摘要
目的通过RNA干扰的方法沉默缝隙连接蛋白43(connexin43,cx43),观察cx43对人视网膜色素上皮(retinal pigment epithelium,RPE)细胞活动的影响。方法培养原代的人RPE细胞,以不同浓度转染针对人cx43基因的小干扰RNA(small interfering RNA,siRNA)特异片段,通过流式细胞仪观察其对细胞周期的影响;通过扫描电镜观察其对细胞形态的影响;采用激光共聚焦和荧光淬灭恢复技术观察荧光恢复速率平均百分率,评价其对细胞间通讯功能的影响;通过制作RPE细胞损伤模型,观察其对损伤修复能力的作用。结果培养24h和48h,流式细胞仪检测S期RPE细胞比率,对照组分别为:(8.85±1.33)%和(15.52±2.60)%;100nmol.L-1、200nmol.L-1、300nmol.L-1siRNA1转染RPE细胞后,24h、48hS期细胞比率分别为:(10.64±2.50)%、(20.98±5.78)%,(19.42±2.88)%、(32.67±4.31)%,(20.31±4.03)%、(27.59±6.95)%,S期细胞比率明显增多,与对照组相比差异均有统计学意义(P均<0.05)。细胞的扫描电镜显示转染siRNA后,细胞表型发生变化,细胞体积增大,表面触手增多。cx43基因被干扰后,细胞荧光恢复速率降低,细胞间通讯功能明显降低;细胞损伤修复能力下降。结论cx43正常表达不仅影响细胞间通讯功能,且对细胞的生长周期、损伤修复多种细胞行为也起着重要作用。
Objective To investigate the effects of connexin 43 （ cx43 ） on cell activity of retinal pigment epithelial（RPE） cells by silencing cx43 through RNA interference. Methods Primary human RPE cells were cultured, and took transfection with different concentrations for specific parts of small interfering RNA （siRNA） in human cx43 gene. The effects of cx43 on cell cycle were observed by flow cytometer, changes of cell morphology were observed by scanning electron microscope, and the mean percentage of fluorescence recovery rate was detected by laser confocal microscopy and fluorescence redistribution after photobleaching technique. The effects of cx43 on cellcell communication were also evaluated,and the effects of cx43 on recovery ability from injury were observed through establishing injury models of RPE cells. Results After culturing for 24 hours and 48 hours, RPE cells ratios at S phase were （8.85 ±1.33） % and （ 15.52 ± 2. 60）% in control group by flow cytometer, respectively, and were （ 10.54 ± 2.50）% and （20.98 ±5.78）% with 100 nmol · L^-1 siRNA for the transfection of RPE ceils, （ 19.42 ± 2.88 ） % and （ 32.67 ± 4.31 ） % with 200 nmol · L^-1 siRNA, and （ 20.31 ± 4.03）% and （27.59±6.95）% with 300 nmol · L^-1 siRNA,respectively. The cell ratios significantly increased at S phase, and there was significant difference compared with control group （ all P 〈 0.05 ）. After the transfection of siRNA, scanning electron microscope showed that cell phenotype changed, cell volume enlarged and tentacles at surface increased. After cx43 interfered, recovery rate of cell fluorescence decreased, cellcell communication significantly lowered, and the cell recovery ability from injury descended. Conclusion The normal expression of cx43 not only influences the cell-cell communication ,but also plays an important role in many cell activities such as cell cycle and recoverv abilitv from iniury.
出处
《眼科新进展》
CAS
北大核心
2009年第9期677-680,共4页
Recent Advances in Ophthalmology
基金
北京市自然科学基金资助(编号:7032046)~~
作者简介
司艳芳，女，1976年9月出生，河南许昌人，博士，主治医师。主要从事视网膜眼底病变和眼底激光的临床工作。联系电话：010-66775081（0）；E—mail：siyanfang309@yahoo．com．cn
通讯作者：王凤翔，E-mail：wangfxiang@263．net
