Primary culture of human blood-retinal barrier cells and preliminary study of APOBEC3 expression: an in vitro study.
نویسندگان
چکیده
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.
منابع مشابه
3D study of capillary network derived from human cord blood mesenchymal stem cells and differentiated into endothelial cell with VEGFR2 protein expression
New blood forming vessels are produced by differentiation of mesodermal precursor cells to angioblasts that become endothelial cells (ECs) which in turn give rise to primitive capillary network. Human cord blood (HCB) contains large subsets of mononuclear cells (MNCs) that can be differentiated into endothelial-like cells in vitro. Human mononuclear progenitor cells were purified from fresh umb...
متن کاملMicroRNA Modulation during the In vitro Culture of Hematopoietic Stem Cells Prior to Transplantation
Background: Human umbilical cord blood (HUCB) is an acceptable and readily accessible source of stem cells. There is an ongoing interest in cord blood stem cell therapies; however, little is known about the possible unfavorable effects of laboratory modifications on the isolated HUCB cells. The involvement of miRNAs in several biological processes has been shown. The aim of this study was to ev...
متن کاملAngiogenesis Following Three-Dimensional Culture of Isolated Human Endometrial Stromal Cells
Background Endometriosis is the presence of endometrial tissue outside of the uterine cavity and is the most common gynecologic disorder in women of reproductive age. We have preliminary evidence that in the presence of a 3-dimensional (3-D) fibrin matrix, human endometrial glands, stroma, and neovascularization can develop in vitro, mimicking the earliest stages of endometriosis. The aim of th...
متن کاملAdipose Stem Cells as a Feeder Layer Reduce Apoptosis and p53 Gene Expression of Human Expanded Hematopoietic Stem Cells Derived from Cord Blood
Introduction: Human hematopoietic stem cells (hHSCs) have been used for transplantation in hematologic failures. Because the number of hHSCs per cord blood unit is limited, the expansion of these cells is important for clinical application. It has been reported that cytokines and feeder layer provide a perspective to in vitro expansion of hHSCs. In this regard, cord blood CD34+ cells ex...
متن کاملI-5: Multicellular Human Testicular Organoid: A Novel 3D In Vitro Germ Cell and Testicular Toxicity Model
Background Background: Mammalian spermatogenesis is regulated through paracrine and endocrine activity, specific cell signaling, and local control mechanisms. These highly specific signaling interactions are effectively absent upon placing testicular cells into two-dimensional primary culture. The specific changes that occur between key cell types and involved spermatogenesis signaling pathways...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Investigative ophthalmology & visual science
دوره 50 9 شماره
صفحات -
تاریخ انتشار 2009