细胞内Ca^2＋及MERTK基因在人视网膜色素上皮细胞吞噬功能中的作用

The role of receptor protein tyrosine kinase MERTK and intracellular Ca^2＋ playing in the phagocytosis of human retinal pigment epithelial cells

目的观察细胞内Ca^2＋及MERTK基因在人视网膜色素上皮（RPE）细胞的吞噬功能中所起的作用及二者的相互关系。方法用视细胞外节膜盘（ROS）于37℃孵育培养的RPE细胞，在孵育不同终止吞噬反应。双重荧光标记法检测RPE细胞吞噬动力学；钙离子荧光探针负载法在荧光显微镜下测定RPE细胞内Ca^2＋的变化；半定量逆转录聚合酶链反应（RT-PCR）法检测相应时间点MERTK基因表达的变化及施加Ca^2＋激活剂（A23187载体）或拮抗剂（verapamile）后MERTK基因表达的变化。结果RPE细胞与ROS孵育过程中，ROS结合于RPE细胞表面发生在15min时，RPE细胞吞噬ROS在24h时达到饱和。细胞内Ca^2＋在孵育15min时升高，并在24h内保持高水平；MERTK基因表达在RPE细胞与ROS孵育5min时增强，并在全部孵育过程中呈现出高表达状态；以A23187载体开放钙通道升高RPE细胞内Ca^2＋后，MERTK基因信使核糖核酸（mRNA）水平升高，并呈剂量依赖性。经A23187预处理后的孵育实验中所检测到的MERTK mRNA水平除3h这一时间点外均高于对照组；verapamile拮抗RPE细胞内Ca^2＋后，MERTK基因表达明显下降并呈剂量依赖性；以verapamile预处理后继续与ROS孵育，所检测到的MERTK基因在24h内均低于对照组。结论MERTK基因及细胞内Ca^2＋发挥着维持RPE细胞吞噬过程的重要作用，MERTK作为上游调控信号启动下游的细胞内Ca^2＋信号来维持RPE细胞对ROS的摄入过程。

Objective To investigate the role of intracellular Ca^2＋ and MERTK in the phagocytosis of human retinal pigment epithelial （RPE） cells, and reveal the relationship between MERTK and intracellular Ca^2＋. Methods The cultured RPE cells were incubated with rod outer segments （ROS） at 37°C, the phagocytosis was terminated at different incubation time points. The concentration of intraeellular Ca^2＋ was assayed by Fluro-3/AM loading methods combined with fluorescence microscope and CCD system, and the mRNA level of MERTK gene was measured by reverse transcription polymerase chain reaction （RT-PCR）. Treating the RPE cells with stimulator （A23187） or inhibitor（verapamile）of intracellular Ca^2＋ to observe the changes of MERTK gene expression. Results ROS adhered to hRPE cells at the 15th minute, and the ingestion saturated at the 24th hour. The concentration of intracellular Ca^2＋ increased at the 15th minute, and kept the high level in 24 hours. The level of MERTK mRNA increased at the 5th minute, and kept the high level duration the whole incubation. When RPE ceils were treated by A23187, the expression of MERTK increased in a dose-dependent manner. After RPE cells was pretreated by A23187, the expression level of MERTK was higher in the proceeding incubation groups than which in the control group except at the 3rd hour. When RPE cells were treated by verapamil, the expression level of MERTK decreased in a dose-dependent manner. After RPE cells were pretreated by verapamil , the expression level of MERTK was lower in all the proceeding incubation groups than which in the control group （P〈0.05）. Conclusion MERTK gene and Ca^2＋ play an important role in sustaining RPE cells phagoeytizing ROS. As an up-stream regulator, the receptor tyrosine kinase MERTK keeps RPE cells phagocytizing ROS by starting the intracellular Ca^2＋.