海鞘（Ciona savignyi）性腺组织细胞、血细胞和生殖干样细胞的体外培养

IOCAS-IR > 海洋环流与波动重点实验室

	海鞘（Ciona savignyi）性腺组织细胞、血细胞和生殖干样细胞的体外培养
其他题名	In vitro culture of gonad cells,hemocytes and germline stem like cells from ascidian (Ciona savignyi)
	张晓明
学位类型	博士
	2008-05-31
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	细胞培养海洋无脊椎动物生殖干样细胞肿瘤坏死因子细菌污染海鞘（ciona Savignyi）
摘要	随着对海洋无脊椎动物免疫、发育以及细胞生物学等方面研究的需要，海洋无脊椎动物细胞培养日益受到关注。然而，由于海洋无脊椎动物的细胞代谢途径以及生长特性与陆生哺乳动物有很大差异，细胞培养难度较大,至今尚未有连续的细胞系建立。海鞘（Ciona savignyi）属于尾索动物亚门(Urochordata)，是典型的被囊类动物。作为无脊椎动物中进化地位较高等的一类动物，海鞘在免疫、胚胎发育、细胞学等各个领域对研究脊椎动物的系统发生都有着非常重要的作用。本文分别以海鞘的性腺组织细胞和血细胞为材料，建立和优化海鞘细胞体外培养方法和条件；另外，还识别了成体海鞘性腺内的生殖样干细胞，并对其进行了体外培养和鉴定，为进一步开展海鞘细胞体外培养最终建立细胞系积累了资料。首先比较了4种基础培养基L-15、M199、DMEM和RPMIl640 与各种培养添加物组合、不同温度和PH值对海鞘性腺组织细胞和血细胞的体外生长的影响。结果表明，20℃，pH6.8，M199基础培养基添加10%胎牛血清最适合海鞘性腺组织细胞生长。同时对海鞘性腺的分离方法即机械解离细胞法和酶学解离细胞法进行了比较，发现机械解离细胞法最适合海鞘性腺组织细胞的分离，分离得到的细胞贴壁率和成活率高。对于海鞘血细胞的培养，20℃，pH6.8条件下，L-15基础培养基并添加10%胎牛血清对血细胞的体外存活和生长效果较好。另外，还成功的利用原代培养的血细胞检测了海鞘肿瘤坏死因子配体家族成员（CsTL）基因的表达变化。论文还研究了海鞘细胞培养中细菌污染的鉴定和控制方法。对于培养过程中的细菌污染，通过细菌分离、培养和纯培养发现两类菌株检出率较高，均为革兰氏阴性菌。经PCR 扩增16S rDNA 基因序列片断，结果显示这两类菌株分别属于弧菌属和施万氏菌属。药敏试验结果表明，亚胺培南和氯霉素等对受检施万氏菌的敏感度较高；而受检弧菌对氯霉素和环丙沙星的敏感度高。为控制培养中的微生物污染，比较了几种抗生素组合的使用效果，其中氯霉素和亚胺培南与双抗的抗生素组合有较好的抑菌效果并对培养细胞的贴壁和生长没有影响。然而，海鞘性腺组织细胞和血细胞在体外的传代培养并未取得成功，本论文对来源于成体海鞘性腺的生殖样干细胞进行了体外培养和鉴定。结果表明，成体海鞘性腺内存在生殖样干细胞，且在体外可以生长，繁殖并且可能具有分化潜能。体外培养的过程中，生长的细胞克隆明显具有类似胚胎干细胞的形态和基因表达特点。本研究克隆了海鞘肿瘤坏死因子配体家族成员（CsTL）基因。CsTL全长995个核苷酸编码281个氨基酸。组织表达结果显示，CsTL在性腺组织的表达水平相对比较高，提示CsTL可能对海鞘性腺的发育或分化等起着一定的作用。利用昆虫杆状病毒表达系统表达纯化了CsTL蛋白。结果显示，重组CsTL对L929细胞显示了明显的细胞毒性作用，说明CsTL是具有生物学活性的重组蛋白。但是尝试用获得的重组CsTL蛋白作为培养添加物培养海鞘性腺组织细胞，但并未检测到CsTL对海鞘性腺组织细胞的生长或凋亡有任何影响。总之，本文筛选到了适合海鞘性腺组织细胞和血细胞生长的培养基，并成功的将这两类细胞在体外进行了原代培养，并且虽然细胞传代未获成功，但为今后继续深入开展海鞘细胞培养研究奠定了基础，另外，海鞘生殖样干细胞的识别和培养也将为海洋无脊椎动物的细胞培养提供一条新途径。
其他摘要	Ascidian (Ciona savignyi) is an important model animal for studies of evolution and comparative biology. The success to culture cells from ascidian will greatly to contribute in vitro approach as to study gene expression and regulation. The purpose of this study is to find out and optimize the culture conditions for the growth of ascidian cells and establish immortalized ascidian cell lines in vitro. The tissues used for primary cultures are gonad and hemocytes. The media selected are L-15，M199, DMEM and RPMI1640. The optimal culture conditions for gonad cells in vitro are: M199 medium containing 10% FBS. It also is found that L-15 supplemented with 10% fetal bovine serum best supports the growth of ascidian hemocytes. And the optimal temperature and pH for the growth of ascidian cells is 20°C and 6.8, respectively. The identification and prevention of bacterial contamination in cell culture were investigated. Two bacterial strains that were contaminated in cell culture were isolated from the bacterial contamination samples. The detected bacterium were isolated and identified as the members of by 16S rDNA sequencing. Two bacterial strains were gram-negative bacterium. In order to control the microbial contamination in cell culture，three antibiotics mixture were compared．It is shown that gentanycin was efective to prevent bacterial contamination. However, primary culture of gonad cells and hemocytes are difficult to proliferate and has a short lifespan in vitro. Germline stem (GS) cell is an adult stem cell population with the capability of self-renewal and differentiation throughout life. GS cell culture may provide a new approach for the successful development of cell cultures from marine invertebrates. In the present study, we report that GS-like cells were derived from the adult gonads of Ciona savagnyi and embryonic stem (ES) -like cells would appear in the cultures during the initiation of gonad cells cultures. These ES-like cells were similar to those previously described pluripotent stem cells in morphology and gene expression and had a potential ability to differentiate in vitro,suggesting that ascidian gonads might retain the ability to generate pluripotent cells. A novel invertebrate TNF ligand was identified and characterized in Ciona savignyi. The CsTL cDNA consisted of 995 nucleotides and encoded 281 amino acids. Expression studies demonstrated that the transcription of CsTL mRNA is higher in gonads tissues. The Recombinant CsTL protein expressed using a baculovirus expression system showed potential cytotoxic activity in L929 cells but no effection on gonad cells.
页数	136
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/341
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	张晓明. 海鞘（Ciona savignyi）性腺组织细胞、血细胞和生殖干样细胞的体外培养[D]. 海洋研究所. 中国科学院海洋研究所,2008.

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