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

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) % ,两组比较差异有显著意

Inhibition of lens epithelial cell cycle and PCNA mRNA expression by an antisense oligonucleotides complementary to the messenger RNA of proliferating cell nuclear antigen

OBJECTIVE: To determine whether antisense oligonucleotides complementary 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 PCNA protein. METHODS: BLECs were cultured in vitro, and the second passage cells were used in this experiment. PCNA ASODN (30 micro mol/L), PCNA SODN (sense oligonucleotides, 30 micro mol/L), basic fibroblast growth factor (bFGF, 10 micro g/L), bFGF (10 ng/ml) + ASODN (30 micro mol/L), bFGF (10 micro g/L) + SODN (30 micro mol/L) were introduced respectively into the medium, and the same amount of PBS was added into the medium as a control. After 24 hours, the cell cycle and the PCNA expression were counted by flow cytometry, and the expression of PCNA mRNA was indicated by Northern ELISA hybridization. RESULTS: PCNA ASODN could decrease the rate of the cells of S phase and down-regulate the expression of PCNA mRNA and PCNA protein. Comparing with the control 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 of PCNA protein from 55.27% to 12.32%. PCNA ASODN could also inhibit the proliferation 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 PCNA ASODN decreases expression of PCNA mRNA and PCNA protein, and stops the cell to enter and progress through S phase. These results provide an important impetus to initiate in vivo studies to determine the feasibility of antisense strategies in the prevention of posterior capsular opacification.