Human β-NGF gene transferred to cat corneal endothelial cells

• 作者: Chuan-Fu Wang Gui-Qiu Zhao Li-Ting Hu Min Liu Wen-Juan Luo Xiang-Ping Liu
• 来源期刊: 国际眼科杂志：英文版
• 学科: 医学
• 关键词: nerve growth factor corneal endothelial cell transfect PROLIFERATION
• 年，卷（期）: gjykzzywb_2016,（7）
• 页码范围: 937-942
• 页数: 6页
• 分类号: R77

摘要

AIM:To transfect the cat corneal endothelial cells(CECs) with recombinant human β-nerve growth factor gene adeno-associated virus(AAV-β-NGF) and to observe the effect of the expressed β-NGF protein on the proliferation activity of cat CECs.METHODS:The endothelium of cat cornea was torn under the microscope and rapidly cultivated in Dulbecco’s modified Eagle’s medium(DMEM) to form single layer CECs and the passage 2 endothelial cells were used in this experiment.The recombinant human AAV-β-NGF was constructed.The recombinant human AAV-β-NGF was transferred into cat CECs directly.Three groups were as following:normal CEC control group,CEC-AAV control group and recombinant CECAAV-β-NGF group.Forty-eight hours after transfection,the total RNA was extracted from the CEC by Trizol.The expression of the β-NGF target gene detected by fluorescence quantitative polymerase chain reaction;proliferation activity of the transfected CEC detected at 48 h by MTT assay;the percentage of G1 cells among CECs after transfect was detected by flow cytometry method(FCM);cell morphology was observed under inverted phase contrast microscope.RESULTS:The torn endothelium culture technique rapidly cultivated 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.Forty-eight hours after transfect,the human β-NGF gene mRNA detected by fluorescence quantitative polymerase chain reaction showed that there was no significant difference between normal CEC control group and CECAAV control group(P>0.05);there was significant difference between two control groups and recombinant CEC-AAV-β-NGF group(P<0.05).MTT assay showed that transfect of recombinant AAV-β-NGF promoted 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 that the percentage of G1 cells in CECAAV-NGF group was 76.8% whi