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采用胰酶消化法获得三角帆蚌(Hyriopsis cumingii)外套膜细胞,用Fluo-3/AM荧光标记技术和激光共聚焦扫描显微镜(confocal laser scanning microscope,CLSM)检测外套膜内外表皮细胞在静息状态下细胞的游离Ca2+浓度及不同浓度维生素D3(VD3)孵育后的外套膜细胞的Ca2+的动态情况。结果表明,未添加VD3(1.25 mmol/L Ca2+)时,外套膜外表皮细胞的Ca2+荧光强度显著高于内表皮细胞内的Ca2+(P<0.05)。在添加不同浓度VD3后,内表皮细胞与外表皮细胞内Ca2+浓度变化趋势一致,表现为细胞内Ca2+沉积量的变化是随着VD3浓度的增大而增大,其中,对照组与添加50 IU/L组差异不显著(P>0.05);当添加浓度为100 IU/L时,与对照组和50 IU/L组差异显著(P<0.05);当添加组浓度达到500IU/L时,与前3组有显著差异(P<0.05);当添加的浓度增大到1 000 IU/L后与500 IU/L组差异不显著(P>0.05),但与其他组均存在显著差异(P<0.05)。本实验为外套膜细胞的钙代谢机制研究提供了实验依据。
The mantle cells of Hyriopsis cumingii were obtained by trypsin digestion. Fluorescence staining with Fluo-3 / AM and confocal laser scanning microscopy (CLSM) were used to detect the inner and outer epidermal cells of the mantle in resting state The free Ca2 + concentration of inferior cells and the dynamic state of Ca2 + in mantle cells incubated with different concentrations of vitamin D3 (VD3). The results showed that the Ca2 + fluorescence intensity of outer mantle epidermal cells was significantly higher than that of the inner epidermal cells without VD3 (1.25 mmol / L Ca2 +) (P <0.05). After adding different concentrations of VD3, the changes of intracellular Ca2 + concentration in endothelium and outer epidermis were the same. The change of intracellular Ca2 + deposition was increased with the increase of VD3 concentration. The control group and the addition of 50 IU / L group (P> 0.05). When the concentration was 100 IU / L, the difference was significant (P <0.05) with the control group and 50 IU / L group (P <0.05). When the concentration was increased to 1 000 IU / L, there was no significant difference between the three groups (P> 0.05), but there was a significant difference (P> <0.05). This experiment provides experimental evidence for the study of calcium metabolism in mantle cells.