【作者】 江海涛；

【导师】 余多慰； 何强泰；

【作者基本信息】 南京师范大学 ， 细胞生物学， 2005， 硕士

【摘要】 本实验以桑黄为实验材料分两个方面进行研究。第一方面主要对桑黄的生物学特性进行研究,主要研究巨原生质体的分离、再生条件以及其显微和亚显微结构的差别。实验结果表明:桑黄在3%的溶壁酶的作用下,37℃酶解7天即可获得大量的巨原生质体,直径在（15±1.7）μm;接种在再生培养基上,25℃培养11天后,即萌发出幼嫩的菌丝;透射电镜发现,桑黄巨原生质体的细胞结构有很大的变化。首先,酶解时间的延长对细胞结构有一定的破坏作用,细胞膜出现部分缺口。其次,巨原生质体最显著的特征就是液泡体积明显增大。最后,与普通原生质体相比,巨原生质体线粒体的数目和体积也有相应的增多和变大,在本实验中未发现细胞核增多这个现象。第二方面主要研究桑黄多糖的分离、纯化和生物学活性。桑黄子实体经过热水提取,减压浓缩,酒精沉淀,脱蛋白,乙醚、丙酮洗涤,真空干燥,得到桑黄水溶性粗多糖,经透析,SephadexG-200柱层析分离纯化得到桑黄水溶性多糖纯品PIPS1和PIPS2。两者经柱层析鉴定均为单一对称峰,醋酸纤维薄膜电泳以及聚丙烯酰胺凝胶电泳和琼脂糖凝胶电泳均为单一条带。根据洗脱体积与分子量的对数呈线性关系,本实验测得PIPS1的分子量为4.7×10⁵KD,PIPS2的分子量为8.6×10⁴KD。经过提取纯化的多糖具有良好的生物学活性,能够提高机体内细胞因子IL-2的表达;能够提高机体抗氧化酶的活性,提高机体清除自由基的能力,减轻自由基对机体的损伤,能够改善肝脏局部的微循环,及时修复损伤的肝细胞,减少谷草、谷丙转氨酶向血中的释放,对CCl₄引起的化学性肝损伤具有很好的保护作用;本研究中,桑黄多糖还初步显示对高脂饲料引起的脂肪肝具有一定的抑制作用,主要是通过降低胆固醇和甘油三酯,提高高密度脂蛋白的表达来实现;体外抗肿瘤实验发现,桑黄多糖对体外培养的宫颈癌细胞具有很明显的抑制效果,表现出良好的细胞毒性作用,对肝癌细胞的生长也有一定的抑制作用。以上结果均说明,桑黄多糖具有良好的生物学活性。更多还原

【Abstract】 This experiment includes two parts based on Phellinus igniarisu.In the first part, biological characteristic of gigant protoplast separated from Phellinus igniarius was studied. We observed that isolation、 regeneration of giant protoplast and its ultrastructural process.The results showed that Phellinus igniarius could yield large numbers of giant protoplast dealed with 3% lywallzyme for seven days within 37 ℃. Its diameter ranged from 13 to 20μm.The mecylia formed when giant protoplast were inoculated on regeneration medium and cultured for eleven days at 25°C .Compared with general protoplast,great changes happened on giant protoplast by TEM.At first,cell structure of giant protoplast were affected by long time and the gap came into being between cell membrane;secondly,vacuole in the giant protoplast became obviously larger than before;at last,numbers and vacuum of mt also became more and larger.In addition, we didn’ t discover the phenomenon of nucleolus abnormal.ln the second part, water-solubilable polysaccharide was extracted by boiling water、 isolated and purified by all varieties of bio-chemical techonologies.Two main apex were separated by SephadexG-200 gel coloum.Gel electrophoresis experiments showed that each polysaccharide apex was very pure and their molecular weight were forty-seven hundred thousand and one hundred thousand respectively.Polysaccharide had very strong biological activities after it was purified.It could improve the expression of cytokines like IL-2 in the body of rat,could increase antioxidant enzyme activity and enhance the ability of body to scanvage free radicals and reduce the damage caused by free radicals,meanwhile it could prompt microcircles of liver and restore liver cells,decrease the release of ALT and ALT,so the polysaccharide had protective effect on chemical liver injury caused by CCl4.During the study,we observed that polysaccharide could inhibit the appearance of fatty liver.ln vitro antitumor experiment proved that polysaccharide possessed direct cell toxicity because of its inhibition effect on Hela tumor cell.更多还原