CAJ | 학술논문

目的研究慢病毒载体介导绿色荧光蛋白（GFP）转染犬脂肪干细胞（ADSCs）的理想条件，检测转染后细胞的生物学特性及分化能力。方法酶消化法获取ADSCs ，流式细胞技术检测细胞表面抗原，慢病毒载体介导GFP以50、100、150和200的感染复数（ MOI）作用24 h转染ADSCs，荧光显微镜及流式细胞检测荧光强度和转染效率，CCK-8法检测转染对ADSCs生长和增殖的影响，将转染ADSCs经成肌诱导后，免疫荧光检测成肌细胞特异性抗原结蛋白（desmin）和α-平滑肌肌动蛋白（α-SMA）表达情况。结果流式细胞仪检测结果CD90、CD44、CD105表达均为阳性，CD34、CD45表达均为阴性，MOI为50、100、150和200，转染效率分别为75.88％、90.99％、97.42％和99.17％。 MOI为150时，转染对ADSCs增殖无明显影响。病毒转染ADSCs经成肌诱导分化后，表达desmin和α-SMA阳性。结论慢病毒载体介导的GFP能高效标记ADSCs，且标记后细胞的增殖分化能力无明显影响。
목적연구만병독재체개도록색형광단백（GFP）전염견지방간세포（ADSCs）적이상조건，검측전염후세포적생물학특성급분화능력。방법매소화법획취ADSCs ，류식세포기술검측세포표면항원，만병독재체개도GFP이50、100、150화200적감염복수（ MOI）작용24 h전염ADSCs，형광현미경급류식세포검측형광강도화전염효솔，CCK-8법검측전염대ADSCs생장화증식적영향，장전염ADSCs경성기유도후，면역형광검측성기세포특이성항원결단백（desmin）화α-평활기기동단백（α-SMA）표체정황。결과류식세포의검측결과CD90、CD44、CD105표체균위양성，CD34、CD45표체균위음성，MOI위50、100、150화200，전염효솔분별위75.88％、90.99％、97.42％화99.17％。 MOI위150시，전염대ADSCs증식무명현영향。병독전염ADSCs경성기유도분화후，표체desmin화α-SMA양성。결론만병독재체개도적GFP능고효표기ADSCs，차표기후세포적증식분화능력무명현영향。
Objective To study the optimal condition to transfect green fluorescent protein gene ( GFP) to canine adipose-derived stem cells ( ADSCs ) by lentiviral vectors and to determine the biological properties and differentiation potency of transfected ADSCs .Methods ADSCs were harvested by collagen enzyme and flow cytometric analysis was carried out to detect the immunophenotypes of ADSCs ,ADSCs were transfected by lentiviral vector carrying GFP for 24 h at different multiplicity of infection (MOI=50,100,150,and 200).The fluorescence intensity and transfection efficiency were analyzed by fluorescent microscopy and flow cytometric analysis .The proliferation and growth of transfected ADSCs were detected by CCK-8 assay.After the transfected ADSCs were induced into myoblasts ,Immunofluorescence was utilized to detect the expression of myoblast specific antigens desmin and α-SMA.Results Flow cytometry demonstrated that the ADSCs expressed CD 90,CD44 and CD105,but not CD34 or CD45.The efficiency of transfection at MOI 50,100,150 and 200 was 75.88%,90.99%,97.42% and 99.17%,respectively .Compared with ADSCs without transfection , the transfection had no significant effect on the proliferation of ADSCs transfected at MOI 150 .The transfected ADSCs expressed desmin and α-SMA after differentiating into myoblasts .Conclusions The transfected ADSCs by lentiviral vector expressed GFP with much higher efficiency and hardly affected the ability of proliferation and differentiation .