脊椎动物源细胞因子对合浦珠母贝外套膜细胞培养及其矿化功能的影响

Effects of vertebrate cell factors on cell culture and biomineralization ability of mantle cells from Pinctada fucata

筛选出能促进合浦珠母贝外套膜细胞培养及矿化功能的细胞因子,以作为优化本物种细胞培养基的参考,挑选三龄合浦珠母贝,获取其外套膜进行原代细胞培养。向各实验组细胞培养基中分别添加表皮生长因子(EGF)、内皮细胞生长添加剂(ECGS)、胰岛素样生长因子-1(IGF-1)和碱性成纤维细胞生长因子(b FGF),通过比较细胞活性、贴壁能力、迁移能力和4种基质蛋白基因(pif80、n16、msi7及accbp)表达水平的变化,来评判这些因子对细胞培养及细胞矿化功能的影响。结果显示:(1)EGF能显著提高原代培养细胞的活性、贴壁能力和迁移能力,并促进pif80、n16和accbp基因的表达;(2)ECGS能增强细胞的贴壁能力和迁移能力,并大幅提高pif80、n16和msi7基因的表达水平;(3)IGF-1能显著增强细胞活性、贴壁能力、迁移能力和msi7的基因表达水平,但对pif80、n16和accbp基因的表达有一定抑制作用;(4)碱性成纤维细胞生长因子能增强细胞的活性及贴壁能力,并显著提升pif80、n16和accbp基因的表达水平。研究表明,脊椎动物源细胞因子具有延长细胞培养时间、增强细胞活性及促进矿化相关基因表达的作用,能够用于优化合浦珠母贝外套膜细胞的培养基。

The establishment of marine mollusks permanent cell lines is one of the scientific problems that has not been conquered. The main reasons are lacking appropriate culture medium and cell factors. In this study,we investigate the effects of vertebrate cell factors on primary cell culture and biomineralization ability of mantle cells from the pearl oyster Pinctada fucata. Mantle tissues of 3-year-old 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（ b FGF） were added separately into the culture medium. The effects were evaluated by comparing the cell viability,adherence capacity,migration ability and expression levels of four matrix protein genes（ pif80,n16,msi7 and accb p）.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 abilities of 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 levels of msi7 were increased by IGF-1 treatment. However the expression level of pif80,n16 and accbp decreased after cells were treated by this factor. After b FGF treatment,the cell viability and adherence capacity were improved. The expression levels of pif80,n16 and accbp were also increased by this factor. These results suggested that vertebrate cell factors may be used as cell culture supplements,which were able to increase cell viability and biomineralization ability of mantle cells in Pinctada fucata. This study therefore may provide some theoretical references for the establishment of marine mollusks permanent cell lines.