Cationic liposome-mediated bcl-xl gene transfection into human keratocytes.
The efficiency and safe range of Lipofectamine2000 (LF2000)/bcl-xl applied in human keratocytes, the optimal ratio of LF2000/bcl-xl and the bcl-xl gene expression in human keratocytes were investiaged. By using trypan-blue staining, the effects of LF2000 and bcl-xl on the survival rate of the cultured human keratocytes were measured respectively. By using semi-quantitative RT-PCR, the efficiency and the expression of LF2000-mediated bcl-xl transfection into keratocytes were examined. The results showed that the survival rate of human keratocytes had no signficant change in the presence of LF2000 (20 microg/ml) or bcl-xl (10 microg/ml) for 24 h. LF2000 could effectively mediate the transfection of exogenous gene bcl-xl into human keratocytes. The best transfection efficiency could be obtained when the ratio of bcl-xl/LF2000 was 1:8. One day after transfection, the positive cells for bcl-xl could be detectable, and the positive rate reached the peak-on the posttransfection day 3 (48.3%), then gradually decreased. Fifteen days after transfection, there were few positive cells. It was suggested that LF2000 could effectively transfer the exogenous gene bcl-xl into human keratocytes without obvious toxicity during a concentration range. LF2000/bcl-xl may be likely to play an important role in gene therapy of human keratocytes.
Liu
Lei
L
Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Li
Xinyu
X
Zhu
Xuefei
X
Li
Guigang
G
D002412:Cations / Q000008:administration & dosage; D003315:Cornea / Q000166:cytology; D015316:Genetic Therapy; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D008081:Liposomes; D014162:Transfection; D051020:bcl-X Protein / Q000096:biosynthesis / Q000235:genetics*
D002412:Cations; D008081:Liposomes; D051020:bcl-X Protein