CAJ | 학술논문

目的构建携带绿色荧光蛋白(GFP)和靶向干扰早期生长反应基因1(Egr-1)短发夹RNA(shRNA)共表达的慢病毒载体,转染小鼠视网膜组织,观察其对Egr-1基因的干扰效率.方法针对已经筛选确定的Egr-1基因shRNA有效靶序列,构建携带GFP和靶向干扰Egr-1 shRNA的慢病毒载体LV-shRNA(Egr-1).15日龄C57BL/6小鼠完全随机法分为实验组和阴性对照组,每组10只.LVshRNA(Egr-1)慢病毒载体经玻璃体腔注射转染至实验组小鼠右眼内,不针对任何特异基因的LV-NC 慢病毒载体通过同样的转染途径转染至阴性对照组小鼠右眼内,实验组及阴性对照组小鼠左眼不做任何处理设为空白对照组.2周后荧光显微镜下观察转染情况,实时定量PCR(RQ-PCR)、免疫印迹(Western blot)、免疫荧光检测Egr-1的表达,观察Egr-1基因的干扰效率.结果慢病毒载体经玻璃体腔注射途径转染小鼠视网膜后,GFP广泛分布于视网膜全层,包括视网膜色素上皮层.与空白对照组和阴性对照组注射眼比较,RQ-PCR检测显示实验组注射眼Egr-1 mRNA表达明显下调(0.290+0.074比1.006±0.033、1.010±0.086,均P＜0.001),抑制率为71.29%;免疫印迹显示实验组注射眼内Egr-1蛋白表达明显下调(0.224±0.035比0.674±0.050、0.688±0.049,P＜0.001),抑制率为67.44%.免疫荧光检测发现实验组注射眼在视网膜除内核层有少许Egr-1阳性细胞分布外,没有荧光表达.结论成功构建携带绿色荧光蛋白和靶向干扰Egr-1基因shRNA的慢病毒载体,经玻璃体腔注射转染小鼠眼内其转染效率高,分布范围广,且对小鼠视网膜Egr-1基因抑制效率高.
목적 구건휴대록색형광단백(GFP)화파향간우조기생장반응기인1(Egr-1)단발협RNA(shRNA)공표체적만병독재체,전염소서시망막조직,관찰기대Egr-1기인적간우효솔.방법 침대이경사선학정적Egr-1기인shRNA유효파서렬,구건휴대GFP화파향간우Egr-1 shRNA적만병독재체LV-shRNA(Egr-1).15일령C57BL/6소서완전수궤법분위실험조화음성대조조,매조10지.LVshRNA(Egr-1)만병독재체경파리체강주사전염지실험조소서우안내,불침대임하특이기인적LV-NC 만병독재체통과동양적전염도경전염지음성대조조소서우안내,실험조급음성대조조소서좌안불주임하처리설위공백대조조.2주후형광현미경하관찰전염정황,실시정량PCR(RQ-PCR)、면역인적(Western blot)、면역형광검측Egr-1적표체,관찰Egr-1기인적간우효솔.결과 만병독재체경파리체강주사도경전염소서시망막후,GFP엄범분포우시망막전층,포괄시망막색소상피층.여공백대조조화음성대조조주사안비교,RQ-PCR검측현시실험조주사안Egr-1 mRNA표체명현하조(0.290+0.074비1.006±0.033、1.010±0.086,균P＜0.001),억제솔위71.29%;면역인적현시실험조주사안내Egr-1단백표체명현하조(0.224±0.035비0.674±0.050、0.688±0.049,P＜0.001),억제솔위67.44%.면역형광검측발현실험조주사안재시망막제내핵층유소허Egr-1양성세포분포외,몰유형광표체.결론 성공구건휴대록색형광단백화파향간우Egr-1기인shRNA적만병독재체,경파리체강주사전염소서안내기전염효솔고,분포범위엄,차대소서시망막Egr-1기인억제효솔고.
Objective To construct the lentivirus vector containing green fluorescent protein (GFP)and early growth response factor-1 short hairpin RNA(Egr-1 shRNA),and to determine the efficiency of interference on Egr-1 gene in lentivirus vector-transfected mouse retinal tissue.Methods Based on the target sequences of Egr-1 shRNA screened and identified previously,the LV-shRNA(Egr-1)containing GFP and Egr-1 shRNA Was constructed.Twenty 15-day-old C57BL/6 mice were randomly divided into experimental group and negative control group(n=10 each).LV-shRNA(Egr-1)lentivirus vector Was transfected into the right eyes in the experimental group with intravitreal injection.LV-NC lentivirus vector that did not target at any specific gene Was also transfected into the right eyes in the negative control group,while the left eyes were untreated in both two groups as the blank control group.After 2 weeks,the transfection of lentiviral vector was examined under fluorescence microscope.Moreover,real-time quantitative polymerase chain reaction (RQ-PCR), Western blot and immunofluorescence were used to detect the expression of Egr-1 gene for evaluation of the interference efficiency. Results After lentiviral vector was intravitreally injected and transfected into the mouse retinal tissue, GFP was found to be widely distributed in the whole layer of the retina including the retinal pigment epithelium. RQPCR showed significantly down-regulated expression of Egr-1 mRNA in the injected eyes of experimental group compared with blank or negative controls (0.290±0.074 vs 1.006±0.033, 1.010± 0.086, all P＜0.001) , with the inhibition rate being 71.29%. Western blot demonstrated that the expression of Egr-1 protein was remarkably down-regulated in the injected eyes of experimental group (0.224±0.035 vs 0.674±0.050, 0.688±0.049, all P＜0.00l) , with the inhibition rate being 67.44%. Furthermore, immunofluorescence revealed no expression of fluorescence protein, except sparse distribution of Egr-1 positive cells in the inner nuclear layer of injected eyes of the experimental group. Conclusions The lentivirus vector containing GFP and Egr-1 shRNA is successfully constructed, and it may have high transfection efficiency and a wide distribution in the intravitreally injected mice eyes. Furthermore, the lentivirus vector has a high inhibition efficiency of Egr-1 gene in the mouse retina.