Research On Cell Culture And Biomineralization Roles Of Mantle Tissue Cells From The Pearl Oyster Pinctada Facata

Previous studies generally believe that mantle cells of Pinctada fucata mediate shell mineralization through secreting matrix proteins. However, whether mantle cells directly involved in the biomineralization process has yet to be proven. The first part of this research is commited to study the influence of the mantle cells on CaCO3 crystal nucleation and growth. According to the results, in the control groups without any mantle cells, no crystals could be observed. In the experiment groups with mantle cells, large amounts of CaCO3 crystals could be observed. These crystals were piled up by highly ordered units, showing the typical characteristics of biomineralization products. After dissolved by EDTA, a thin layer of organic framework could be observed. Some of the crystals had etched surfaces, which appeared quite smoother than the other parts. Mantle cells could be observed attaching on some of the smooth surfaces, suggesting that they may be involved in the crystal remodeling process. In the other experiment groups containing substances secreted by mantle cells CaCO3 crystals did not precipitate. However in the in vitro CaCO3 crystallization experiments, the cultured medium could change the morphology of natural calcite crystals, accelerate crystal growth and promote aggregation of the crystals. These results indicate that mantle cells may be directly involved in the nucleation and remodeling process of the CaCO3 crystalization.Soluble substances including matrix proteins, which are secreted by mantle cells, are not able to independently mediate crystal nucleation, but can accelerate the crystal growth, promote crystal fusion, and influence the crystal morphology. These findings may contribute to an overall understanding of shell mineralization process.The establishment of marine mollusks permanent cell lines is one of the scientific problems that has not been conquered. Lacking appropriate culture medium and cell factors may be the main reasons. The second part of this research is committed to investigate the effects of vertebrate cell factors on the primary cell culture and biomineralization ability of mantle cells from Pinctada fucata. Mantle tissues of 3-yearold pearl oysters were selected to culture the primary cells. Epidermal growth factor(EGF), endothelial cell growth supplement(ECGS), insulin-like growth factor-1(IGF-1) and basic fibroblast growth factor(bFGF) were added separately into the cell culture medium. The effects were evaluated by comparing the cell viability, adherence capacity, and migration ability and expression level of four matrix protein genes(pif80, n16, msi7 and accbp). According to the results, the cell viability, adherence and migration ability were significantly improved by EGF. The expression levels of pif80, n16 and accbp all increased under the stimulation of this factor. After ECGS treatment, the adherence and migration ability of the primary cells were improved, and the expression levels of pif80, n16 and msi7 were increased significantly. The cell viability, adherence, migration ability as well as the expression level of msi7 were increased by IGF-1 treatment. However the expression level of pif80, n16 and accbp decreased after cells treated by this factor. After bFGF treatment, the cell viability and adherence capacity were improved. The expression levels of pif80, n16 and accbp were also increased by this factor. The results suggestded that vertebrate cell factors may be used as cell culture supplements, which can increase cell viability and biomineralization ability of mantle cells from Pinctada fucata. This study therefore may provide some theoretical references for the establishment of marine mollusks permanent cell lines.