Primary culture of human blood-retinal barrier cells and preliminary study of APOBEC3 expression: an in vitro study.
OBJECTIVE
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.
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Haotian
H
Lin
Zhenping
Z
Zhang
Hui
H
Zhang
Pisong
P
Yan
Qilin
Q
Wang
Ling
L
Bai
D000071478:APOBEC Deaminases; D000328:Adult; D001813:Blood-Retinal Barrier; D015153:Blotting, Western; D002196:Capillaries; D018929:Cell Culture Techniques; D002469:Cell Separation; D003564:Cytidine Deaminase; D043525:Cytosine Deaminase / Q000235:genetics* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D007124:Immunoenzyme Techniques; D007371:Interferon-gamma / Q000494:pharmacology; D020286:Pericytes / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*; D012171:Retinal Vessels / Q000166:cytology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation / Q000187:drug effects
D012333:RNA, Messenger; D007371:Interferon-gamma; D043525:Cytosine Deaminase; D000071478:APOBEC Deaminases; C502889:APOBEC3 proteins, human; D003564:Cytidine Deaminase