长牡蛎免疫系统的发生和发育机制的初步研究

IOCAS-IR > 实验海洋生物学重点实验室

	长牡蛎免疫系统的发生和发育机制的初步研究
	宋小瑞
学位类型	博士
导师	宋林生
	2016-05-13
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	长牡蛎个体发生免疫系统造血作用母源免疫
摘要	长牡蛎是世界范围内重要的海水养殖品种，也是软体动物门，冠轮动物超门最具代表性的物种之一。然而近年来长牡蛎大规模死亡事件频发，特别是夏季苗种繁育时期稚贝的大量死亡，逐渐成为制约养殖业健康持续发展的一个限制性因素。本论文主要采用分子免疫学等技术，分析了长牡蛎幼虫在早期个体发育过程中的免疫防御能力，探讨了造血相关转录因子在个体发育过程中的表达模式，并据此对长牡蛎发育过程中的造血作用进行了初步分析。研究结果进一步深化了无脊椎动物免疫系统发生和发育的研究，同时对贝类病害防控具有重要的指导意义。一、长牡蛎早期发育过程中免疫能力的检测在早期 D 形幼虫（17 hpf）中观察到吞噬现象发生于面盘区域。此外，模式识别分子（integrin β-1、caspase-3 和 C-type lectin 3）和免疫效应分子（IL17-5）的蛋白定位在面盘和消化腺区域，推测它们为免疫系统发生的潜在位点。孵化期（9 hpf）为胚胎期和浮游期之间的过渡阶段。抗氧化酶（EC-SOD 和 CAT）和溶菌酶的活性，以及 12 个候选免疫因子的 mRNA 表达水平在孵化期处于发育过程中的最低水平。随后在担轮幼虫期（15 hpf），3 种酶的活性和 7 个候选免疫因子的 mRNA 表达量均开始上升。个体发育至 D 形幼虫期（50 hpf），6 个候选免疫因子的 mRNA 表达水平出现上升趋势，壳顶幼虫期（120 hpf），12 个候选免疫因子的 mRNA 表达水平均出现稳定的上升趋势。灿烂弧菌刺激后，D 形幼虫中 12 个候选免疫因子的 mRNA 表达水平均显著上调。上述结果表明幼虫在孵化期时免疫能力相对较低，当发育至担轮和 D 形幼虫期，免疫系统开始发生发育，在病原菌的刺激下，免疫系统能够被进一步激活。壳顶时期的幼虫已经具备较为完善的免疫系统，能够对外界刺激产生有效的免疫应答。二、长牡蛎 Cg-SCL 基因的鉴定与功能分析克隆获得长牡蛎造血相关转录因子 Cg-SCL 基因的 cDNA 序列，发现其含有一个保守的 bHLH 结构域，并与脊椎动物及海胆中的同源 SCL 基因具有较高的相似性。Cg-SCL 在牡蛎成体血细胞、鳃和外套膜组织中有较高的 mRNA 和蛋白表达水平。在长牡蛎早期个体发育过程中，Cg-SCL 的 mRNA 表达水平从受精后缓慢上升，担轮幼虫早期（10 hpf）达到峰值，然后下降，直至个体发育至壳顶幼虫期（120 hpf）。整体免疫荧光定位显示 Cg-SCL 最先出现在囊胚期（4 hpf），呈现为近似对称性的两点，并且这一表达模式持续到原肠胚期（8 hpf），至担轮幼虫期 Cg-SCL 分布于背部区域并呈现为清晰的环形结构，D 形幼虫期，Cg-SCL 蛋白在背部区域特化为两个窦穴状结构，同时以纤维状贯穿整个幼虫体内。灿烂弧菌刺激后，D 形幼虫和壳顶幼虫中 Cg-SCL mRNA 表达水平均出现显著上调。在成体长牡蛎中，Cg-SCL 的干扰会引起血细胞特异性基因（Integrin 和 EcSOD）和造血相关转录因子（GATA3，C-Myb 和 c-kit）的显著下调。上述结果说明 Cg-SCL 可以作为长牡蛎造血作用的标记分子，从其表达模式推测长牡蛎中造血作用类似于果蝇，存在胚胎和幼虫两次造血过程。胚胎造血发生于囊胚时期，幼虫造血则发生于担轮幼虫时期，Cg-SCL 可能在两次造血过程中均发挥重要的作用，可以作为长牡蛎造血作用的潜在标记分子。三、长牡蛎 Cg-Runt 基因的鉴定及其时空表达模式克隆获得长牡蛎造血相关转录因子 Cg-Runt 基因的 cDNA 序列，生物信息学分析发现它含有一个保守的 Runt 结构域，同源于 Runx 家族分子中的 Runx1 基因。在血细胞、鳃和外套膜组织中检测到较高的 Cg-Runt mRNA 和蛋白表达水平。在长牡蛎早期个体发育过程中，Cg-Runt 在受精卵和胚胎期均有表达，且在担轮幼虫早期（10 hpf）达到表达峰值，但进入 D 形幼虫期后，Cg-Runt mRNA 表达水平显著降低。整体免疫荧光定位显示 Cg-Runt 蛋白在囊胚期（4 hpf）不规律地分布于整个胚胎，原肠胚期（8 hpf）分布于植物极，在担轮幼虫期（15 hpf）则分布于背部区域并呈现清晰的环形结构，在 D 形幼虫早期（22 hpf），Cg-Runt 蛋白在消化腺区域特化为窦穴状结构，D 形幼虫期（50 hpf）以后蛋白信号呈纤维状贯穿整个幼虫体内，并且在壳顶幼虫（120 hpf）期，信号更加强烈。研究结果表明 Cg-Runt 参与幼虫造血过程，此外还可能在个体发育早期和幼虫后期参与细胞分化和组织形成。四、长牡蛎 Cg-GATA-2/3 基因的鉴定与功能分析克隆获得长牡蛎造血相关转录因子 Cg-GATA-2/3 基因的 cDNA 序列，生物信息学分析发现它含有两个串联重复的 ZnF_GATA 锌指结构域，与脊椎动物中的 GATA-2 和 GATA-3，以及海洋动物中的同源 GATA 基因聚为一个大的类群。在血细胞、鳃和外套膜组织中检测到较高 Cg-GATA-2/3 的 mRNA 和蛋白表达水平。整体免疫荧光定位显示 Cg-GATA-2/3 蛋白在囊胚（4 hpf）和原肠胚期（8 hpf）不规律地分布于整个胚胎。随着胚胎不断发育在担轮幼虫期（15 hpf）迁移至背部区域并呈现为清晰的环形结构，在 D 形幼虫早期（22 hpf）在背部区域特化为两个窦穴状结构，并持续到 D 形幼虫中期。随着个体发育为壳顶幼虫（120 hpf）， Cg-GATA-2/3 蛋白的表达增多，集中分布于背部区域及面盘处。在成体长牡蛎中，Cg-GATA-2/3 的干扰引起血细胞特异性基因 EcSOD 和造血相关转录因子 C-Myb 的显著下调，同时导致循环血细胞和鳃组织中的新生细胞数目显著减少。以上研究结果表明 Cg-GATA-2/3 参与了胚胎和幼虫两次造血，对长牡蛎血细胞的生成具有重要的调控作用，可能主要通过调控 Cg-SCL 而间接参与胚胎造血，并协同 Cg-SCL、Cg-Runt 共同调控幼虫造血。综上所述，在长牡蛎的早期个体发育过程中，母源免疫物质在胚胎早期为个体提供重要的免疫保护作用。个体免疫系统在担轮幼虫期开始发生发育，胚胎造血和幼虫造血相继发生在囊胚期和担轮幼虫期，其中转录因子 Cg-SCL 和 Cg-GATA-2/3 可能参与胚胎造血，Cg-SCL 、Cg-Runt 和 Cg-GATA-2/3 共同参与幼虫造血作用；随着幼虫的发育，D 形幼虫的血细胞开始具有吞噬能力，免疫系统在壳顶幼虫时期基本发育完善。
其他摘要	Pacific oyster Crassostrea gigas is one of the most important aquaculture species worldwide, and one of the most representative species among the mollusc, Lophotrochozoa. Along with the enlargement of the cultivation scale, high mortalities of hatchery-reared, juvenile oysters, has long been threatening the healthy and sustainable development of the oyster aquciculture industry. In the present study, using molecular immunology methods, the immunocompetence of different developmental stages during the ontogeny of oyster larvae was examined, and the ontogenesis and development of hematopoietic system was explored based on the expression pattern of conserved hematopoietic transcription factors. These results will deepen the study on the ontogenesis and development of immune system in marine invertebrates, as well as improve the diseases research work on the aquaculture development. 1. The immunological capacity in the larvae of Paciﬁc oyster C. gigas In the present study, the development of immune system and its response against bacteria challenge were investigated in order to characterize the repertoire of immunological capacity of Paciﬁc oyster C. gigas during the ontogenesis. The phagocytosis was ﬁrstly observed in the early D-veliger larvae (17 hpf), especially in their velum site, which indicated the appearance of functional hemocytes during early D-veliger larvae stage. The whole-mount immunoﬂuorescence assay of three pattern recognition receptors (integrin β-1, caspase-3 and C-type lectin 3) and one immune effector gene (IL17-5) was performed in blastula, early D-veliger and umbo larvae, suggested that velum and digestive gland were the potential sites of immune system in the larvae. The lowest activities of antioxidant enzymes (superoxide dismutase and catalase) and hydrolytic enzyme (lysozyme), as well as descended expression levels of 12 immune genes at the transition between embryogenesis and planktonic, indicated that the larvae at hatching (9 hpf) were in hypo-immunity. While the ascending activities of enzymes and expression levels of seven immune genes during the trochophore stage (15 hpf) suggested the initiation of immune system. The steadily increasing trend of all the 12 candidate genes at the early umbo larvae (120 h) hinted that the immune system was well developed at this stage. After bacterial challenge, some immune recognition (TLR4) and immune effector (IL17-5 and defh2) genes were activated in blastula stage (4 hpf), and other immune genes were up regulated in D-veliger larvae, indicating that the zygotic immune system could respond earlier against the bacterial challenge during its development. These results overall indicated that the cellular and humoral immune components appeared at trochophore stage, and the cellular immune system was activated with its occurrence, while the humoral immune system executed until the early umbo larval stage. The immune system emerged earlier to aid larvae in defending bacterial challenge during the early stages of oyster development. 2. The identification and functional analysis of Cg-SCL In the present work, a conserved haematopoietic transcription factor Tal-1/Scl (Stem Cell Leukemia) was identiﬁed in Paciﬁc oyster (Cg-SCL), and it was evolutionarily close to the orthologs in deuterostomes. In the adult oyster Cg-SCL was highly expressed in the hemocytes as well as gill and mantle. Among the larval developmental stages, the mRNA transcripts of Cg-SCL gradually increased after fertilization and peaked at early trochophore larvae stage (10 hpf, hours post fertilization), then sharply decreased in late trochophore larvae stage (15 hpf) before resuming in umbo larvae (120 hpf). Whole-mount immunoﬂuorescence assay further revealed that the immunoreactivity of Cg-SCL appeared in blastula larvae with two approximate symmetric spots, and this expression pattern lasted in gastrula larvae. By trochophore, the immunoreactivity formed a ring around the dorsal region and then separated into two remarkable spots at the dorsal side in D-veliger larvae. After bacterial challenge, the mRNA expression levels of Cg-SCL were signiﬁcantly up-regulated in the D-veliger and umbo larvae, indicating the available hematopoietic regulation in oyster larvae. The hemocyte speciﬁc genes Integrin, EcSOD and haematopoietic transcription factors GATA3, C-Myb, c-kit, were down-regulated when Cg-SCL was interfered by dsRNA. These results demonstrated that Cg-SCL could be used as haematopoietic speciﬁc marker to trace embryonic hematopoiesis of oyster, which occurred early in blastula stage and maintained until D-veliger larvae. 3. The identification and functional analysis of Cg-Runt A conserved hematopoietic transcription factor gene Cg-Runt was cloned from oyster C. gigas, which contains conserved runt homology domain, belonging to the Runx family. Cg-Runt was highly expressed in the hemocytes, gill, and mantle at both mRNA and protein level. Among the larval developmental stages, the mRNA transcripts of Cg-Runt could be detected after fertilization and reached to peak expression level at early trochophore larvae stage (10 hpf), then exhibited decreased trend after entered into D-veliger stage. Whole-mount immunoﬂuorescence assay further revealed that the immunoreactivity of Cg-Runt expressed throughout the whole blastula larvae, and gathered into vegetal pole in the gastrula larvae. By trochophore, the expression of Cg-Runt could be detected as a ring structure around the dorsal region, and finally formed sinus structure in the digestive gland region in the early D-veliger larvae. At the later D-veliger, the immunoreactivity of Cg-Runt exhibited three-bifurcation structure throughout the whole larvae, and the expression pattern lasted into the umbo larvae, with stronger immunoreactivity. The results suggested that Cg-Runt involved into the larval hematopoiesis. Besides, it maybe functions in the cell differentiation as well as histogenesis during the embryo period and later larval stages. 4. The identification and functional analysis of Cg-GATA-2/3 A conserved hematopoietic transcription factor Cg-GATA-2/3 was cloned from the oyster C. gigas. Cg-GATA-2/3 contains conserved two tandem repeat zinc finger GATA domains, belonging to the GATA-123 family. Cg-GATA-2/3 was highly expressed in the hemocytes, gill, and mantle both at the mRNA and protein level. Whole-mount immunoﬂuorescence assay further revealed that the immunoreactivity of Cg-GATA-2/3 expressed throughout the whole blastula and gastrula larvae. By trochophore, the immunoreactivity formed a ring around the dorsal region and then separated into two remarkable spots at the dorsal side in D-veliger larvae, which was similar with Cg-SCL. The distribution Cg-GATA-2/3 was also specialized into velum region with development into umbo larvae. When Cg-GATA-2/3 was interfered by dsRNA, the hemocyte speciﬁc gene Ec-SOD and haematopoietic transcription factor C-Myb were down-regulated, and the number of newborn cells was remarkably decreased both in the circulating hemocytes and gill tissue. These results suggested that Cg-GATA-2/3 played critical role in the production of hemocytes and the two waves of hematopoiesis in the oyster. In the embryonic hematopoiesis, it was speculated that Cg-GATA-2/3 indirectly functioned through the regulation of Cg-SCL, and directly functioned in the larval hematopoiesis together with Cg-SCL and Cg-Runt. In conclusion, during the early ontogenesis of larvae oyster, the maternal transferred immunity provides immune protection for the embryo of oyster, and the embryonic hematopoiesis occurred as early as the blastula stage. The occurrence of immune system started at the trochophore stage, followed by the larval hematopoiesis. Along with the development of the oyster larvae, the hemocytes in D-veliger larvae become functional and show signs of phagocytosis. The immune system should be well developed by the umbo larval stage.
学科领域	地球科学 ; 海洋科学
语种	英语
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/112527
专题	实验海洋生物学重点实验室
作者单位	1.中国科学院大学 2.中国科学院海洋研究所
推荐引用方式 GB/T 7714	宋小瑞. 长牡蛎免疫系统的发生和发育机制的初步研究[D]. 北京. 中国科学院大学,2016.

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