人视网膜色素上皮细胞吞噬过程中MERTK基因mRNA表达水平与蛋白激酶C关系的研究

目的 探讨人视网膜色素上皮(hRPE)细胞吞噬过程中MERTK基因mRNA表达水平与蛋白激酶C(PKC)的相互作用.方法 对照实验研究.采用1×1010个/L视细胞外节膜盘(ROS),于37℃下孵育体外培养的正常hRPE细胞,在孵育的不同时间终止吞噬反应.用双重荧光标记法,检测hRPE细胞的吞噬能力.用液闪记数γ-32P放射活性法,检测不同吞噬时间点的PKC活性.以逆转录聚合酶链反应(RT-PCR)法,检测MERTK基因的mRNA水平变化情况.以PKC激活剂和拮抗剂处理hRPE细胞后,再行MERTK基因mRNA表达水平检测.采用SPSS 13.0统计学软件进行数据分析,实验组与对照组hRPE细胞吞噬ROS能力、PKC活性比较采用Student't检验,MERTK基因mRNA表达水平比较采用单因素方差分析.结果 ROS孵育后的hRPE细胞结合及吞入ROS的数量逐渐增多,至24 h时,hRPE细胞吞噬ROS的数量达到高峰,为(2.85±0.11)×106个,与对照组(0.00±0.00)×106个比较,差异有统计学意义(t=47.64,P＜0.05).ROS孵育后的hRPE细胞胞质PKC活性降低,至24 h时,PKC活性降至最低,细胞质的PKC活性为(151.13±17.67)nmol·g-1·min-1,细胞膜的PKC活性为(152.45±64.83)nmol·g-1·min-1;对照组细胞质的PKC活性为(329.63±14.26)nmol·g-1·min-1,细胞膜的PKC活性为(467.67±68.87)nmol·g-1·min-1;两组间PKC活性比较,差异有统计学意义(细胞质:t=89.66,P＜0.05;细胞膜t=10.31,P＜0.05).在hRPE细胞与ROS不同时段的孵育过程中,MERTK基因mRNA均呈现出高表达状态,孵育至90 min时,MERTK基因mRNA表达灰度值为1.8853±0.0077,与对照组灰度值0.7246±0.0062相比,差异有统计学意义(F=16 060.2167,P＜0.05);孵育至24 h时,MERTK基因mRNA表达灰度值为0.5946±0.0082,与对照组灰度值0.3343±0.0064比较,差异有统计学意义(F=919.8421,P＜0.05).上调hRPE细胞的PKC活性后,再行不同时段的ROS孵育,短时与长时孵育的MERTK基因mRNA表达灰度值均低于对照组(短时孵育:F=17 142.2331,长时孵育:F=1886.4614;P＜0.05).拮抗hRPE细胞的PKC活性后,再行不同时段的ROS孵育,30 min内MERTK基因mRNA表达灰度值为4.4670±0.0092至5.7034±0.0095范围,均高于对照组灰度值0. 9117±0.0021(F=199 012.9138,P＜0.05).结论 PKC的低活性和MERTK基因mRNA的高表达可促进hRPE细胞吞噬ROS的过程.PKC活性和MERTK基因mRNA表达水平作为上游调控信号,通过两条不同的信号通路,以负相关的方式调节hRPE细胞吞噬ROS的功能.

The relationship between MERTK gene and protein kinase C in the phagocytic process of human retinal pigment epithelial cells

Objective To investigate relationship of the expression of MERTK gene and the activity of protein kinase C (PKC) in the phagocytic process of human retinal pigment epithelial (hRPE) cells.Methods Cultured hRPE cells were incubated with rod outer segments (ROS) suspension (containing ROS 1 × 1010/L) at 37 ℃, then cells were rinsed at different times to terminate the phagocytosis. The kinetic of phagocytosis was measured by double-fluorescent labeling. The activity of PKC and the expression level of MERTK gene were measured by counting γ-32p radio-activity with liquid scintillation and RT-PCR respectively. Change of MERTK gene expression was measured after hRPE was treated cells with stimulator or inhibitor of PKC. Statistical analysis was performed by SPSS 13.0 software. Results The phagocytic assay showed that the quality of bound and ingested ROS by hRPE cells increased. The quality of ingested ROS by hRPE cells at 24 hours was ( 2. 85 ± 0. 11 ) × 106, which reached maximum contrast with control (0. 00 ± 0. 00 ) × 106 ( t = 47.64, P ＜ 0. 05 ). The activity of PKC ( both in cytoplasm and on membrane )decreased during all the incubation periods compared with control cytoplasm: (329. 63 ± 14. 26) nmol · g-1 · min - 1 and on membrane: ( 467.67 ± 68. 87 ) nmol · g- 1 · min - 1 ], and reached the minimum at 24 h cytoplasm:( 151.13 ± 17.67) nmol · g-1 ·min-1 and on membrane: ( 152.45 ±64. 83) nmol · g-1 min-1 ;cytoplasm t = 89.66 and membrane t = 10. 31 ,P ＜0. 05＝. The level of MERTK mRNA increased in pulse-chase and long-time incubation test. The gray level for 90 min was 1. 8853 ± 0. 0077, contrasted with control 0. 7246 ±0. 0062, F = 16060. 2167 and P ＜ 0. 05. The gray level for 24 h was 0. 5946 ± 0. 0082,contrasted with control 0. 3343 ± 0. 0064, F = 919. 8421 and P ＜ 0. 05. When up-regulating the activity of PKC in hRPE cells, the level of MERTK mRNA was decreased in the proceeding incubating with ROS contrasted with control ( pulse-chase group F = 17 142. 2331,long time group F = 1886. 4614;P ＜ 0. 05 ＝. After down-regulating the activity of PKC in hRPE cells, the level of MERTK mRNA waved between 4. 4670 ± 0. 0092 and 5.7034 ±0. 0095 in the first 30 min of incubating with ROS,which lower than control 0. 9117 ± 0. 0021 (F = 199 012. 9138 ,P ＜0.05). Conclusions The lower activity of PKC and the higher expression MERTK gene are very important for sustaining phagocytic process of ROS by hRPE cells. MERTK gene and PKC both as up-stream regulators are negative-correlated in the phagocytic process of hRPE.