褐牙鲆三种细胞系的建立、鉴定及dnmt3基因表达模式初步分析

IOCAS-IR > 实验海洋生物学重点实验室

	褐牙鲆三种细胞系的建立、鉴定及dnmt3基因表达模式初步分析
	彭丽敏
学位类型	博士
导师	尤锋
	2015-05-27
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	海洋生物学
关键词	褐牙鲆性腺肌肉细胞系 Dnmt3 Real-time Rt-pcr
摘要	自Wolf和Quimby在1962年建立第一个永生细胞系，即虹鳟鱼（Onchorynchus mykiss）性腺细胞系RTG-2，随后越来越多的鱼类细胞系被陆续建立。截止到2011年，全世界报道的鱼类细胞系已有275株，但性腺来源的体细胞系和肌肉来源细胞系十分稀少。鱼类性腺体细胞主要包括卵巢的间质细胞、颗粒细胞和鞘膜细胞，以及精巢的Sertoli细胞、Leydig细胞等。性腺体细胞系对于生殖细胞正常的分裂、分化和性腺组织的结构组成等方面具有决定性的作用，尤其是近几年对于生殖干细胞研究的热潮，使得性腺体细胞的体外培养成为发展必然。肌肉是鱼类主要的食用组织，而肌肉细胞系的建立则为进一步研究鱼类肌肉的生长、分化和发育提供了平台，也有助于养殖鱼类快速生长发育机制的研究和应用。鱼类细胞系已广泛应用于病毒学、遗传学、基因调控和功能分析、生理学、内分泌学、资源保护等领域。因此，开展鱼类细胞培养及细胞系建立的研究具有深远意义。鱼类性别决定和性腺分化机制研究一直是热点，而相关的表观遗传修饰-DNA甲基化对其调控作用研究还处于起步阶段。无论在海水鱼还是淡水鱼中，对于DNA甲基转移酶基因dnmt3的研究十分匮乏，环境因子如温度、外源激素等对于甲基化模式的影响更是知之甚少。而dmrt1基因被认为是迄今唯一保守的性别相关基因，近年来其在各种鱼类雌雄个体差异表达谱研究较多，但其甲基化表观遗传修饰在性腺分化中的研究报道还很薄弱。褐牙鲆（Paralichthys olivaceus），是名贵的海产养殖品种，在相同养殖成本下，雌性个体比雄性个体生长快、体积大，因而其性别决定和分化是国内外研究的热点。然而由于缺少合适的性腺细胞系，其性别相关形成机制尤其是细胞水平的机理几乎还是空白。且研究证明褐牙鲆三倍体生长明显快于二倍体，2010年已成功实现褐牙鲆三倍体诱导并达到批量化，然而到目前为止，还没有关于褐牙鲆三倍体肌肉生长发育机制的任何研究进展，更没有褐牙鲆三倍体肌卫星细胞体外培养的任何研究报道。本研究首先建立、鉴定了褐牙鲆精巢、卵巢、三倍体褐牙鲆肌肉3个细胞系，并分别对其进行外源质粒转染，以期将这3种细胞系作为体外遗传操作模型运用于外源基因载体的应用研究。同时，克隆得到褐牙鲆甲基转移酶基因dnmt3a和dnmt3b的cDNA全长。利用雌核发育褐牙鲆幼鱼为材料，通过28^oC、雌二醇处理，获得遗传雌性（XX）表型雄性或雌性的褐牙鲆。之后检测了dnmt3a和dnmt3b保守区域与dmrt1在雌二醇刺激和性逆转褐牙鲆的表达关联，以期探讨甲基化作用对雄性相关基因dmrt1的调控作用。具体内容及研究结果如下：褐牙鲆精巢细胞系、卵巢细胞系，分别命名为POSC和POOC，目前分别传代到55代和34代。两细胞系都用DMEM/F12基本培养基，FBS添加量为20%，对于EGF生长因子POSC添加15 ng/ml，POOC添加20 ng/ml。POSC和POOC主要细胞类型均为成纤维样；线粒体707bp COI序列分析结果与NCBI褐牙鲆COI序列一致性为99%，证明2种细胞系均来自于褐牙鲆；染色体分析显示POSC和POOC染色体众数都为48，都具有褐牙鲆正常二倍体核型；精巢支持细胞特异抗体Fas-L免疫组化实验证明POSC主要为精巢支持细胞，POOC对卵巢间质特异抗体P450_SCC和3β-HSD反应呈现阳性，证明POOC为卵巢间质细胞；脂质体2000分别成功将pEGFP-N3转染到POSC和POOC细胞系，转染效率分别为10%和15%，说明2种细胞系均适宜作为有效的外源基因转化载体，都有望应用于体外遗传操作。三倍体褐牙鲆骨骼肌肌肉细胞系命名为POMSC_S(3n)，经过一年的时间目前已传代66代。POMSC_S(3n)适宜的生长培养基为MEM，添加20% FBS。其细胞类型主要为梭形或纺锤体样；染色体分析显示POMSC_S(3n)的染色体众数为72，具有褐牙鲆正常三倍体核型；肌卫星特异标记基因pax7b RT-PCR扩增和Desmin特异抗体免疫组化实验均证明POMSC_S(3n)为骨骼肌肌卫星细胞；通过利用含2%马血清的分化培养基DM诱导POMSC_S(3n)细胞分化，再利用DAPI染核发现，分化组（DM培养基）相对于对照组（GM培养基）含有较多的2核甚至3-4核的多核纤维样细胞，证实POMSC_S(3n)细胞具有分化潜能，且分化能力强；同样脂质体2000成功将pEGFP-N3转染到该细胞系，转染效率高达20%，说明该细胞系适宜成为有效的外源基因载体，有望成为遗传操作的有利工具。克隆获得褐牙鲆dnmt3a和dnmt3b基因cDNA全长，分别得到dnmt3a 基因4个转录本，dnmt3b 基因3个转录本，转录本之间相似性较高，都具有甲基转移酶保守结构域PWWP。以雌核发育褐牙鲆幼鱼为材料，通过雌二醇（E2）和高温28^oC处理分别得到100%遗传雌表型雌褐牙鲆和100%遗传雌表型雄性逆转褐牙鲆，与预期结果一致。分析E2处理和28^oC处理条件下，dmrt1与dnmt3a和dnmt3b表达模式，发现dmrt1与dnmt3a和dnmt3b表达有关联，且dnmt3a与dmrt1关系更为密切，暗示雄性相关基因dmrt1基因表达受甲基转移酶影响，进一步推测褐牙鲆性别决定和分化过程受甲基化作用调控。
其他摘要	Since the first permanent cell line (RTG-2) of rainbow trout (Oncorhynchus mykiss) established by Wolf and Quimby in 1962, more than 275 fish cell lines have been reported by 2011 in the world. However gonad or muscle-derived cell lines are rarely established. Fish gonadal somatic cells, such as ovarian stromal cells, granulosa cells and sertoli cells, leydig cells of the testes etc., play crucial roles in the process of normal division and differentiation of germ cells, and in the gonadal structure. Then, the research of gonadal somatic cells become inevitable. As is known, the delicious fish food was mainly derived from the tissue of fish muscle, and the cell line from fish muscle will become a platform for the further study in growth, differentiation and development of fish muscle, and which will be helpful for the study and application of fast growth mechanism in the cultured fish. Furthermore, fish cell lines have been widely used in the theory and applied research of virology, genetics, gene regulation and functional analysis, fish physiology, endocrinology, fish resources protection and so on. Therefore, the research on fish cell culture and the establishment of fish cell lines will be of profound significance. The research on fish sex determination and gonad differentiation has been a hot spot, while study on DNA methylation of fish sex determination and gonadal differentiation is currently only in its infancy. The study on DNA methyltransferase such as dnmt3 is very scarce and the influence of environmental factors such as temperature, exogenous hormones on the methylation patterns is poorly understood. Furthermore, dmrt1 is considered to be the only conservative sex related gene, while research about it is more focused on freshwater fish. Research reports on methylation epigenetic modifications of dmr1 in gonad is still very weak, so it is urgent to carry out relevant research. Olive flounder (Paralichthys olivaceus), is an important marine aquaculture species in Japan, Korea and China. The females grow faster and larger than males when cultured in the same environment, so there has been considerable attention given to all-female production and the mechanisms of flounder sex determination and gonadal differentiation. However, little was known in particular at the cellar level due to the lack of appropriate gonadal cell lines to study the mechanism. Studies have shown that triploid ones of flounder grow faster than the diploid obviously. Furthermore triploid induction of flounder was succeed in 2010 and mass production has been achieved. So far, however, there is no research about the mechanism of growth and development of triploid muscle, neither report about in vitro culture of satellite cells from triploid muscle. Therefore, this study established and characterized three new fish cell lines from the testis, ovary and triploid muscle of P. olivaceus, and transfected exogenous plasmid to these three cell lines as in vitro systems for genetic operation. In addition, we cloned and got the full cDNA length of methyltransferase gene dnmt3a and dnmt3b. The gynogenetic flounder fry were cultured at 28^oC or were treated by estradiol (E2) in their critical transition period and we got the pseudo-male juveniles and all female juveniles. Expression levels of dnmt3a, dnmt3b and dmrt1 during differentiation developmental stage of gonads of gynogenetic and its pseudo-male juveniles were detected by real-time PCR to analyze the regulation of methylation on sex-related dmrt1 gene. The main details and results were as follows: The cell lines POSC and POOC respectively derived from testis and ovary tissues of flounder have been subcultured 55 and 34 times. The two cell lines were optimally maintained at 25^oC in DMEM/F12 supplemented with 20% fetal bovine serum, and 15 ng/ml EGF for POSC and 20 ng/ml for POOC. POSC and POOC are mainly composed of fibroblast-like cells. COI were amplified by using RT-PCR method and subsequent comparative analysis demonstrated a 99% match with the known mitochondrial COI sequence of P. olivaceus, the results showed that POSC and POOC cells indeed originated from P. olivaceus. Chromosome analysis revealed that most POSC and POOC cells maintained the normal diploid karyotype with chromosome number of 48 respectively, which demonstrated that the two cell lines are normal flounder diploid cells. POSC cells were strongly positive for Fas-L and POOC cells were positive for P450_SCC and 3β-HSD, which suggested that POSC were mainly testicular sertoli cells and POOC were ovarian stromal cells. Both POSC and POOC cells were transfected with pEGFP-N3 plasmid by lipofectamine^TM 2000 and the transfection efficiency was approximately 10% and 15% respectively. The skeletal muscle satellite cell line POMSC_S(3n) from triploid flounder has been subcultured 66 times for about a year. POMSC_S(3n) cells were optimally maintained at 25^oC in MEM supplemented with 20% FBS. POMSC_S(3n) were mainly composed of fusiform or spindle-like cells. Chromosome analysis revealed that most POMSC_S(3n) cells maintained the normal triploid karyotype with chromosome number of 72, which demonstrated that the cell line are triploid cells. POMSC_S(3n) cells expressed the muscle satellite cell gene marker pax7b by RT-PCR detecting and were strongly positive for Desmin, which both suggested that POMSC_S(3n) were skeletal muscle satellite cells. The differentiation experiment showed that at 48 h after changed to DM, long fibrous cells could be observed in POMSC_S(3n) compared with the control group all along with GM. What’s more, certain number of multinucleated myotubes with about 2 or 3-4 nuclei per cell after DAPI staining were found in the DM group. The results revealed that POMSC_S(3n) cells have the differentiation ability to differentiate into small multinucleated myosatellite fibres. Similarly, POMSC_S(3n) cells were transfected with pEGFP-N3 plasmid by lipofectamine^TM 2000 and the transfection efficiency was approximately 20%. The full lengths of dnmt3a and dnmt3b cDNA sequences were cloned by using RT-PCR and RACE techniques. Four transcripts of dnmt3a and three transcripts of dnmt3b were obtained with high similarity, respectively. Among them, there all have the conservative PWWP domain. In order to compare the expression patterns between dnmt3 and dmrt1, the gynogenetic flounder fry were separately treated by E2 and cultured at 28^oC in their critical transition period and finally we got the expected 100% ( genetic and phenotypic) female flounders and 100% pseudo-male juveniles. Expression patterns of dnmt3a, dnmt3b and dmrt1 during gonadal differentiation developmental stage of gynogenetic respectively treated by E2 and 28^oC were detected by real-time PCR to analyze the regulation of methylation on sex-related dmrt1 gene. The results revealed that the expression of dmrt1 was regulated by both dnmt3a and dnmt3b, and dnmt3a meight play more important role than dnmt3b during the process. Then we implied that the sex determination and gonad differentiation of P. olivaceus could also be regulated by DNA methylation.
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/22764
专题	实验海洋生物学重点实验室
作者单位	中科院海洋所
第一作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	彭丽敏. 褐牙鲆三种细胞系的建立、鉴定及dnmt3基因表达模式初步分析[D]. 北京. 中国科学院大学,2015.

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