三角帆蚌外套膜细胞体外培养优化及体内植入培养对细胞生长的影响

Impact of in vitro optimization and in vivo implantation culture on the growth of Hyriopsis cumingii mantle cells

以DMEM为基础培养基,通过优化改良培养基及缓冲液的配方,对三角帆蚌(Hyriopsis cumingii Lea)外套膜进行细胞培养,并且以显微观测、RNA/DNA的比值作为该细胞增殖的评价指标,分别对体外培养细胞的迁出时间、速度、数量以及增殖活力进行测定。分别取体外培养第24、108、120小时的细胞进行Hoechst DNA荧光标记,然后将3组标记细胞植入三角帆蚌外套膜中在蚌体内培养,在植入后第24、72、120、168、216小时运用RNA/DNA指标检测活体培养的细胞增殖活力。结果表明,经过优化后的缓冲液和培养基更有利于细胞从外套膜组织中迁出,迁出速度和细胞数量显著增加(P<0.05),且细胞的活力随着培养时间的延长而逐渐增大,培养至108 h时,细胞活力达到最大,RNA/DNA比值为24.53,在108 h后显著下降(P<0.05),显微观测的细胞生长状况与RNA/DNA指标测定相吻合。对体外培养的细胞植入蚌体外套膜中再进行培养时发现,对体外培养活力较好的细胞,活体内环境可增大细胞的活力,RNA/DNA比值最高达到25.45,但在活体培养168 h后活力显著下降(P<0.05);而对体外培养活力较差的细胞,活体内环境对细胞活力影响不显著(P>0.05)。本研究旨在为三角帆蚌外套膜细胞增殖的深入研究及建株提供基础性资料。

In China, Hyriopsis cumingii is the shellfish of choice for cultivating freshwater pearls;however, there has been very little research on cell and mantle tissue culture. Based on Dulbecco’s modified Eagle’s medium （DMEM）, this study attempted to optimize modified formula of medium and buffer for mantle tissue and cell culture. The ratio of RNA/DNA as an evaluation index of cell proliferation was determined and, using microscopic observations, tissue cul-ture cells versus time, speed and in vitro cell energy were established. Hoechst fluorescent staining of DNA was carried out using 24-, 108-and 120-h cells of in vitro culture, and then the three marked cell groups were implanted into the mantle for culture in vivo. The results show that, after optimization, the buffer and medium improved cell migration from the mantle, the velocity of migration and the significantly increased cell number （P〈0.05）.Cell vitality also in-creased with incubation time. Cell vitality was maximal for culture in vitro at 108 h, with a RNA/DNA ratio of 24.53, but was followed by a significant decline （P〈0.05）.Microscopic observations of cells were in agreement with the deter-mined RNA/DNA index values. When in vitro cultured cells were implanted into the mantle a second time, in vitro ac-tivity improved and culture conditions increased cell activity, with RNA/DNA values up to 25.45, but vitality declined significantly after 168 h when injected into the cell for in vivo culture （P〈0.05）. Regarding the poor in vitro activity of cells, it would appear that a living environment has no significant effect on cell viability. Our study provides basic but valuable information for further research on mantle cell proliferation and the establishment of cell lines.