合浦珠母贝贝壳珍珠层一直是生物矿化研究的热点，外套膜是贝壳珍珠层形成直接作用的矿化器官，外套膜合成和分泌几乎所有与珍珠形成相关的基质蛋白。论文主要从合浦珠母贝外套膜组织、外套膜细胞以及外套膜分泌的基质蛋白Pif80三个方面研究外套膜细胞对珍珠层形成作用的机理。我们通过平板珍珠实验研究了外套膜分泌的基质蛋白在珍珠层起始生物矿化中的作用。在贝壳内层膜系统中，我们发现文石晶体在第5天就开始成核，而对照组中第5天方解石晶体先出现，第10天有机质膜覆盖方解石，随后才开始沉积文石，这直接导致贝壳内层膜系统的珍珠层片状结构比对照组中提前5天出现，与基质蛋白的表达最高峰密切相关。基质蛋白（Nacrein、N16、N19、MSI60和Pif80）在贝壳内层膜系统中有类似的表达模式控制着珍珠层的生长变化，而在对照组中这些基质蛋白同样有着类似的表达模式控制着珍珠层的生长。结果显示外套膜分泌的基质蛋白与贝壳内层膜协同控制珍珠层的生长。通过原代培养外套膜细胞，我们首次发现细胞内无定形碳酸钙的形成。利用Real-time PCR和Western-blot分析显示体外培养的外套膜细胞保持了体内合成和分泌基质蛋白（ACCBP、Pif80和nacrein）的能力，另外细胞也保持着与贝壳形成相关的碱性磷酸酶和碳酸酐酶的高活性。偏振光显微镜和电子扫描显微镜发现细胞内分布大量的晶体颗粒物，FTIR和XRD技术证实该晶体颗粒物是无定形碳酸钙，锶离子标记显示外套膜细胞从头合成无定形碳酸钙，我们的结果显示外套膜细胞通过无定形碳酸钙进行生物矿化活动，可能直接参与合浦珠母贝贝壳的形成。Pif80能够促进碳酸钙晶体的结晶速度，在方解石结晶体系中Pif80能够诱导方解石向球文石发生转化，并具有浓度依赖性。在文石结晶体系中Pif80抑制文石生长，破坏镁离子对文石的促进作用。另外，Pif80能够抑制外套膜细胞和成骨细胞MC3T3-E1的矿化活性。我们推测Pif80是一个双调控蛋白，既能抑制文石生长，又能抑制方解石生长，促进贝壳珍珠层精美结构的正确组装。综合以上研究发现：外套膜细胞直接参与了珍珠层的生物矿化过程，细胞在珍珠层形成中作用研究对全面了解生物矿化的细胞机制具有重要的意义。

The nacre from the shell has become one of the most intensively studied biological structures in the field of biomineralization. The mantle tissue is responsible for nacre biomineralization and secrets almost all of matrix proteins involving in nacre growth. In this study, we have explored the mechanism of nacre biomineralization from three angles of the mantle tissue, the mantle cells and the matrix protein Pif80.A flat pearl method in the presence of the inner-shell film was conducted to evaluate the role of matrix proteins in the initial stages of nacre biomineralization in vivo. We examined the crystals deposited on a substrate and the expression patterns of the matrix proteins in the mantle facing the substrate. The aragonite crystals nucleated on the surface at 5 days in the inner-shell film system. In the film-free system, the calcite crystals nucleated at 5 days, a new organic film covered the calcite, and the aragonite nucleated at 10 days. This meant that the nacre lamellae appeared in the inner-shell film system 5 days earlier than that in the film-free system, timing that was consistent with the maximum level of matrix proteins during the first 20 days. In addition, matrix proteins in the inner-film system had similar expression patterns in controlling the sequential morphologies of the nacre growth, while the matrix proteins in the film-free system also had similar patterns of expression. These results suggest that matrix proteins regulate aragonite nucleation and growth with the inner-shell film in vivo.The growth of molluscan shell crystals is usually thought to be initiated from the extrapallial fluid by matrix proteins. However, the cellular mechanisms of biological crystallization pathway remain unknown. We first reported amorphous calcium carbonate crystallization by cellular biomineralization in primary mantle cell cultures of Pinctada fucata. Real-time PCR and western blot showed that the mantle cells retained the synthesis and secretion of ACCBP, Pif80 and nacrein in vitro. In addition, mantle cells also maintained the high activities of alkaline phosphatase and carbonic anhydrase. On the basis of polarized light microscopy and scanning electron microscopy, we observed in vitro production of intracellular mineralized crystals in the mantle cell cultures, and FTIR and XRD analyses revealed the crystals to be amorphous calcium carbonate, then de novo biomineralization was confirmed by following the incorporation of Sr into calcium carbonate. Our results demonstrate the ability of the mantle cells to undergo fundamental biomineralization processes via amorphous calcium carbonate, and they may be derectly involved in shell formation of the pearl oysters.Pif80 could increase the rate of calcium carbonate deposition in the in vitro crystallization assay, and induce the growth of vaterite and inhibit the formation of aragonite crystals. In addition, pif80 was identified as a negative regulatory factor inhibiting the function of mantle cells and MC3T3-E1 cells in biomineralization. Taken together, mantle cells are derectly involved in shell formation of the pearl oysters, this would provide a valuable complementary to the research of the cellular mechanisms in nacre biomineralization