摘要
目的探讨不同加样方法对逆转录病毒转染软骨细胞效率的影响。方法将构建并包装好的逆转录病毒重组基因PLNCX2-IL-1Ra—GFP分别采用以下加样转染方法进行转染并作对照研究，a）第1组：先加病毒上清，6h后补充培养液．b）第2组：先加病毒上清，隔夜补充培养液；c）第3组：先加病毒上清，6h后更换成培养液，隔夜培养后弃液，重复先加病毒上清，6h后更换成培养液；d）第4组：先加病毒上清，6h后补充培养液，隔夜培养后弃液，重复先加病毒上清，6h后补充培养液；e）第5组：经典转染法，同时加病毒上清液和培养液。转染2d后免疫荧光显微镜下观察荧光显色，4周稳定转染后分别检测各组细胞培养液中NO含量，并用ELISA法检测上清液中hIL-1Ra的表达情况。结果酶切鉴定重组基因正确；免疫荧光显微镜观察可见绿色荧光显色；1～5组NO含量以第4组为最高，第5组为最低；ELISA检测1～5组培养液中hIL-1Ra的含量以第4组为最高，第5组为最低。结论四种不同加样转染方法的转染效率均高于经典法，其中以第4组为最好。
Objective To study effect of retrovirus transfected efficience on articular chondrocytes with several adding-sample methods. Methods With following adding, methods,using constructed and packaged retrovirus carrier PLNCX2-IL-1Ra-GFP transfect articular chondrocytes ,and making control study. 1 ,adding virus supernatant ,culture medium was complemented after 6 hours. 2,adding virus supernatant firstly,complementing culture medium on the second day. 3, adding virus supernatant, replaceing the virus supernatant with culture medium after 6 hours,quiting medium after culturing overnight, and then adding virus supernatant, replaceing the virus supernatant with culture medium. 4,adding virus supernatant firstly,complementing culture medium after 6 hours ,quitting medium after culturing overnight,and then adding virus supernatant,culture medium was complemented after 6 hours. 5,the classical transfected method：adding virus supernatant and culture medium on the same time. To observe fluorescence color with immunofluorescence microscope after transfecting 2 days. Testing the NO content of each group＇s culture medium after transfecting stably about 4 weeks ,and detecting expression of hIL-1Ra in supemate by ELISA. Results Enzyme digestion identificated the recombination was correct. To observe green fluorescence with immunofluorescence microscope. Among the NO content from 1 to 5 group,the highest is 4 group and the lest is 5 group,There is statistical significance in comparison between any two means. Among the hIL-1Ra content from 1 to 5 group ,the highest is 4 group and the lest is 5 group. There is statistical significance in comparison between any two means. Conclusion Four adding methods＇transfecting efficiency was higher than classical method.
出处
《实用骨科杂志》
2008年第12期733-735,共3页
Journal of Practical Orthopaedics
基金
国家自然科学基金项目(30500515)
山西省自然科学基金项目(2006021045)
关键词
转染方法
逆转录病毒
转染效率
transfect methods
retrovirus
transfecting efficiency
