视网膜色素上皮细胞吞噬功能与MERTK-Ras-肌球蛋白通路关系的研究

Study on relation between the phagocytic fuction of retinal pigment epithelial cells and MERTK-Ras-myosin signal pathway

目的：研究视网膜色素上皮（retinal pigment epithelium,RPE）细胞吞噬功能与MERTK受体及其下游信号通路Ras-MEK-MLCK-肌球蛋白的关系。方法：用视细胞外节膜盘（rod outer segments,ROS）于37益孵育离体培养的3~5代C57小鼠RPE细胞,在孵育0、30、60、120、180、240min终止吞噬反应。双重荧光标记法检测RPE细胞吞噬动力学;以MERTK及Ras抗体、磷酸化MEK及MLCK抗体应用Western-Blot方法检测不同孵育时间（30、60、120、180min）MERTK、Ras、MEK及MLCK的激活状态;以瞬时转染质粒方法抑制Ras及MERTK基因表达后,再次以Western-Blot方法检测对应时间MERTK-Ras通路激活状态及吞噬功能的变化。结果：C57小鼠RPE细胞吞入ROS发生在孵育30min,并在3h达到饱和。在吞噬过程中,随着孵育时间的延长,RPE细胞MERTK、Ras、MEK和MLCK的蛋白表达水平不断增多（与对照组相比,P〈0.05）。Ras及MERTK干扰后的RPE细胞与ROS共孵育（30、60、120、180min）的全过程中,MERTK、Ras、MEK和MLCK的蛋白表达及ROS的吞入数量始终维持较低水平,仅在孵育180min见到少量ROS吞入;与未转染RPE细胞相比,si Ras-RPE细胞及si MERTK-RPE细胞与ROS共孵育120、180min时,MERTK、Ras、MEK及MLCK的蛋白表达量均明显减少（P〈0.05）。结论：Ras-MEK-MLCK-肌球蛋白通路是鼠RPE细胞吞噬过程中MERTK受体激活的下游信号通路。

AIM：To investigate relation between the phagocytic fuction of retinal pigment epithelial（RPE）cells and the signal transduction pathway of MERTK-Ras-extracellular signal regulated kinase kinase（MEK）-myosin light chain kinase（MLCK）-myosin. METHODS： Cultured 3～5 passage RPE cells of C57BL/6 mouse were incubated with rod outer segments（ROS）suspension（containing ROS 1×107 /ml）at 37℃, then cells were rinsed at different times（30,60,120,180,240min）to terminate the phagocytosis. The kinetics of phagocytosis was measured by double-fluorescent labeling. The activity levels of MERTK, Ras, MEK and MLCK at different incubation times were measured by Western Blot with antibodies of MERTK, Ras and phospho-antibodies of MEK and MLCK, respectively. To repeat the measurement of the phagpcytic kinetics and activity levels of MERTK, Ras, MEK and MLCK at different incubation times after interference to Ras and Mertk gene in RPE cells by plasmid transfection. RESULTS： The phagocytic kinetics showed that the ingestion occurred at 30min of incubation. Ingested ROS by RPE cells increased until saturated at 180min. The protein levels of MERTK, Ras, MEK and MLCK in RPE cells increased during all the incubation periods compared with control group（P〈0.05）. After interference to Ras and MERTk gene in RPE cells by plasmid transfection, the protein levels of MERTK, Ras, MEK and MLCK and the quality of ingested ROS sustained at the lower levels during all the incubation periods, only a few ingested ROS were seen at 180 min. Compared with untransfected RPE cells, the protein levels of MERTK, Ras, MEK and MLCK and the quality of ingested ROS in siRas-RPE cells and siMERTK-RPE cells decreased distinctly at 120 min and 180 min during incubation（P〈0.05）.CONCLUSION： Ras-MEK-MLCK-myosin signal pathway is the downstream of MERTK receptor in the phagocytic process of RPE cells from mice.