CAJ | 학술논문

目的观察重组腺相关病毒(rAAV)-多聚嘧啶序列结合蛋白相关剪接因子(PSF)对氧诱导视网膜新生血管的抑制作用.方法 7日龄C57BL/6J小鼠18只,随机分为正常组、rAAV-PSF注射组、rAAV注射组,各组均6只.蛋白免疫印迹法(Western blot)检测各组小鼠视网膜组织中PSF的表达.7日龄C57BL/6J小鼠48只随机分为正常组、氧诱导视网膜病变(OIR)模型组、OIR rAAV-PSF治疗组、OIR rAAV治疗组,各组均为12只.除正常组外,其余各组小鼠与哺乳母鼠共同置于氧浓度为(75±2)％的氧箱内饲养5d后转移至正常环境中饲养5d,建立OIR模型.12日龄时,OIR rAAV-PSF治疗组小鼠玻璃体腔注射病毒滴度为5×1013pfu/ml的rAAV-PSF重组载体2μI; OIR rAAV治疗组小鼠玻璃体腔注射相同病毒滴度的单纯rAAV载体2μl.OIR模型组小鼠出氧箱后不作任何处理.石蜡切片苏木精-伊红染色并计数突破视网膜内界膜的血管内皮细胞核;Western blot检测视网膜中VEGF蛋白表达含量.结果正常组小鼠视网膜PSF蛋白表达与rAAV-PSF注射组比较,差异有统计学意义(F=16.05,P=0.001),与rAAV注射组比较,差异无统计学意义(F=16.05,P=0.890).正常组、OIR模型组、OIR rAAV-PSF治疗组、OIR rAAV治疗组突破内界膜的视网膜新生血管内皮细胞核数比较,正常组与OIR模型组差异有统计学意义(F=101.00,P=0.007);rAAV-PSF治疗组与OIR模型组差异有统计学意义(F=101.00,P=0.002);OIR rAAV治疗组与OIR模型组差异无统计学意义(F=101.00,P=0.550).各组视网膜VEGF蛋白表达比较,正常组与OIR模型组差异有统计学意义(F=13.20,P=0.005),OIR模型组与OIR rAAV-PSF治疗组差异有统计学意义(F=13.20,P=0.001);OIR模型组与OIR rAAV组差异无统计学意义(F=13.20,P=0.071).结论 rAAV-PSF玻璃体腔注射可以抑制OIR小鼠视网膜VEGF表达,从而抑制其新生血管生成.
목적 관찰중조선상관병독(rAAV)-다취밀정서렬결합단백상관전접인자(PSF)대양유도시망막신생혈관적억제작용.방법 7일령C57BL/6J소서18지,수궤분위정상조、rAAV-PSF주사조、rAAV주사조,각조균6지.단백면역인적법(Western blot)검측각조소서시망막조직중PSF적표체.7일령C57BL/6J소서48지수궤분위정상조、양유도시망막병변(OIR)모형조、OIR rAAV-PSF치료조、OIR rAAV치료조,각조균위12지.제정상조외,기여각조소서여포유모서공동치우양농도위(75±2)％적양상내사양5d후전이지정상배경중사양5d,건립OIR모형.12일령시,OIR rAAV-PSF치료조소서파리체강주사병독적도위5×1013pfu/ml적rAAV-PSF중조재체2μI; OIR rAAV치료조소서파리체강주사상동병독적도적단순rAAV재체2μl.OIR모형조소서출양상후불작임하처리.석사절편소목정-이홍염색병계수돌파시망막내계막적혈관내피세포핵;Western blot검측시망막중VEGF단백표체함량.결과 정상조소서시망막PSF단백표체여rAAV-PSF주사조비교,차이유통계학의의(F=16.05,P=0.001),여rAAV주사조비교,차이무통계학의의(F=16.05,P=0.890).정상조、OIR모형조、OIR rAAV-PSF치료조、OIR rAAV치료조돌파내계막적시망막신생혈관내피세포핵수비교,정상조여OIR모형조차이유통계학의의(F=101.00,P=0.007);rAAV-PSF치료조여OIR모형조차이유통계학의의(F=101.00,P=0.002);OIR rAAV치료조여OIR모형조차이무통계학의의(F=101.00,P=0.550).각조시망막VEGF단백표체비교,정상조여OIR모형조차이유통계학의의(F=13.20,P=0.005),OIR모형조여OIR rAAV-PSF치료조차이유통계학의의(F=13.20,P=0.001);OIR모형조여OIR rAAV조차이무통계학의의(F=13.20,P=0.071).결론 rAAV-PSF파리체강주사가이억제OIR소서시망막VEGF표체,종이억제기신생혈관생성.
Objective To observe the inhibitory effect of intraocular injection of recombinant adenoassociated virus-polypyrimidine tract-binding protein-associated splicing factor (rAAV-PSF) in oxygen induced retinopathy mice model.Methods Eighteen C57BL/6J mice were divided into 3 groups randomly,including normal group,rAAV-PSF injection group,rAAV injection group.Western blot analysis was applied to detect the transfection expression level of PSF.The other 48 C57BL/6J mice were randomly divided into 4 groups:normal group,ischemia-induced retinopathy (OIR) group,rAAV-PSF treated OIR group,rAAV treated OIR group,12 mice in each group.Placed all mice (excepted the mice in control group) in cages of (75±2) ％ oxygen concentration environment for 5 days then moved to a normal environment for 5 days to induced the OIR model.At the 12th day the OIR rAAV-PSF treated OIR group was intravitreal injected with 2 μl 5× 1013 pfu/ml rAAV-PSF and the rAAV treated OIR group was treated with intravitreal injection of 2 μl 5 × 1013 pfu/ml rAAV.The mice in OIR group were left intact after moved out of oxygen cages.Five days after injection,the eyeballs were harvested and retinal sections were stained to count the nuclear of retinal endothelium cells.Western blot analysis was applied to detectthe protein level of vascular endothelial growth factor (VEGF) in retina.Results There was a significant difference of the expression PSF between normal group and rAAV-PSF treated group (F=16.05,P=0.001).There was no significant difference of the PSF expression between normal group and rAAV treated group(F=16.05,P=0.890).There was a significant difference of the number of retinal endothelium cells nuclear between normal group and OIR group (F=101.00,P=0.007).There is a significant difference of the number of retinal endothelium cells nuclear between rAAV-PSF treated group and OIR group (F=101.00,P=0v002).There was no significant difference of the number of retinal endothelium cells nuclear between OIR rAAV treated group and OIR group (F=101.00,P=0.550).There was a significant difference of VEGF protein expression between normal group and OIR group (F=13.20,P =0.005),OIR group and rAAV treated OIR group (F=13.20,P=0.001).There was no difference of VEGF protein expression between OIR rAAV treated group and OIR group (F=13.20,P=0.071).Conclusion The rAAV-PSF vitreous injection can inhibit the expression of VEGF in OIR mice,hence to restrain the proliferation of neovascularization.