【摘要】 中国南海小轴海绵(Axinella sp.)体内含有2种活性生物碱:debromohymenialdisine(DBH)和hymenialdisine(HD),这2种生物碱位于小轴海绵中的特定类型细胞——小球细胞中。针对该类细胞建立1种特异性的染色标记,可以为细胞分化提供指示,并应用于细胞体外培养的代谢调控研究中。3,3′–二氨基联苯胺(DAB)染色是一种常用的过氧化物酶染色法,本研究通过对小轴海绵实施细胞分离并对不同类别的细胞进行DAB染色,证明了小球细胞具有颗粒状着染的特征;对各类细胞和海绵组织的蛋白提取物进行了非变性聚丙烯酰胺凝胶电泳(native-PAGE),考察各组分中与DAB作用的过氧化物酶条带;通过电镜观察揭示了小球细胞的2种内含体,发现其中的大内含体与光镜下的染色颗粒在形态和数量上存在对应关系;另外,在细胞体外培养体系(细胞团培养)中应用了DAB染色,在20 d的培养期间内,颗粒状着染细胞与小球细胞的比例同步变化,小球细胞的特征着染率保持在93%以上。综合上述结果,可认为小轴海绵组织和细胞体外培养过程中的小球细胞均具有DAB颗粒状着染的显著特征,该染色法对于该类细胞而言是一种可靠的细胞化学染色标记方法。更多还原

【Abstract】 Debromohymenialdisine(DBH) and hymenialdisine(HD) are two bioactive alkaloids which has been isolated from marine sponge Axinella sp.living in the South China Sea,and these two alkaloids are located particularly in spherulous cells in Axinella sp..Establishment of a specific marker for this type of cells by cytochemical staining can provide an indication method for cell differentiation,and can be applied in the metabolism regulation in in vitro cell culture.The 3,3′-diaminobenzidine(DAB) staining is commonly used to detect peroxidase activity.In this study,sponge cells were fractionated and stained by DAB.A distinguishing feature of dark brown granules in cytoplasm was observed in spherulous cells.Native-PAGE was carried out with protein extracts from fresh tissue and different cell fractions of marine sponge to examine the peroxidase responsible for DAB staining.By transmission electron microscope(TEM),two kinds of inclusions were found inside the spherulous cells,the larger ones of which showed consistency in dimen-sion and amount with the positive stained granules under light microscopy.In addition,this staining method for spheru-lous cell identification was also applied in in vitro cell culture(primmorph culture).The proportion of both granularly positive-stained cells and the spherulous cells synchronously varied during a 20-day culture period,and the ratio of positive-stained spherulous cells was kept above 93%.The above results suggested that the cytochemical method of DAB staining was reliable and could be applied in both fresh-tissue dissociation and in vitro cell culture,with the dis-tinctive characteristic of positive stained granules in cytoplasm marking spherulous cells in sponge Axinella sp..更多还原