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Originally published In Press as doi:10.1167/iovs.08-3169 on April 15, 2009
 (Investigative Ophthalmology and Visual Science. 2009;50:4436-4443.)
© 2009 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.08-3169
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Primary Culture of Human Blood–Retinal Barrier Cells and Preliminary Study of APOBEC3 Expression: An In Vitro Study

Haotian Lin,1 Zhenping Zhang,1 Hui Zhang,2 Pisong Yan,1 Qilin Wang,1 and Ling Bai3

1From the State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China; the 2Department of Medicine, Division of Infectious Diseases, Center for Human Virology, Thomas Jefferson University, Philadelphia, Pennsylvania; and the 3Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, China.

PURPOSE. To develop methods for primary culture of human blood–retinal barrier (BRB) cells and to explore the expression of APOBEC3 (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3) family gene, novel host-defense factors to HIV-1.

METHODS. Cellular components of human BRB (human retinal capillary endothelial cells [HRCECs], human retinal capillary pericytes, and human retinal pigment epithelial cells) were isolated separately and subjected to primary culture according to procedures modified in our laboratory. Immunocytochemistry and immunofluorescence were used to identify specific markers of the primary cells and to analyze their purity by flow cytometry. RNA of the three different cells was isolated, and primers were designed to probe expression of the APOBEC3 gene by reverse transcription–polymerase chain reaction (RT-PCR) and real-time PCR. For further confirmation, APOBEC3F and APOBEC3G proteins were detected in the cultured cells and fresh retina tissue through Western blot analysis. In the end, HRCECs were treated with IFN-{gamma}, and change of APOBEC3G expression was displayed.

RESULTS. Pure BRB cells (>95% purity) were primary cultured according to procedures modified in our laboratory. Qualitative test of RT-PCR and semiquantitative examination of real-time PCR demonstrated the presence of APOBEC3B, -3C, -3F, and -3G genes and the absence of APOBEC3A and -3D genes in all cellular components of the BRB. Finding of the APOBEC3G and APOBEC3F proteins expressed in the three primary cultured cells and different layers of retinal tissue by Western blot analysis further confirmed the PCR results. Moreover, IFN-{gamma} could upregulate the expression of APOBEC3G in HRCECs.

CONCLUSIONS. Major cellular components of human BRB could be primary cultured in vitro according to procedures optimized in our laboratory. Different expression of APOBEC3 in human blood–retinal barrier gives a clue to further research in intrinsic antiviral immunity in HIV-1–related retinopathy.






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