视网膜下液致视网膜色素上皮细胞增殖过程中蛋白激酶C活性的变化  

Changes of protein kinase C for the proliferation of retinal pigment epithelium cells induced by subretinal fluid

作　　者：孙兆义[1] 洪晶[1] 

机构地区：[1]中国医科大学附属第一医院眼科,沈阳110001

出　　处：《中华眼科杂志》2006年第8期738-743,共6页Chinese Journal of Ophthalmology

基　　金：国家自然科学基金资助项目(30471865)

摘　　要：目的探讨视网膜下液(SRF)能否引起体外培养的视网膜色素上皮(RPE)细胞增殖及在其增殖过程中是否出现蛋白激酶C(PKC)的激活和转位,进一步明确RPE细胞增殖与RPE细胞中PKC信号系统变化的关系及PKC抑制剂的作用。方法实验对象为体外培养的RPE细胞;刺激因素为提取的增生性玻璃体视网膜病变(PVR)为B、C级患者的SRF和来自角膜移植后的供体眼球所提供的正常玻璃体成分;PKC特异激活剂佛波酯(PMA)为阳性对照;DMEM培养液作为空白对照。用3H-胸腺嘧啶脱氧核苷(3H-TdR)掺入法测定各自的RPE细胞增殖情况。用B、C级SRF、正常玻璃体成分、PMA、DMEM培养液分别在不同的时间刺激RPE细胞,通过细胞裂解和离心获取细胞质和细胞膜蛋白粗提液,用同位素32P标记和液体闪烁计数法检测细胞质和细胞膜PKC活性水平。选用PKC特异抑制剂N,N-二甲基鞘氨醇预处理各组细胞后,再分别观察各组RPE细胞中PKC活性表达水平及增殖情况。结果用B、C级SRF和PMA处理过的RPE细胞出现高增殖;SRF和PMA都可以激活RPE细胞质中的PKC,并使其由胞质向胞膜转位,但SRF作用于RPE细胞时,胞膜上PKC活性峰值出现的时间较PMA明显延长。其中,B级SRF作用于RPE细胞时,胞膜上PKC活性峰值出现的时间较C级长且峰值低,增殖程度也低;正常玻璃体成分和DMEM培养液组未出现PKC活性变化和高增殖。用PKC特异抑制剂预处理各组细胞后,未出现PKC活性和细胞增殖的改变,组间比较差异无统计学意义(P>0．05)。结论SRF可促进RPE细胞增殖,RPE细胞中的PKC是以激活和转位方式参与细胞增殖的过程;使用PKC特异抑制剂可阻止此过程发生。Objective To study the effects of subretinal fluid （SRF） on the proliferation of retinal pigment epithelium （RPE） cells and on the activation and translocation of protein kinase C （PKC）; to investigate the relationship between the proliferation and the changes of PKC in the RPE cells; and to study the effect of PKC inhibitor. Methods RPE cells were harvested to measure the PKC activity in cytoplasm and cellular membrane after being treated with subretinal fluid （SRF） obtained from PVR patients with different degrees （B grade and C）, PKC specific activator PMA （positive control） or normal vitreous. RPE cells cultured with DMEM culture medium only were used as the negative controls. PKC activity in cytoplasm and cellular membrane was measured with radioactive isotope ^32P label method. For further study, the PKC specific inhibitor N, N-dimethyl was used to pretreat the RPE cells before the administration of SRF, PMA or normal vitreous, and then the activity of PKC was observed and recorded. ^3 H-TdR was used to measure the proliferation of RPE cells with or without the activation and translocation. Results SRF and PMA could promote the proliferation of RPE cells and activate PKC in the cytoplasm of RPE cells, and then promoted the PKC translocated from the cytoplasm to cellular membrane. The peak of PRC on the cell membrane appeared later in cells treated with SRF when compared with those treated with PMA. The appearance of peak of PKC in cells treated with SRF from grade B PVR was later than those treated with SRF from grade C PVR, the stimulating effect on the proliferation of RPE cells by the SRF B was also less than those from the SRF C. No activation of PKC and increased proliferation were observed in RPE cells treated with normal vitreous or DMEM culture medium. Pre-treatment with PKC inhibitor could block the PKC-activating effects and proliferation-stimulating effects of SRF and PMA on the RPE cells, therefore no significant difference could be detected between different groups. Conc

关 键 词：视网膜 蛋白激酶C 色素上皮 眼 细胞分裂 

分 类 号：R774.1[医药卫生—眼科] R774.12[医药卫生—临床医学]