An Comparison Investigation Of Gene Transfer Into The Isolated Rabbit Corneal Stoma Cells In Vitro Using Adenovirus And Lentivirus Vector

Objective:We use enhanced green fluorescent proteins (EGFP) as a reporting gene to observe the transfection efficiency of Adenovirus vector mediated EGFP (AV-EGFP) and Lentivirus vector mediated EGFP (LV-EGFP) in the isolated rabbit corneal stroma cells in vitro. We want to study the feasibility of the better virus vector as one of the gene therapy vector for corneal disease ,then make preparations for the following experiments about using the better virus vector mediated objective gene transduction and therapy corneal haze。Methods: The primary cultured and subcultured keratocytes from NewZealand white rabbits and cells identification;The cells were exposed to different MOI of LV-EGFP and AV-EGFP in transfective groups and the same quality of blank culture medium in contrast group. We observed the expression of EGFP by inverted fluorescence microscope at different periods and then calculated the transfection efficiency through FCM;To detect the expression of EGFP mRNA by RT-PCR in transfective groups and contrast group;The ultrastructure of transfectived cell was observed with transmission electron microscope;We observed the influence of the cell activity by MTT assay after the two viruses transfected the cells.Results: 1. AV-EGFP transfected stromal cells 24-48 hours from the beginning of the green fluorescence can be seen clearly,earlier than LV-EGFP. When the MOI was 1000, the transfection efficiency of LV-EGFP was above 61% and AV-EGFP was 40% three days after. At the same MOI, the transfection efficiency of LV-EGFP was specially higher than AV-EGFP and the difference was statistically significant(p<0.05). 2.The two transfection groups were the mRNA expression of EGFP and the contrast group was not. Compared with LV-EGFP transfection group, AV-EGFP transfected group reduced the expression of EGFP mRNA, and the difference was statistically significant(p<0.05). 3.The result showed that when MOI was 1000 in LV-EGFP transfection group and when MOI was 104 in AV-EGFP transfection group, apoptosis of cell was detected by transmission electron microscope. when MOI was 500, The ultrastructure of LV-EGFP transfective cell was nonsignificant abnormalities. 4.When the MOI was 1 to 500 in LV-EGFP transfection group and MOI was 1 to 1000 in AV-EGFP transfection group, the influence of cell activity, the transfection group compared with the control group, there was no significant difference. When the MOI was more than 1000 in LV-EGFP and 104 in AV-EGFP, the cell activity compared with the contrast group, the difference was statistically significant (p<0.05). Lentivirus vector inhibited the cell activity, causing cell apoptosis. When the MOI was 104 , the cell activity in LV-EGFP transfection group compared with the AV-EGFP transfection group, the difference was no significant difference (p>0.05). It show that cell survival rate was all droped in LV-EGFP and AV-EGFP transfection groups.Conclusions: 1. All show that the Adenovirus and the Lentivirus vector can be efficient in vitro transfecting rabbit corneal stromal cells,and Lentivirus vector transduction efficiency was higher than Adenovirus; 2. The optimal MOI of Lentivirus vector was 500 and the infection efficiency was up to 50% three days after transfection. The optimal MOI of Adenovirus vector was 1000 and the infection efficiency was up to 40%. 3. Security concentration range of Lentivirus vector transfecting rabbit corneal stromal cells was no more than 500 and Adenovirus vector was no more than 1000.