Gene transfer to human trabecular meshwork cells in vitro and ex vivo using HIV-based lentivirus

作     者：Yan Xiang Bin Li Jun-Ming Wang Gui-Gang Li Hong Zhang Anne Manyande Xue-Bi Tian 

作者机构：Department of OphthalmologyTongji HospitalTongji Medical CollegeHuazhong University of Science and Technology School of PsychologySocial Work and Human SciencesUniversity of West London Department of AnesthesiologyTongji HospitalTongji Medical CollegeHuazhong University of Science and Technology 

出 版 物：《International Journal of Ophthalmology(English edition)》 (国际眼科杂志（英文版）)

年 卷 期：2014年第7卷第6期

页      面：924-929页

核心收录：

学科分类：1002[医学-临床医学] 100212[医学-眼科学] 10[医学] 

基　　金：Supported by Natural Science Foundation of China(No.30901395) the Doctoral Fund of Ministry of Education of China(No.20090142120012,20110142120021) 

主　　题：gene transfer trabecular meshwork HIV-based lentivirus glaucoma 

摘      要：AIM: 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 ***: 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 ×108transducing unit(TU) virus in perfusion liquid. The intraocular pressure was recorded every 8h for 21 d. 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 ***: 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 *** 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).CONCLUSION: EGFP gene could be efficiently transferred into HTM cells both in vitro and ex vivo by using HIV-based lentivirus.