Xiang Yan, Li Bin, Wang Jun-Ming, Li Gui-Gang, Zhang Hong, Manyande Anne, Tian Xue-Bi
Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China.
School of Psychology, Social Work and Human Sciences, University of West London, London W5 5RF, UK.
Int J Ophthalmol. 2014 Dec 18;7(6):924-9. doi: 10.3980/j.issn.2222-3959.2014.06.02. eCollection 2014.
To investigate whether the enhanced green fluorescent protein (EGFP) reporter gene could be transferred into human trabecular meshwork (HTM) cells by a HIV-based lentivirus both in vitro and ex vivo.
The HIV-based lentivirus that contains an EF1-α promoter driving EGFP expression cassette was constructed following the standard molecular cloning methods. The cultured HTM cells were transduced at a range of multiplicity of infection (MOI) with HIV-based lentivirus. EGFP positive cell populations were detected by flow cytometry. Human anterior eye segments were cultured with perfusion culture system and transfected by HIV-based lentivirus with a 1×10(8) transducing unit (TU) virus in perfusion liquid. The intraocular pressure was recorded every 8h for 21d. The expression of EGFP in the anterior segment of the human eye was detected by fluorescence microscopy. Furthermore, the distribution of EGFP expression was confirmed by anti-EGFP immunohistochemical staining.
The HIV-based lentivirus which contains an EF1-α promoter driving EGFP expression cassette was constructed successfully. After HTM cells were transduced with HIV-based lentivirus containing EGFP in vitro, the ratio of EGFP positive cells to the total cell number reached 92.3%, with the MOI of 15. After the lentivirus containing EGFP were used to transduce human anterior eye segments, the EGFP could be directly detected by fluorescence microscopy in vivo. Immunohistochemistry staining revealed that 88.19% EGFP-positive trabecular meshwork (TM) cells were observed in the human anterior segment. Nevertheless, the intraocular pressure in the lentivirus-transduced group kept constant when compared with control group (P>0.05).
EGFP gene could be efficiently transferred into HTM cells both in vitro and ex vivo by using HIV-based lentivirus.
研究基于HIV的慢病毒能否在体外和离体条件下将增强型绿色荧光蛋白（EGFP）报告基因转入人小梁网（HTM）细胞。
按照标准分子克隆方法构建含EF1-α启动子驱动EGFP表达盒的基于HIV的慢病毒。用基于HIV的慢病毒以一系列感染复数（MOI）转导培养的HTM细胞。通过流式细胞术检测EGFP阳性细胞群体。用人眼前节灌注培养系统培养，并在灌注液中用1×10(8)转导单位（TU）的基于HIV的慢病毒进行转染。每8小时记录眼压，共记录21天。通过荧光显微镜检测人眼前节中EGFP的表达。此外，通过抗EGFP免疫组织化学染色确认EGFP表达的分布。
成功构建了含EF1-α启动子驱动EGFP表达盒的基于HIV的慢病毒。体外将含EGFP的基于HIV的慢病毒转导HTM细胞后，EGFP阳性细胞与总细胞数的比例在MOI为15时达到92.3%。用含EGFP的慢病毒转导人眼前节后，可通过荧光显微镜在体内直接检测到EGFP。免疫组织化学染色显示，在人眼前节中观察到88.19%的EGFP阳性小梁网（TM）细胞。然而，与对照组相比，慢病毒转导组的眼压保持恒定（P>0.05）。
利用基于HIV的慢病毒可在体外和离体条件下将EGFP基因有效转入HTM细胞。
