增殖细胞核抗原反义寡核苷酸对牛晶状体上皮细胞增殖的影响  

Inhibition of lens epithelial cell cycle and PCNA mRNA expression by an antisens e oligonucleotides complementary to the messenger RNA of proliferating cell nucl ear antigen

作　　者：翁景宁[1] 张惠蓉[2] 

机构地区：[1]福建医科大学附属协和医院眼科,福州350001 [2]北京大学第三医院眼科

出　　处：《中华眼科杂志》2003年第2期98-101,共4页Chinese Journal of Ophthalmology

基　　金：福建省自然科学基金资助项目 (C9810 0 33)

摘　　要：目的　探讨增殖细胞核抗原 (proliferatingcellnuclearantigen ,PCNA)反义寡核苷酸对牛晶状体上皮细胞增殖的影响。方法　取体外培养的第 2代牛晶状体上皮细胞用于实验。A～F组分别加入PBS、30 μmol/LPCNA反义寡核苷酸、30 μmol/LPCNA正义寡核苷酸、10 μg/L碱性成纤维细胞生长因子 (basicfibroblastgrowthfactor,bFGF)、10 μg/LbFGF及 30 μmol/L反义寡核苷酸、10 μg/LbFGF及30 μmol/L正义寡核苷酸 ;常规培养 2 4h后 ,使用流式细胞仪检测细胞周期的变化和PCNA蛋白的含量。提取细胞mRNA ,采用地高辛标记PCNA探针和Northen酶联吸附免疫杂交法 ,在酶标仪上测定吸光度 (A值 )以表达PCNAmRNA含量。结果　牛晶状体上皮细胞S期的细胞数量占细胞总数的百分比A组为 ( 15 7± 1 8) % ,B组为 ( 7 9± 0 4) % ,两组比较差异有非常显著意义 (P <0 0 1) ;PCNAmRNA含量A组A值为 0 2 0 6± 0 0 0 4,B组A值为 0 173± 0 0 14,两组比较差异有显著意义 (P<0 0 5 ) ;PCNA蛋白表达率A组为 ( 5 5 2 7± 2 6 4) % ,B组为 ( 12 32± 1 82 ) % ,两组比较差异有非常显著意义 (P <0 0 1)。牛晶状体上皮细胞S期的细胞数量占细胞总数的百分比D组为 ( 2 3 4± 2 8) % ,E组为( 19 9± 2 3) % 。Objective To determine whether antisense oligonucleotides compl ementary to the messenger RNA of proliferating cell nuclear antigen (PCNA ASODN) inhibit the proliferation of bovine lens epithelial cell (BLEC) by changing the cell cycle and down-regulating the expression of PCNA mRNA and PC NA protein. WT5'HZ Methods BLECs were cultured in vitro, and the second passage cells were used in this experiment. PCNA ASODN (30 μmol/L), PCNA SODN (sense oligonucleoi des, 30 μmol/L), basic fibroblast growth factor (bFGF, 10 μg/L), bFGF (10 ng/ml) + ASODN (30 μmol/L), bFGF (10 μg/L) + SODN (30 μmol/L) were introduced respectivel y into t he medium, and the same amount of PBS was added into the medium as a control. Af ter 24 hours, the cell cycle and the PCNA expression were counted by flow cytome try, and the expression of PCNA mRNA was indicated by Northern ELISA hybridizati on. WT5'HZ Results PCNA ASODN could decrease the rate of the cells of S phase and dow n-regulate the expression of PCNA mRNA and PCNA protein. Comparing with the con t rol group, after 24 hours, the rate of cells of S phase was decreased from 15.67 % to 7.96%, the expression of PCNA mRNA from 0.266 to 0.176 and the expression o f PCNA protein from 55.27% to 12.32%. PCNA ASODN could also inhibit the prolifer ation of BLEC induced by bFGF, comparing with the bFGF group, the rate of cells of S phase was decreased from 23.4% to 19.9%, the expression of PCNA mRNA from 0 .576 to 0.357 and the expression of PCNA protein from 76.4% to 35 .48%. Conclusions Our results demonstrate that the PCNAASODN decreases expression of PCNA mRN A and PCNA protein, and stops the cell to enter and progress through S phase. Th ese results provide an important impetus to initiate in vivo studies to determin e the feasibility of antisense strategies in the prevention of posterior capsula r opacification.PCN

关 键 词：增殖细胞核抗原 反义寡核苷酸类 上皮细胞 细胞分裂 晶体 白内障 

分 类 号：R779.66[医药卫生—眼科]