应用Fura-2/AM测定培养人视网膜色素上皮细胞内游离钙浓度的方法

The determination of intracellular free Ca 2+ concentration in culcured human retinal plgment epithelial cells by fura 2/AM.

为探索应用Ｆｕｒａ－２／ＡＭ测定培养人视网膜色素上皮细胞（ＲＰＥ）内游离钙的方法。选用３—５代培养人ＲＰＥ细胞，用０．１％胰蛋白酶消化后，应用Ｆｕｒａ－２／ＡＭ负载进行荧光测定。结果：培养的人ＲＰＥ细胞具有细胞存活力高，细胞数量充足的优点。在３７℃水浴条件下，Ｆｕｒａ－２／ＡＭ负载３０分钟，ＤＭＥＭ室温温育１０分钟，测定结果快捷、稳定。结论：在静息状态下，ＲＰＥ细胞内钙浓度为１９２±３０．４３ｎｍｏｌ／Ｌ，此值在文献报道范围内，加入乙酰胆碱后钙浓度为２６０．７±２４．４３ｎｍｏｌ／Ｌ，说明乙酰胆碱通过与胆碱能受体结合调节细胞内钙的浓度。

To establish the method for determining the intracellular free Ca 2+ concentration[Ca 2+ ]in culcured human retinal pigment epithelial cells by using fluorescent indicator Fura 2/AM.To choise 3 5 passage culcured human RPE cells and to prepare RPE cell suspensions with 0 1% Trpysin and then to be loaded with Fura 2/AM followed by fluorescence determination.RESULT:The culcured human RPE cells had more cellular viability and more number than primary RPE cells Loading the RPE cells suspensions with Fura 2/AM 30 minutes at 37C and then incubating for 10 minutes at room temperature were proved to be a stable and quick method.conclusions:The resting[Ca 2+ ] of cultured human RPE cells was 192±30 43 nmol/L which was withen the expected range of[Ca 2+ ] level,Acetycholine could increase[Ca 2+ ]to 260.7+24 43 nmol/L,it proved that Acetylcholine combinted with muscarinic cholinergic receptors indiating changes in intracellular calcium levels.