中TNF-α水平结果表明,与阴性对照相比,CPHP在100 mg/kg和200 mg/kg给药后,荷瘤小鼠血清中的TNF-α水平明显增加,并且随着给药浓度的增加,血清中TNF-α水平越高。说明CPHP可以促进S180荷瘤小鼠TNF-α的生成。更多还原

【Abstract】 The polysaccharide of CPHP was isolated from Paecilomyces hepialis by hot water.The water-extracted polysaccharide CPHP was fractionated by frozen-thawed, deproteinated by Sevag method, and purified by anion-exchange chromatography on a column of DEAE-Sepharose CL-6B and gel filtration chromatography on a column of Sepharose CL-6B. Then fraction were obtained and named as PHP.HPLC analysis showed that the polysaccharide of PHP was homogeneous components, and the average molecular weights was 28kDa.GC analysis showed that PHP was composed of Man,Gla,Glc,and small content of Rha. The molar ratio was 13.36:8.18:3.59:1. The partial acid hydrolysis of PHP showed that the main chain of PHP was composed of Man and Gal, The end of sugar chain or branched-chain was composed of Glc,Gal and Rha. Periodate oxidation and Smith degradation analysis suggested that PHP was mainly contained 1→、1→6 bond type.In addition, there were 1→、1→2、1→2,6、1→4、1→4,6 in PHP.The methylation analysis applied for PHP show that there were six bond type in PHP.Man was detected in 1→2 and 1→6;Gal was detected in 1→and 1→2,6;Glc was detected in 1→6; Rha was detected in 1→2.All of them could be Oxidized by Periodate. The molar ratio of Man,Gal,Glc and Rha was 2.90:1.73:0.69:0.19.It was are in agreement with GC analysis.CPHP was cultivated with lymphocyte cells.Between 100μg/ml to 400μg/ml, CPHP could induce lymphoctes proliferation,and the higher the concentration, the effect is more obvious. At the dose of 100 and 200μg/ml, CPHP can promote the ConA-induced proliferation of T lymphocytes, However, it did not affect the LPS-induced proliferation of B lymphocytes.At the dose of 50,100,and 200 mg/kg,CPHP could inhibit tumor growth in S180 tumor-bearing mice .tumor inhibition rates were 0.32±0.15,0.44±0.18,and 0.34±0.24.they are lower than the positive control group of Cyclophosphamide.meanwhile, Spleen index of CPHP treated group was significantly higher than the negative control group at the dose of 100mg/kg.however, Among the dose of 50,100,and 200 mg/kg,thymus index of CPHP treated group was lower than the negative control group.ELISA analysis of TNF-αshowed that CPHP could increase the expression of tumor necrosis factor-alpha (TNF-α) in serum at the dose of 100mg/kg and 200mg/kg,and the higher the concentration, the effect is more obvious. These results indicated that the tumor inhibitory activity of the CPHP may be related to the stimulation of immune system, which has a great significance in therapeutics of the tumor growth.更多还原