海洋生态与环境科学重点实验室知识库

仿刺参Apostichopus japonicus，又名刺参，是我国重要的海洋经济物种，隶属棘皮动物门海参纲，后口动物，是无脊椎动物中与脊索动物最为相似的类群，占据特殊分类地位。仿刺参在经历刺激、环境胁迫等不利条件时，在神经调控下遵循固定的模式发生吐脏再生现象，消除了人工损伤导致的个体差异性，保持了再生的同步性和重现性，因此，仿刺参肠道再生是探究单个器官生成的良好模型。目前已明确肠道再生分为伤口愈合、原基形成、肠腔形成、肠道分化与生长五个阶段，再生14天时肠腔完全连通成肠管，完成新肠道生成。仿刺参吐脏后，食道残基与撕裂的肠系膜是再生起点，但大多数研究聚焦于肠系膜再生过程的变化，而忽视了残基端响应损伤及启动再生的特征。

本研究以健康正常仿刺参肠道为对照组，以再生6小时、1天、3天和7天的新生肠道为实验组，基于石蜡切片和透射电镜切片观察伤口愈合期和原基形成早、中、晚期肠道的组织细胞学特征。通过转录组测序探究吐脏后的伤口愈合期（再生6小时）及启动再生期（再生1天、3天和7天）组织水平的基因表达变化。基于10× Genomics scRNA-seq技术构建不同再生时间的肠道原基细胞谱系，明确再生原基的细胞异质性，筛选存在潜在分化关系的细胞类群并进行细胞分化轨迹分析。基于组织细胞学和bulk RNA-seq结果，联合单细胞数据，筛选仿刺参再生相关特异表达基因，继而开展下游功能验证实验，拟查明仿刺参再生关键基因的调控功能，最终明确再生原基细胞的演化特征及相关驱动因子。具体研究结果如下：

1、仿刺参肠道再生原基的组织细胞学特征

对吐脏后的仿刺参肠道断裂面进行H&E染色以观察组织结构变化，结果显示：再生6小时的肠道未见明显的组织层变化，肌肉层变薄，粘膜层出现空泡状结构；再生1天的肠道横切面中看不到明显的肌肉层，结缔组织降解；再生3天的肠道原基愈合成盲端，与肠系膜相连，未见明显的组织层分化；再生7天的新生肠道开腔部分可见肠壁组织层分化，由内到外为粘膜层-结缔组织-体腔上皮层，粘膜层已分化出多种细胞类型，在结缔组织中可见正在迁移的细胞。透射电镜观察到肌上皮细胞去分化，再生肠上皮中，神经内分泌样细胞的分泌颗粒增多。利用EdU和DAPI对增殖细胞和所有细胞进行标记，结果表明：正常肠道和再生6小时肠道几乎无正在增殖的细胞；再生1天肠道中存在零星的增殖细胞，主要分散在粘膜层；再生3天肠道中增殖细胞分散分布在粘膜层和体腔上皮层；再生7天的增厚肠系膜原基中，增殖细胞遍布体腔上皮层和内部结缔组织。利用TUNEL染色标记凋亡细胞，发现在正常肠道的体腔上皮和肠腔上皮分布有少量凋亡细胞；仿刺参吐脏后至再生3天时，凋亡持续增加；再生7天的增厚肠系膜原基中，凋亡细胞遍布体腔上皮层，结缔组织中有少量分布；在再生7天的肠道开腔部位，仅有少量凋亡细胞存在于粘膜层。说明凋亡现象遍布再生原基体腔上皮，但在肠道开腔后结束，凋亡是起始再生的重要事件。

2、仿刺参响应应激及启动肠道再生时组织水平的转录调控特征

为探究仿刺参吐脏后肠道响应应激及启动再生的基因表达特征，对正常肠道，再生6小时、1天、3天和7天的新生肠道进行转录组测序。差异基因的功能富集分析结果表明钙离子和神经活性配体-受体参与损伤信号传递，VP/OT型神经肽信号系统可能在仿刺参肠道伤口愈合阶段起重要调控作用。早期伤口愈合和启动再生过程中发生的主要分子事件是自噬、去粘附、迁移和关闭摄食信号。仿刺参特化再生基因家族AjPSP94有15条串联重复基因，AjPSP94-10在对照组表达量最高，AjPSP94-7在再生1天时表达量最高，其余AjPSP94基因均在再生6小时高表达，随后表达量下降。加权基因共表达网络分析结果表明，AjPSP94-2、AjPSP94-3、AjPSP94-4、AjPSP94-6、AjPSP94-9、AjPSP94-12、AjPSP94-13、AjPSP94-15与伤口愈合期（再生6小时）高度相关。

3、肠道原基细胞异质性

通过单细胞转录组测序，构建了正常肠道与再生肠道的单细胞图谱，基于各细胞类群的基因表达特征，鉴定到成纤维细胞、上皮细胞、间充质细胞、神经细胞、内皮细胞、巨噬细胞、神经内分泌细胞、神经胶质细胞、肌细胞、肠上皮细胞、免疫细胞等细胞类型。发现仿刺参特有的再生基因家族AjPSP94-like特异性表达于伤口愈合期和再生1天、3天的肠道上皮细胞，利用拟时序分析揭示了上皮细胞的潜在干细胞特性。为探究调控上皮细胞活动的神经信号来源，分析发现色素分散因子（pigment-dispersing factor-like precursor, PDF）基因在再生样本的神经内分泌细胞中显著表达。对PSP94-11、PSP94-12以及神经内分泌细胞的特征基因NC2和PDF进行多克隆抗体制备，蛋白免疫印记结果显示PDF蛋白在再生1天、3天、7天的表达量逐渐升高，且PSP94的蛋白表达量也呈现逐渐升高的趋势。在正常肠道中，神经内分泌细胞分布在体腔上皮层，在再生时期向结缔组织迁移。PSP94-12蛋白在正常肠道中主要表达在体腔上皮层，吐脏后迅速响应，在各组织层均有分布。可能是由于损伤导致的神经信号快速应答，从而调控了PSP94基因的上调表达，进而启动了上皮细胞的分化过程。

4、PDF神经肽调控上皮细胞中PSP94-like基因家族参与上皮间充质转化

PDF基因编码两条PDF成熟肽，为进一步探究PDF神经肽在再生原基的功能，鉴定了仿刺参中PDF的功能受体，系统解析了受体基因的分子特征、神经肽与受体的信号传递。仿刺参基因组中两条PDFR均含有7tmB1_PDFR结构域，通过亚细胞定位明确两个受体均表达在细胞膜上。对PDFR的系统发育分析显示，仿刺参的PDFR1和PDFR2均与玉足海参中PDFR聚为一支。成熟肽PDF1/2均能激活PDFR2并引起受体内吞，导致胞内cAMP积累。PDF1/2结合PDFR2激活了MAPK信号级联途径，提高了ERK1/2磷酸化水平，并呈浓度依赖，同时通过抑制AKT磷酸化调控PI3K-AKT信号通路。通过注射siRNA和成熟肽以减少或增加PDF神经肽在体内的含量，发现PDFR2的基因表达变化与PDF神经肽含量的变化保持一致。同时，本研究还观察到与伤口愈合阶段高度相关的AjPSP94基因表达发生了变化，且其表达模式与PDFR2的变化一致，表明PDF神经肽激活PDFR2，并进一步通过胞内信号传递途径调控了PSP94基因的表达。

The sea cucumber Apostichopus japonicus, is an important marine economic species in China, belonging to the phylum Echinodermata, class Holothuroidea, Deuterostomia. It is the most similar group of invertebrates to chordates and occupies a special taxonomic position. When the sea cucumber experiences adverse conditions such as stimulation and environmental stress, the phenomenon of evisceration and regeneration occur under the innervation of the nerve. Evisceration follows a fixed pattern, eliminates the individual differences caused by artificial injury, and maintains the synchronization and reproducibility of regeneration. Therefore, sea cucumber intestinal regeneration is a good model to explore the generation of individual organs. After evisceration, the esophageal residues and the torn mesentery are the starting point of regeneration. At present, it has been clear that intestinal regeneration is divided into wound healing stage, blastema formation stage, lumen formation stage, intestinal differentiation stage and growth stage. By the 14th day of regeneration, the intestinal lumen becomes fully patent, culminating in the formation of a continuous intestinal tube, thereby marking the completion of the neointestinal generation. Most studies focus on mesentery and explore the changes of regeneration process, while ignoring the characteristics of residue end responding to injury and initiating regeneration.

In this study, the healthy and normal intestines were used as the control group, and the newly regenerated intestines at 6 hours, 1, 3, and 7 days post evisceration were used as the experimental group. Based on paraffin sections and transmission electron microscopy sections, the histocytological and cytological characteristics of the intestine in wound healing stage and the early, middle, and late stages of blastema formation stages were observed. Transcriptome sequencing was used to explore the changes of gene expression at the tissue level during wound healing (6 hours post evisceration) and initiation of regeneration (1 day, 3 days and 7 days post evisceration). Based on 10x Genomics scRNA-seq technology, the cell lineages of intestinal blastema at different regeneration time were constructed. The cell heterogeneity of regenerative blastema was clarified, and the cell groups with potential differentiation relationship were screened for cell differentiation trajectory analysis. Based on histocytological results and bulk RNA-seq results, combined with single cell RNA-seq data, specific expression genes were screened for downstream functional verification experiments to identify the regulatory functions of key genes. Finally, the evolution characteristics and driving factors of regenerative blastema cells were clarified. The specific research results are as follows:

1. Histocytological characteristics of intestinal regenerative blastema in Apostichopus japonicus

H&E staining was performed on the intestinal fracture surface after evisceration to observe the changes of tissue structure. There was no obvious tissue layer change at 6hpe, the muscle layer became thinner, and the mucosal layer initiated an inflammatory response. There was no obvious muscle layer and connective tissue degradation in the intestinal transverse section at 1dpe. At 3dpe, the intestinal blastema healed into a blind end, which was connected to the mesentery, and no obvious tissue layer differentiation was observed. At 7dpe, the tissue layer differentiation was observed in the lumen of the regenerated intestine. From the inside to the outside, it was mucosa-connective tissue-coelomic epithelium. A variety of cell types have differentiated in the mucosal layer, and the migrating cells can be seen in the connective tissue. Dedifferentiation of myoepithelial cells was observed by transmission electron microscopy. In the regenerated intestinal epithelium, the secretory granules from neuroendocrine-like cells increased. Using EdU and DAPI to label proliferating cells and all cells, the results showed that there were almost no proliferating cells in the normal intestine and at 6hpe. There are sporadic proliferative cells in the intestine at 1dpe, mainly scattered in the mucosal layer. At 3dpe, the proliferative cells in the intestine were dispersed in the mucosal layer and the coelomic epithelium. In the thickened mesenteric blastema at 7dpe, proliferating cells spread throughout the coelomic epithelium and internal connective tissue. TUNEL staining showed that a small number of apoptotic cells were distributed in the coelomic epithelium and luminal epithelium of the normal intestine. The apoptosis continued to increase until 3dpe. In the thickened mesenteric blastema at 7dpe, apoptotic cells were distributed throughout the coelomic epithelium, and a small number of apoptotic cells were distributed in connective tissue. Only a small number of apoptotic cells were found in the mucosal layer at the site of intestinal opening at 7dpe. It is indicated that apoptosis occurs throughout the coelomic epithelium of the regenerated blastema, but it ends after the intestinal lumen is opened. Apoptosis is an important event in the initiation of regeneration.

2. Transcriptional regulation at tissue level of Apostichopus japonicus in response to stress and initiation of intestinal regeneration

In order to explore the gene expression characteristics of intestinal response to stress and regeneration after evisceration of Apostichopus japonicus, transcriptome sequencing was performed on the normal intestine and the regenerated intestine at 6hpe, 1dpe, 3dpe, and 7dpe.The results of functional enrichment analysis of differentially expressed genes indicate that calcium ions and neuroactive ligand-receptors are involved in injury signal transmission, and VP/OT neuropeptide signaling system may play an important role in the intestinal wound healing stage of Apostichopus japonicus. The main molecular events that occur during early wound healing and initiating regeneration are autophagy, deadhesion, migration, and switching off feeding signals. There were 15 tandem repeats in the specific regenerated gene cluster AjPSP94.The expression level of AjPSP94-10 and AjPSP94-7 were the highest at the control group and 1dpe. The other AjPSP94 genes were highly expressed at 6hpe, and then the expression level decreased. The results of weighted gene co-expression network analysis showed that AjPSP94-2, AjPSP94-3, AjPSP94-4, AjPSP94-6, AjPSP94-9, AjPSP94-12, AjPSP94-13, AjPSP94-15 were highly correlated with wound healing period (Aj6hpe).

3. The heterogeneity of intestinal blastema

Using single-cell transcriptomic sequencing, we construct single-cell atlases of both normal and regenerated intestines. Based on the gene expression characteristics of each cell cluster, various cell types were identified, including fibroblasts, epithelial cells, mesenchymal cells, neurocytes, endothelial cells, macrophages, neuroendocrine cells, neurogliocytes, myocytes, enterocyte, immune cells etc. The AjPSP94-like gene cluster was specifically expressed in epithelial cells during wound healing and regeneration at 1dpe and 3dpe. The potential stem cell characteristics of epithelial cells were revealed by pseudotime analysis. The pigment-dispersing factor (PDF) gene was significantly expressed in the neuroendocrine cells of the regenerated samples. Polyclonal antibodies were prepared for the genes NC2 and PDF of neuroendocrine cells, as well as PSP94-11 and PSP94-12. The results of western-blot showed that the expression of PDF protein increased gradually from 1dpe to 7dpe, and the expression of PSP94 protein also increased gradually. In the normal intestine, neuroendocrine cells are distributed in the coelomic epithelium and migrate to connective tissue during regeneration. In normal intestine, PSP94-12 protein is mainly expressed in the coelomic epithelium, which responds quickly after evisceration and is distributed in all tissue layers. It may be due to the rapid response of nerve signals caused by injury, which regulates the up-regulated expression of PSP94 gene, and then initiates the differentiation process of epithelial cells.

4. PDF neuropeptide regulates the PSP94-like gene family in epithelial cells to participate in epithelial-mesenchymal transition

The PDF gene expresses two mature PDF peptides. In order to further explore the function of PDF neuropeptides in the early stage of regeneration, functional receptors in Apostichopus japonicus were identified, the molecular characteristics of receptor genes, and the signal transmission between neuropeptide and receptor were systematically analyzed. The two tandemly arranged PDFRs contained the 7tmB1_PDFR domain, and the subcellular localization confirmed that both receptors were expressed on the cell membrane. The phylogenetic analysis of PDFRs reveals that PDFR1 and PDFR2 of A. japonicus form a clade with PDFRs from Holothuria leucospilota. PDF encodes two mature peptides, both of which can activate PDFR2 and cause ingestion in vivo, resulting in intracellular cAMP accumulation. PDF1/2 binding to PDFR2 activated the MAPK signaling cascade pathway and increased the level of ERK1/2 phosphorylation in a concentration-dependent manner. The PI3K-AKT signaling pathway is regulated by inhibiting AKT phosphorylation. The content of neuropeptides in vivo was reduced or increased by injection of siRNA and mature peptide, and the changes in gene expression of PDFR2 were consistent with the changes in the content of neuropeptide in vivo. Meanwhile, we examined the expression changes of AjPSP94 genes highly related to the wound healing stage. The gene expression changes of AjPSP94 were consistent with the expression changes of PDFR2. The results indicated that PDF neuropeptides could activate PDFR2 and regulate PSP94 gene expression through intracellular signaling.

第1章 绪论 1

1.1 动物再生       1

1.1.1 动物再生模式    1

1.1.2 再生细胞来源    2

1.2 棘皮动物再生的研究进展   3

1.2.1 细胞与分子机制 3

1.2.2 神经信号在棘皮动物再生中的研究      4

1.3 海参肠道再生的研究进展   5

1.3.1 吐脏后的早期事件    6

1.3.2 再生关键基因    8

1.4 研究目的、意义及研究思路      11

1.4.1 研究背景与科学问题       11

1.4.2 研究目的与意义 12

1.4.3 技术路线    12

1.4.4 预期成果    13

第2章 仿刺参肠道再生原基组织细胞学特征      15

2.1 研究背景       15

2.2 材料与方法   16

2.2.1 动物处理与样品采集       16

2.2.2 石蜡切片及H&E染色      16

2.2.3 透射电镜切片观察    16

2.2.4 EdU细胞增殖检测     16

2.2.5 TUNEL细胞凋亡染色 17

2.3 实验结果       17

2.3.1 肠道再生初期组织结构变化   17

2.3.2 肠道再生初期细胞学变化       19

2.3.3 肠道再生初期细胞增殖情况   20

2.3.4 细胞凋亡特征    21

2.4 讨论 23

2.4.1 仿刺参启动肠道再生的组织细胞学事件      23

2.4.2 细胞增殖及凋亡在肠道再生初期的作用      24

2.5 本章小结       25

第3章 肠道再生原基的转录调控特征   27

3.1 研究背景       27

3.2 材料与方法   28

3.2.1 实验材料与样品采集       28

3.2.2 文库构建及测序 28

3.2.3 参考基因组比对及基因表达定量   29

3.2.4 基因差异表达分析    29

3.2.5 WGCNA分析      29

3.2.6 GO和KEGG功能富集分析      29

3.2.7 特征基因的热图分析       29

3.2.8 AjPSP94基因家族的鉴定及基因结构分析    30

3.3 实验结果       30

3.3.1 转录组测序概况 30

3.3.2 差异表达基因分析    32

3.3.3 差异表达基因的功能富集分析       33

3.3.4 加权基因共表达网络分析       41

3.3.5 目标模块及hub基因筛选      43

3.3.6 AjPSP94基因结构及表达特征 44

3.4 讨论 45

3.4.1 多种神经信号参与仿刺参肠道伤口愈合      45

3.4.2 关键生物学过程在肠道再生初期的表达模式       46

3.4.3 肠道再生初期的基因响应特征       47

3.5 本章小结       48

第4章 基于scRNA-seq解析仿刺参肠道再生的细胞异质性       49

4.1 研究背景       49

4.2 材料与方法   50

4.2.1 样品采集与单细胞悬液制备   50

4.2.2 10× Genomics文库构建与测序      50

4.2.3 数据质控、过滤及参考基因组比对      50

4.2.4 细胞分群及细胞类型注释       51

4.2.5 拟时序分析 51

4.2.6 关键基因免疫组化验证    51

4.3 实验结果       52

4.3.1 单细胞转录组测序质控结果   52

4.3.2 仿刺参肠道再生初期单细胞图谱   53

4.3.3 细胞类型鉴定    56

4.3.4 细胞分化轨迹分析    59

4.3.5 再生关键基因的表达特征       61

4.4 讨论 64

4.4.1 仿刺参肠道细胞类型多样性   64

4.4.2 AjPSP94-like基因家族是上皮细胞的主效基因       65

4.5 本章小结       65

第5章 PDF神经肽对肠道再生原基的调控特征   67

5.1 研究背景       67

5.2 材料与方法   68

5.2.1 序列特征及系统发育分析       68

5.2.2 体内神经肽注射 68

5.2.3 RNAi实验    68

5.2.4 总RNA提取及反转录      69

5.2.5 实时荧光定量PCR    69

5.2.6 PDFR(1/2)-EGFP和PDFR(1/2)-pcDNA3.1(+)表达载体构建及质粒扩增  69

5.2.7 HEK293细胞复苏及培养  70

5.2.8 细胞瞬时转染及荧光染料染核和染膜   70

5.2.9 蛋白免疫印记分析    70

5.2.10 cAMP含量检测       71

5.3 实验结果       71

5.3.1 PDF及PDFR序列特征与系统发育分析 71

5.3.2 PDF-RNAi结果   73

5.3.3 体内注射PDF成熟肽对PDFR及AjPSP94基因表达的影响      74

5.3.4 PDF1/2引起PDFR2受体内吞  75

5.3.5 PDF1/2激活PDFR2介导胞内cAMP积累和蛋白磷酸化       78

5.4 讨论 80

5.4.1 PDF1/2激活PDFR2   80

5.4.2 PDF1/2可能调控肠道再生      82

5.5 本章小结       82

第6章 总结与展望    83

6.1 研究总结       83

6.2 主要创新点   84

6.3 存在问题       84

6.4 研究展望       84

参考文献       87

附录一  用于RNAi和RT-PCR的引物   105

附录二  mRNA数量、表达量、线粒体RNA比例的QC小提琴图   106

致  谢   107

作者简历及攻读学位期间发表的学术论文与其他相关学术成果   109