鲨鱼软骨粘多糖的提取、分离与功能性研究
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摘要
粘多糖是由糖苷键连接起来的醛糖或酮糖组成的天然分子，几乎存在于所有的有机体中，包括动物、植物、微生物(细菌和真菌)。粘多糖具有广泛的生理活性，如抗病毒、抗肿瘤、抗凝血、降血糖、降血脂、抗溃疡、抗感染等多种生理功能。本实验就鲨鱼软骨粘多糖进行了提取和分离，筛选了最佳提取方案和分离纯化措施。结果显示0.3mol／L的氢氧化钠(60～70℃)粘多糖的得率较高。采用阴离子树脂D-241和DEAE-纤维素联合作用分离粘多糖效果较好，重复使用一次或两次DEAE-纤维素可以使纯化的效果更佳。
文章也对鲨鱼软骨粘多糖的功能性进行了研究，此研究主要是针对抗肿瘤和抗衰老两方面的功能展开的。本文首先通过体外培养对鲨鱼软骨粘多糖的抗肿瘤活性进行了研究，将对数生长期的细胞制成细胞悬液，调整细胞浓度为5×10~4个／ml，接种于24孔培养板，每孔加100μl细胞悬液。实验组加入受试物(鲨鱼软骨粘多糖)，并且设2个浓度梯度。培养72小时后观察实验结果。实验结果证明鲨鱼软骨粘多糖对血癌细胞K562的生长具有一定抑制作用，并且其作用效果和浓度存在一定的相关性，粘多糖的浓度在5％时对肿瘤细胞的抑制作用要比在2.5％时的作用强；另外由于活细胞对台盼蓝染料具有排斥作用，不易着色，而死细胞则没有此功能从而被染料着色。染色结果说明：在添加粘多糖的实验组，活细胞率大大下降，所以鲨鱼软骨粘多糖能在一定程度上抑制和杀死K562细胞。
文章采用果蝇作为抗衰老的实验动物对鲨鱼软骨的抗衰老活性进行了探讨，试验组果蝇的平均寿命增长5.12—6.65天，最高寿命增长5.40—6.17天，此结果表明鲨鱼软骨粘多糖能明显的延长实验果蝇的平均寿命和最高寿命。果蝇头部的蛋白质电泳结果说明，在果蝇的衰老过程中一些特定的蛋白质发生了变化，对照组的蛋白带变化较明显而实验组的变化与对照组相比不太明显，由于这些蛋白质的改变是由于特定基因表达的结果，从而认为：在果蝇食用鲨鱼软骨粘多糖后，一方面，体内的多糖环境抑制或减缓了某些跟衰老相关的基因的表达；另一方面，由于机体的免疫力增强，从而能有效的抵制体内外不良的环境的侵袭，在此环境下相应的长寿基因得到较好的表达，同时也抑制了衰老基因的表达。但是鲨鱼软骨粘多糖对雌性和雄性果蝇的影响存在一定的差异。
Mucopolysaccharide was a kind of natural molecular, which was basically consisted of aldoses linked by indicant bond, and was almost found in all organisms including animal, plant and microbe. Mucopolysaccharide was of extensive physiological function and was widely used for the purpose of antivirus, anticoagulation, antiulcer, antiinfection, blood sugar decreasing and lipid decreasing research. In this paper, mucopolysaccharide in shark cartilage was extracted and separated and the optimum extraction scheme and isolation method were screened. The results indicated that the harvested rate of mucopolysaccharide by using 0.3mol/L sodium hydroxide under 60-70℃ was relatively high. Negative ion resin D-241 combined with diehlaminoeth could extract mucopolysaccharide desirably and the result can be bettered by repeating diehlaminoeth one or two more times.
In addition, the function of mucopolysaccharide from shark cartilage was examined in this paper. And the research mainly focused on its activity in antitumor and antisenity. The antitumor activity of mucopolysaccharide from shark cartilage was studied in vitro culture. Leukemia K562 cells in logarithm growth stage were made into suspension and cell density was adjusted to 5X 104 cells/ml. They were incubated in 24-well plate and 100μlcell suspense per well. Two concentration levels were set to examine its antitumor activity and the results were checked after 72h culture. Experimental results suggested that mucopolysaccharide from shark cartilage could inhibit the growth of Leukemia K562 cells to some degree. There was some correlationshrp between inhibition effect and mucopolysaccharide concentration and the inhibition effection of 5% mucopolysaccharide was relatively greater than that of 2.5% mucopolysaccharide. Viability of the cells was measured by a trypan blue exclusion test. The results indicated that
viability of Leukemia K562 cells was reduced greatly by mucopolysaccharide treatment and mucopolysaccharide could inhibit and kill Leukemia K562 cells to some degree.
Drosophilae was used as a model animal to study antisenity activity of mucopolysaccharide from shark cartilage. The experimental results suggested that the longevity and maximum longevity of Drosophilae treated by mucopolysaccharide could be elongated by 5.12 to 6.65 days and by 5.4 to 6.17 days
respectively. The protein from Drosophilae head was analyzed by electrophoresis. Protein electrophoresis results indicated that compared with control, some protein of Drosophilae has significantly changed during the course of decrepitude, which may be caused by the expression of some gene. The possible mechanism of antisenity of mucopolysaccharide from shark cartilage was also discussed in this paper. Amylose environment in drosophilae's body may inhibit or retard the expression of some gene related to decrepitude. On the other hand, the immunity ability of organism was improved which may be favorable to resist adverse environment stress in body and out of the body. There was certain difference in the effecteion of mucopolysaccharide between male and female drosophilae.
文章也对鲨鱼软骨粘多糖的功能性进行了研究，此研究主要是针对抗肿瘤和抗衰老两方面的功能展开的。本文首先通过体外培养对鲨鱼软骨粘多糖的抗肿瘤活性进行了研究，将对数生长期的细胞制成细胞悬液，调整细胞浓度为5×10~4个／ml，接种于24孔培养板，每孔加100μl细胞悬液。实验组加入受试物(鲨鱼软骨粘多糖)，并且设2个浓度梯度。培养72小时后观察实验结果。实验结果证明鲨鱼软骨粘多糖对血癌细胞K562的生长具有一定抑制作用，并且其作用效果和浓度存在一定的相关性，粘多糖的浓度在5％时对肿瘤细胞的抑制作用要比在2.5％时的作用强；另外由于活细胞对台盼蓝染料具有排斥作用，不易着色，而死细胞则没有此功能从而被染料着色。染色结果说明：在添加粘多糖的实验组，活细胞率大大下降，所以鲨鱼软骨粘多糖能在一定程度上抑制和杀死K562细胞。
文章采用果蝇作为抗衰老的实验动物对鲨鱼软骨的抗衰老活性进行了探讨，试验组果蝇的平均寿命增长5.12—6.65天，最高寿命增长5.40—6.17天，此结果表明鲨鱼软骨粘多糖能明显的延长实验果蝇的平均寿命和最高寿命。果蝇头部的蛋白质电泳结果说明，在果蝇的衰老过程中一些特定的蛋白质发生了变化，对照组的蛋白带变化较明显而实验组的变化与对照组相比不太明显，由于这些蛋白质的改变是由于特定基因表达的结果，从而认为：在果蝇食用鲨鱼软骨粘多糖后，一方面，体内的多糖环境抑制或减缓了某些跟衰老相关的基因的表达；另一方面，由于机体的免疫力增强，从而能有效的抵制体内外不良的环境的侵袭，在此环境下相应的长寿基因得到较好的表达，同时也抑制了衰老基因的表达。但是鲨鱼软骨粘多糖对雌性和雄性果蝇的影响存在一定的差异。
Mucopolysaccharide was a kind of natural molecular, which was basically consisted of aldoses linked by indicant bond, and was almost found in all organisms including animal, plant and microbe. Mucopolysaccharide was of extensive physiological function and was widely used for the purpose of antivirus, anticoagulation, antiulcer, antiinfection, blood sugar decreasing and lipid decreasing research. In this paper, mucopolysaccharide in shark cartilage was extracted and separated and the optimum extraction scheme and isolation method were screened. The results indicated that the harvested rate of mucopolysaccharide by using 0.3mol/L sodium hydroxide under 60-70℃ was relatively high. Negative ion resin D-241 combined with diehlaminoeth could extract mucopolysaccharide desirably and the result can be bettered by repeating diehlaminoeth one or two more times.
In addition, the function of mucopolysaccharide from shark cartilage was examined in this paper. And the research mainly focused on its activity in antitumor and antisenity. The antitumor activity of mucopolysaccharide from shark cartilage was studied in vitro culture. Leukemia K562 cells in logarithm growth stage were made into suspension and cell density was adjusted to 5X 104 cells/ml. They were incubated in 24-well plate and 100μlcell suspense per well. Two concentration levels were set to examine its antitumor activity and the results were checked after 72h culture. Experimental results suggested that mucopolysaccharide from shark cartilage could inhibit the growth of Leukemia K562 cells to some degree. There was some correlationshrp between inhibition effect and mucopolysaccharide concentration and the inhibition effection of 5% mucopolysaccharide was relatively greater than that of 2.5% mucopolysaccharide. Viability of the cells was measured by a trypan blue exclusion test. The results indicated that
viability of Leukemia K562 cells was reduced greatly by mucopolysaccharide treatment and mucopolysaccharide could inhibit and kill Leukemia K562 cells to some degree.
Drosophilae was used as a model animal to study antisenity activity of mucopolysaccharide from shark cartilage. The experimental results suggested that the longevity and maximum longevity of Drosophilae treated by mucopolysaccharide could be elongated by 5.12 to 6.65 days and by 5.4 to 6.17 days
respectively. The protein from Drosophilae head was analyzed by electrophoresis. Protein electrophoresis results indicated that compared with control, some protein of Drosophilae has significantly changed during the course of decrepitude, which may be caused by the expression of some gene. The possible mechanism of antisenity of mucopolysaccharide from shark cartilage was also discussed in this paper. Amylose environment in drosophilae's body may inhibit or retard the expression of some gene related to decrepitude. On the other hand, the immunity ability of organism was improved which may be favorable to resist adverse environment stress in body and out of the body. There was certain difference in the effecteion of mucopolysaccharide between male and female drosophilae.
引文
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