合浦珠母贝是重要的海水珍珠养殖贝类，其贝壳结构与珍珠类似，是具有良好机械性能的生物矿物。因此了解其贝壳的形成机理有助于提高人工养殖珍珠的产量和品质，并为制造仿生材料提供指导。本课题以合浦珠母贝为研究对象，探讨外套膜组织和血细胞在贝壳生长过程中的调控作用。在贝壳损伤修复过程中，外套膜在空间上的位置和形态直接决定了再生贝壳的微观结构。同时，外套膜可以通过上调有机基质的分泌来增加碳酸钙的成核位点，从而加速贝壳的再生。在平板珍珠的形成过程中，套膜区外表皮细胞的组成和基因的表达都发生了显著的变化；而在间液腔中插入弧形玻片时，只有贴近外套膜的一面有碳酸钙沉积，说明外套膜在碳酸钙的沉积中起到了直接的指导作用。进一步探究外套膜分泌贝壳组分的过程表明外套膜通过分泌泡将有机基质直接分泌到贝壳的生长界面上。间液蛋白的质谱分析表明外套膜分泌的大部分基质蛋白并不以游离的形式存在于间液中。我们推测基质蛋白可能是从外套膜细胞上直接被分泌到矿化界面，从而调控贝壳的生长。血细胞作为主要的免疫细胞，被认为参与了贝壳的矿化过程。我们的研究结果表明血细胞可以出入间液腔，其组成与循环系统中的血细胞一致。当间液腔中存在大量的微生物时，珍珠层的沉积会受到严重影响，并导致贝壳疾病的发生。而血细胞可以凝集并吞噬病原微生物，通过发挥细胞免疫和体液免疫来维持间液腔的微环境，保证贝壳沉积的顺利进行。在受到免疫刺激和贝壳缺刻损伤刺激后，血细胞显著上调钙代谢相关的基因。同时我们也发现了一类特殊的颗粒血细胞，这些血细胞能够富钙元素，并且能够快速迁移，可能是钙的运输载体。这表明血细胞可以通过在贝壳形成过程中起到了免疫保护和潜在的钙库的作用。此外，我们还探索了合浦珠母贝中棱柱层的多边形排列的演变规律和形成机理。我们在棱柱层中发现了两种不同的生长模式，即水平扩张时的Voronoi分布和垂直生长时的二维泡沫演变。这两种方式都是由热力学规律驱动的。有机基质的异质性分布则为棱柱层按照特定规律进行生长确定了边界条件。本课题研究结果阐明了外套膜直接调控了贝壳的生长过程主导作用，而血细胞起到了保障和促进贝壳生长的辅助作用，这加深了我们对贝壳生长的上游细胞调控过程的理解，为珍珠的科学养殖提供了一定的理论支撑。
The pearl oyster Pinctada fucata is an economically important species for the aquacultured seawater pearls. Its shell structure is similar to that of pearls and exhibits outstanding mechanical properties. Therefore, understanding the formation mechanism of the shells will improve the yield and quality of the cultured pearls and provide guidance for the manufacture of biomimetic materials. In this study, we studied the regulation mechanism of the mantle tissue and hemocytes in the process of shell growth.In the process of shell repair, the spatial position and morphology of the mantle directly determined the microstructure of the regenerated shell. Simultaneously, the mantle tissue could promote calcium carbonate nucleation by upregulating the organic matrix secretion, thereby accelerating the shell regeneration. During the formation of flat pearl, it was found that the composition of the epidermal cells and their gene expressions were dramatically changed; when a curved glass was inserted into the extrapallial space (EPS) as an explant, calcium carbonate deposits were only found on the outer surface of the explant which was could directly contact with the mantle cells, further supporting that the mantle directly controls the shell deposition process. Further investigation of the process of secreting shell components from the mantle revealed that the mantle directly deposited the organic matrix onto the growing interface of the shell via secreting vesicles. However, proteomic analysis of the extrapallial fluids showed that most of the matrix proteins secreted by the mantle were not present as a free form in the EPS. Therefore, we speculate that the mantle cells directly secrete matrix proteins to the mineralization interface, thus regulating the shell growth in a close manner.As the main immunocomponent, hemocytes are supposed to be involved in shell mineralization. Our results suggest that hemocytes can migrate in and out of the EPS, and their composition is similar to hemocytes in the circulatory system. When a large amount of microorganisms was present in the EPS, the deposition of nacre was severely affected, and it even led to shell disease. Hemocytes agglutinates and phagocytoses the pathogenic microorganisms, and maintained the microenvironment of the EPS by exerting cellular immunity and humoral immunity, therefore ensuring the successful deposition of the shell. After immune stimulation and shell notch damage, hemocytes significantly upregulate genes involved in calcium metabolism. At the same time, we found that some hemocytes might be involved in the transport of calcium ions, and these cells were rich in calcium ions and moved quickly, which were beneficial for calcium transportion. This suggests that hemocytes plays a role in immune protection and serves as a potential calcium pool in shell formation.In addition, we explored the evolution and formation mechanism of the polygonal arrangement of prismatic layers. We found two different growth modes in the prismatic layer, namely the Voronoi distribution during horizontal expansion and the two-dimensional foam evolution during vertical growth. These two processes are governed by thermodynamic rules. The heterogeneity distribution of the organic matrix determines the boundary conditions for the prism layer to grow under mathematics and physics laws.Our results indicate that the mantle tissue plays a leading role in shell growth and directly control the shell deposition, while hemocytes play an auxiliary role by ensuring and promoting the shell growth, which deepens our understanding of the upstream cellular regulation process of shell growth and will provide a theoretical support for the aquaculture of the pearls.
