摘要
目的探讨通过腺病毒,慢病毒,脂质体2000三种方法转染THP-1巨噬细胞的最佳方法。方法采用腺病毒,慢病毒,脂质体2000三种方法分别转染THP-1巨噬细胞,并用荧光显微镜观察细胞转染荧光表达情况,流式细胞仪检测THP-1细胞转染效率,台盼蓝染色检测细胞病死率。结果腺病毒对THP-1巨噬细胞转染效率可在对细胞损伤较小的情况下达较高水平,但可能对细胞免疫状态影响大。MOI=200时,转染效率较低,仅为(29.81±2.50)%,细胞病死率为(5.17±0.44)%;MOI=800,转染效率最高,EGFP阳性率可达(86.49±6.11)%,细胞病死率为(8.80±0.54)%;MOI升至1200后,EGFP阳性率为(77.88±5.77)%,细胞病死率上升明显(14.50±0.77)%(P<0.05);慢病毒转染效率高及细胞毒性较腺病毒低,但因其病毒产量低,实验消耗病毒量大,因而影响其应用。当MOI=10时,EGFP阳性率仅为(28.09±1.67)%,细胞病死率为(4.74±0.31)%;MOI=60时,转染率最高,EGFP阳性率为(91.96±1.66)%,细胞病死率升至(7.25±0.50)%,此时病死率才有统计学意义(P<0.05)。脂质体2000对THP-1巨噬细胞毒性较大,细胞死亡率高,转染效率极低,不适合转染。结论腺病毒与慢病毒转染THP-1巨噬细胞各存在一定优劣,在实验中需酌情选择,脂质体2000不适宜转染THP-1巨噬细胞。
Objective Three methods were used for transfection of THP-1 macrophage,which included adenovirus,lentivirus and liposome 2000. To research the optimal method for the transfection of THP-1 macrophage. Methods Adenovirus,lentivirus and liposome 2000 were used for transfection of THP-1macrophage, fluorescence expression conditions of cell transfection were observed with a fluorescence microscope,the transfection efficiency of THP-1 cell was detected with a flow cytometry,and cell mortality rate was detected by trypan blue staining method. Results Adenovirus exhibited high transfection efficiency of THP-1 macrophage,and low cellular damage. But it may have strong effects on cellular immune conditions.MOI = 200,transfection efficiency is lower（ 29. 81 ± 2. 50） %,cell mortality is（ 5. 17 ± 0. 44） %. MOI = 800,transfection efficiency with the highest rate,EGFP positive rate is（ 86. 49 ± 6. 11） %,cell mortality is（ 8. 80 ±0. 54） %. When MOI = 1200,EGFP positive rate is（ 77. 88 ± 5. 77） %,cell mortality increased significantly（ 14. 50 ± 0. 77） %（ P 0. 05）. Lentivirus with higher transfection efficiency and lower cellular damage than adenovirus,but low output and high consumption make it can＇ t be applied widely. When MOI = 10,EGFP positive rate is（ 28. 09 ± 1. 67） %,cell mortality is（ 4. 74 ± 0. 31） %. MOI = 60,transfection efficiency with the highest rate,EGFP positive rate is（ 91. 96 ± 1. 66） %,cell mortality is（ 7. 25 ± 0. 50） %,P 0. 05. Lipid some 2000 had strong cytotoxic effects on THP-1 macrophage,cell mortality rate was high,and transfection efficiency was low,so the method is not suitable for transfection. Conclusions Adenovirus and lentivirus have their special advantages and disadvantages for transfection of THP-1 macrophage,selection of transfection method should according to special conditions of researches,generally,liposome 2000 is not suitable for transfection of THP-1 macrophage.
出处
《中华肺部疾病杂志（电子版）》
CAS
2015年第1期7-11,共5页
Chinese Journal of Lung Diseases(Electronic Edition)
基金
国家自然科学基金(81270064)
