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| The study of human β-NGF gene transferred to cat corneal endothelial cells |
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| 作者：罗文娟 文章来源：青岛大学医学院附属医院,眼科 点击数228 更新时间：2012/9/13 文章录入：毛进 责任编辑：毛进 |
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Object To transfect the cat corneal endothelial cells(CEC) with recombinant humanβ-nervegrowth factor gene adeno-associated virus(AAV-β-NGF) and to observe the effect of the expressedβ-NGFprotein on the proliferation activity of cat CEC.
Methods The endothelium of cat cornea was torn under the microscope and rapidly cultivated in DMEM medium to form single layer corneal endothelial cells and the passage 2 endothelial cells were used in this experiment. The recombinant humanAAV-β-NGFwas constructed. The recombinant human AAV-β-NGFwas transferred into cat CEC directly. Three groups were as following: normal CEC control group, CEC-AAV control group and recombinant CEC-AAV-β-NGFgroup. 48 hours after transfection, the total RNA was extracted from the CEC by Trizol. The expression of theβ-nervegrowth factor(β-NGF) target gene detected by fluorescence quantitative polymerase chain reaction; proliferation activity of the transfected CEC detected at 48 hours by MTT assay; the percentage of G1 cells among CEC after transfect was detected by FCM; cell morphology was observed under inverted phase contrast microscope.
Results The torn endothelium culture technique could rapidly cultivate single layer cat corneal endothelial cells. The self-designed primers for the target gene and reference gene were efficient and special confirmed through electrophoresis analysis and DNA sequencing. 48 hours after transfect, the humanβ-NGFgene mRNA detected by fluorescence quantitative polymerase chain reaction showed: there was no significant difference between normal CEC control group and CEC-AAV control group (P > 0.05); there was significant difference between two control groups and recombinant CEC-AAV-β-NGFgroup (P <0.05). MTT assay showed: transfect of recombinant AAV-β-NGFcould promote the proliferation activity of cat CEC, while there was no significant difference between normal CEC control group and CEC-AAV control group (P > 0.05). FCM result showed: the percentage of G1cells in CEC-AAV-NGFgroup was 76.8% while that in normal CEC control group and CEC-AAV control group was 46.6% and 49.8%.
Conclusion The recombinant AAV-β-NGFcan express bioactiveβ-NGFprotein in cat corneal endothelial cells, which will promote the proliferation activity of the cells. |
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