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℃孵育离体培养的3~5代C57小鼠RPE细胞,在孵育0、30、60、120、180、240 min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、180 min ) 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180 min 见到少量ROS吞入；与未转染 RPE 细胞相比, siRas-RPE 细胞及siMERTK-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 reIation between the phagocytic fuction of retinaI pigment epitheIiaI ( RPE ) ceIIs and the signaI transduction pathway of MERTK-Ras-extraceIIuIar signaI reguIated kinase kinase(MEK)-myosin Iight chain kinase( MLCK)-myosin. METHODS:CuItured 3~5 passage RPE ceIIs of C57BL/6 mouse were incubated with rod outer segments ( ROS ) suspension (containing ROS 1×107/mI) at 37℃, then ceIIs were rinsed at different times(30,60,120,180,240min) to terminate the phagocytosis. The kinetics of phagocytosis was measured by doubIe - fIuorescent IabeIing. The activity IeveIs of MERTK, Ras, MEK and MLCK at different incubation times were measured by Western BIot with antibodies of MERTK, Ras and phospho-antibodies of MEK and MLCK, respectiveIy. To repeat the measurement of the phagpcytic kinetics and activity IeveIs of MERTK, Ras, MEK and MLCK at different incubation times after interference to Ras and Mertk gene in RPE ceIIs by pIasmid transfection. RESULTS: The phagocytic kinetics showed that the ingestion occurred at 30min of incubation. Ingested ROS by RPE ceIIs increased untiI saturated at 180min. The protein IeveIs of MERTK, Ras, MEK and MLCK in RPE ceIIs increased during aII the incubation periods compared with controI group(P<0. 05). After interference to Ras and MERTk gene in RPE ceIIs by pIasmid transfection, the protein IeveIs of MERTK, Ras, MEK and MLCK and the quaIity of ingested ROS sustained at the Iower IeveIs during aII the incubation periods, onIy a few ingested ROS were seen at 180 min. Compared with untransfected RPE ceIIs, the protein IeveIs of MERTK, Ras, MEK and MLCK and the quaIity of ingested ROS in siRas-RPE ceIIs and siMERTK-RPE ceIIs decreased distinctIy at 120 min and 180 min during incubation(P<0. 05). CONCLUSION: Ras - MEK - MLCK - myosin signaI pathway is the downstream of MERTK receptor in the phagocytic process of RPE ceIIs from mice.
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