【作者】 初建松；

【导师】 张士璀；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2004， 博士

【摘要】 文昌鱼是研究进化和比较生物学的重要模式生物。细胞培养的成功，将有助于用离体方法探讨一些基本问题。本实验的目的是建立和优化文昌鱼细胞体外培养条件。分别从文昌鱼头、尾、鳃、肠和腹鳍组织，剪切成约1mm~2小块，放到所选择的培养基L-15、F-12、M199、MEM、DMEM和PRMI1640中开展原代培养。结果表明，在25℃下、含20％FBS的L-15或F-12培养基、补充1.5％NaGl的条件下，文昌鱼尾部和头部组织细胞生长最好。这为进一步开展文昌鱼细胞体外培养最终建立细胞系积累了资料。 肝脏是脊椎动物维持体内代谢平衡(homeostasis)的重要器官，能调节营养代谢，并合成和分泌多种代谢物质。因此，其细胞系是体外研究肝脏代谢、生理、毒理学及肿瘤形成的优良模型，也可以用以生产某些具有天然生物活性的物质。鲨鱼是一种海洋鱼类，肝脏能合成角鲨烯。迄今，有关鲨鱼肝细胞的研究很少。我们以条纹斑竹鲨(Chiloscyllium plagiosum)为材料，进行了肝细胞培养研究，成功地进行了原代培养，发现L-15是较理想培养基。在添加含有20％FBS的L-15培养基中，于22℃培养10天，单层细胞覆盖率可达20％。 我们还利用培养细胞，进行了文昌鱼染色体核型和C带型研究，发现除第1对染色体为亚端部着丝粒外，青岛文昌鱼的其余17对均为端部或近端部着丝粒染色体，证实文昌鱼核型为2n=36，2st+34t，FN=36。C-带分析显示，C-阳性带约占染色体表面的54.3％(32／67)。其中，第2对染色体C带呈现明显差异，推测可能是性染色体。用银染技术处理后，第12号染色体的着丝粒区出现2个NORs。文昌鱼染色体大小和NORs特征都表明其染色体代表脊索动物原始类型。更多还原

【Abstract】 Amphioxus, a cephalochordate, is an important model for studies of evolution and comparative biology. The success to culture cells from amphioxus tissues will greatly to contribute in vitro approach as to study gene expression and regulation, etc. The purpose of this study is to find out and optimize the culture conditions for the proliferation of amphioxus cells in vitro. The tissues used for primary cultures were head, tail, gill, gut and metapleural fold. The media selected were L-15, F-12, M 199, MEM, DMEM and PRMI 1640. The optimal culture conditions for amphioxus cells in vitro are: medium, L-15; temperature, 25癈; and osmolarity 735 mmol kg-1. Among the tissues tested, head and tail cells grew best.Liver is an important organ in maintaining the internal homeostasis in vertebrates, and liver cell lines will be a good model for in vitro study of liver metabolism and physiology as well as for the research of toxicology and carcinogenesis. In addition, liver cell lines could be used for sources of bioactive products like liver oil. Here we have tried to initiate liver cell culture of lip shark. It was found that: (1) L-15 medium supplemented with 20% FBS best supports the growth of the cell, and (2) optimal temperature was 22 .The amphioxus cells cultured in vitro were used to study its chromosome number karyotype and C-banding pattern. It showed that the diploid chromosome number (2n) of amphioxus Branchiostoma belcheri tsingtauense was 36. The first pair of the 36 chromosomes of B. belcheri tsingtauense was subtelocentric and the other 17 pairs telocentric. This is in line with the finding that the karyotype of the amphioxus is 2n=36, 2st+34t, FN=36. C-banding analysis showed that about 54.3% (32/67) of the chromosome surface was positively stained. Around the second pair chromosomes,50% of them exhibited markedly different bands from each other. This broadly support that amphioxus chromosomes No. 2 are a pair of sex-chromosomes. In addition, the position of nucleolar organizer regions was situated on the centromeric regions of No. 12 pair chromosomes with Ag-staining method and the total number of NORs was 2. All these show that amphioxus chromosomes represent primitive types of the chromosomes of the lower chordates.更多还原