摘要
目的通过冷冻培养人视网膜色素上皮（ｒｅｔｉｎａｌｐｉｇｍｅｎｔｅｐｉｔｈｅｌｉｕｍ，ＲＰＥ）细胞，测定冷冻前、后细胞内ｃＡＭＰ和Ｃａ２＋浓度的变化，明确冷冻对ＲＰＥ细胞内ｃＡＭＰ和Ｃａ２＋浓度的影响。方法在－７０℃条件下，培养的人ＲＰＥ细胞被冷冻０、１５、３０、４５、６０秒后，应用Ｆｕｒａ２／ＡＭ荧光负荷技术及内源性蛋白结合的方法测定细胞内Ｃａ２＋和ｃＡＭＰ的浓度。结果冷冻１５秒ＲＰＥ细胞的ｃＡＭＰ和Ｃａ２＋水平与冷冻前比较，差异无显著性（ｔ检验，Ｐ＞０．０５）。冷冻３０、４５、６０秒与冷冻前比较，ｃＡＭＰ浓度明显下降，细胞内游离Ｃａ２＋浓度明显增高，二者呈拮抗关系。结论冷冻降低ＲＰＥ细胞内ｃＡＭＰ的浓度，使游离Ｃａ２＋浓度升高。
Objective To investigate the mechanism of retinal pigment epithelial (RPE) cell migration and proliferation at molecular level by measuring the concentrations of intracellular cyclic 3′, 5′-adenosine monophosphate (cAMP) and calcium ion (Ca^2+ ) level of frozen cultured RPE cells.Methods The culture of human RPE cells was frozen for 0s, 15s, 30s, 45s, 60s respectively at -70℃. The concentrations of cAMP and Ca 2+ in cultured human cells were measured by Fura 2/AM fluorescent technique and endogenous protein combination method.Results After freeze for 15 s, the contents of cAMP and Ca^2+ in RPE cells were not changed significantly compared with the control (no freeze). After freeze for 30s, 45s, 60s respectively, the level of intracellular cAMP was significantly decreased, while the concentration of cytoplasmic free Ca^2+ was markedly increased, showing that the concentration of cAMP and Ca^2+ being antagonistic to each other. With the re vitalization of RPE cells for 60 minutes, the intracellular free Ca^2+ gradually reduced. Conclusions The decreased concentration of cAMP and increased concentration of free Ca^2+ in RPE cells may be the effective mechanism of RPE cell migration and proliferation after cryotherapy, and it possibly plays an important role in the formation of proliferative vitreoretinopathy.
出处
《中华眼科杂志》
CAS
CSCD
北大核心
1998年第1期62-64,共3页
Chinese Journal of Ophthalmology
