色素上皮,眼%吞噬作用%蛋白激酶C%受体蛋白质酪氨酸激酶类
色素上皮,眼%吞噬作用%蛋白激酶C%受體蛋白質酪氨痠激酶類
색소상피,안%탄서작용%단백격매C%수체단백질락안산격매류
Pigment epithelium of eye%Phagocytosis%Protein kinase C%Receptor protein tyrosine kinases
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.
目的 探討人視網膜色素上皮(hRPE)細胞吞噬過程中MERTK基因mRNA錶達水平與蛋白激酶C(PKC)的相互作用.方法 對照實驗研究.採用1×1010箇/L視細胞外節膜盤(ROS),于37℃下孵育體外培養的正常hRPE細胞,在孵育的不同時間終止吞噬反應.用雙重熒光標記法,檢測hRPE細胞的吞噬能力.用液閃記數γ-32P放射活性法,檢測不同吞噬時間點的PKC活性.以逆轉錄聚閤酶鏈反應(RT-PCR)法,檢測MERTK基因的mRNA水平變化情況.以PKC激活劑和拮抗劑處理hRPE細胞後,再行MERTK基因mRNA錶達水平檢測.採用SPSS 13.0統計學軟件進行數據分析,實驗組與對照組hRPE細胞吞噬ROS能力、PKC活性比較採用Student't檢驗,MERTK基因mRNA錶達水平比較採用單因素方差分析.結果 ROS孵育後的hRPE細胞結閤及吞入ROS的數量逐漸增多,至24 h時,hRPE細胞吞噬ROS的數量達到高峰,為(2.85±0.11)×106箇,與對照組(0.00±0.00)×106箇比較,差異有統計學意義(t=47.64,P＜0.05).ROS孵育後的hRPE細胞胞質PKC活性降低,至24 h時,PKC活性降至最低,細胞質的PKC活性為(151.13±17.67)nmol·g-1·min-1,細胞膜的PKC活性為(152.45±64.83)nmol·g-1·min-1;對照組細胞質的PKC活性為(329.63±14.26)nmol·g-1·min-1,細胞膜的PKC活性為(467.67±68.87)nmol·g-1·min-1;兩組間PKC活性比較,差異有統計學意義(細胞質:t=89.66,P＜0.05;細胞膜t=10.31,P＜0.05).在hRPE細胞與ROS不同時段的孵育過程中,MERTK基因mRNA均呈現齣高錶達狀態,孵育至90 min時,MERTK基因mRNA錶達灰度值為1.8853±0.0077,與對照組灰度值0.7246±0.0062相比,差異有統計學意義(F=16 060.2167,P＜0.05);孵育至24 h時,MERTK基因mRNA錶達灰度值為0.5946±0.0082,與對照組灰度值0.3343±0.0064比較,差異有統計學意義(F=919.8421,P＜0.05).上調hRPE細胞的PKC活性後,再行不同時段的ROS孵育,短時與長時孵育的MERTK基因mRNA錶達灰度值均低于對照組(短時孵育:F=17 142.2331,長時孵育:F=1886.4614;P＜0.05).拮抗hRPE細胞的PKC活性後,再行不同時段的ROS孵育,30 min內MERTK基因mRNA錶達灰度值為4.4670±0.0092至5.7034±0.0095範圍,均高于對照組灰度值0. 9117±0.0021(F=199 012.9138,P＜0.05).結論 PKC的低活性和MERTK基因mRNA的高錶達可促進hRPE細胞吞噬ROS的過程.PKC活性和MERTK基因mRNA錶達水平作為上遊調控信號,通過兩條不同的信號通路,以負相關的方式調節hRPE細胞吞噬ROS的功能.
목적 탐토인시망막색소상피(hRPE)세포탄서과정중MERTK기인mRNA표체수평여단백격매C(PKC)적상호작용.방법 대조실험연구.채용1×1010개/L시세포외절막반(ROS),우37℃하부육체외배양적정상hRPE세포,재부육적불동시간종지탄서반응.용쌍중형광표기법,검측hRPE세포적탄서능력.용액섬기수γ-32P방사활성법,검측불동탄서시간점적PKC활성.이역전록취합매련반응(RT-PCR)법,검측MERTK기인적mRNA수평변화정황.이PKC격활제화길항제처리hRPE세포후,재행MERTK기인mRNA표체수평검측.채용SPSS 13.0통계학연건진행수거분석,실험조여대조조hRPE세포탄서ROS능력、PKC활성비교채용Student't검험,MERTK기인mRNA표체수평비교채용단인소방차분석.결과 ROS부육후적hRPE세포결합급탄입ROS적수량축점증다,지24 h시,hRPE세포탄서ROS적수량체도고봉,위(2.85±0.11)×106개,여대조조(0.00±0.00)×106개비교,차이유통계학의의(t=47.64,P＜0.05).ROS부육후적hRPE세포포질PKC활성강저,지24 h시,PKC활성강지최저,세포질적PKC활성위(151.13±17.67)nmol·g-1·min-1,세포막적PKC활성위(152.45±64.83)nmol·g-1·min-1;대조조세포질적PKC활성위(329.63±14.26)nmol·g-1·min-1,세포막적PKC활성위(467.67±68.87)nmol·g-1·min-1;량조간PKC활성비교,차이유통계학의의(세포질:t=89.66,P＜0.05;세포막t=10.31,P＜0.05).재hRPE세포여ROS불동시단적부육과정중,MERTK기인mRNA균정현출고표체상태,부육지90 min시,MERTK기인mRNA표체회도치위1.8853±0.0077,여대조조회도치0.7246±0.0062상비,차이유통계학의의(F=16 060.2167,P＜0.05);부육지24 h시,MERTK기인mRNA표체회도치위0.5946±0.0082,여대조조회도치0.3343±0.0064비교,차이유통계학의의(F=919.8421,P＜0.05).상조hRPE세포적PKC활성후,재행불동시단적ROS부육,단시여장시부육적MERTK기인mRNA표체회도치균저우대조조(단시부육:F=17 142.2331,장시부육:F=1886.4614;P＜0.05).길항hRPE세포적PKC활성후,재행불동시단적ROS부육,30 min내MERTK기인mRNA표체회도치위4.4670±0.0092지5.7034±0.0095범위,균고우대조조회도치0. 9117±0.0021(F=199 012.9138,P＜0.05).결론 PKC적저활성화MERTK기인mRNA적고표체가촉진hRPE세포탄서ROS적과정.PKC활성화MERTK기인mRNA표체수평작위상유조공신호,통과량조불동적신호통로,이부상관적방식조절hRPE세포탄서ROS적공능.
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.
