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Protein kinase C? downregulation via siRNA-PKC? released from foldable capsular vitreous body in cultured human retinal pigment epithelium cells.

ABSTRACT: We previously found that downregulation of protein kinase C? (PKC?) can inhibit retinal pigment epithelium (RPE) cell proliferation involved in the development of proliferative vitreoretinopathy (PVR). In this study, we tested whether PKC? could be downregulated via small interfering RNA (siRNA)-PKC? released from foldable capsular vitreous body (FCVB) in cultured human RPE cells. SiRNA-PKC? content, determined by ultraviolet (UV) spectrophotometer, was released from FCVB containing 200, 300, 400, 500, and 600 nm siRNA-PKC? in a time-dependent manner from 1 to 96 hours and a dose-dependent manner at five concentrations. The content (y) had a good linear relationship with time (x), especially in the 600 nm siRNA-PKC? group (y = 16.214x, R(2) = 0.9809). After treatment with siRNA-PKC? released from FCVBs, the PKC? was significantly decreased by RT-PCR, Western blot, and immunofluorescence analysis in RPE cells. These results indicate that PKC? was significantly downregulated by siRNA-PKC? released from FCVB in human RPE cells and provide us with a new avenue to prevent PVR.

SUBMITTER: Chen X

PROVIDER: S-EPMC3131196 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Protein kinase Cα downregulation via siRNA-PKCα released from foldable capsular vitreous body in cultured human retinal pigment epithelium cells.

Chen Xiaoqing X Liu Yaqin Y Jiang Zhaoxin Z Zhou Lian L Ge Jian J Gao Qianying Q

International journal of nanomedicine 20110622

We previously found that downregulation of protein kinase Cα (PKCα) can inhibit retinal pigment epithelium (RPE) cell proliferation involved in the development of proliferative vitreoretinopathy (PVR). In this study, we tested whether PKCα could be downregulated via small interfering RNA (siRNA)-PKCα released from foldable capsular vitreous body (FCVB) in cultured human RPE cells. SiRNA-PKCα content, determined by ultraviolet (UV) spectrophotometer, was released from FCVB containing 200, 300, 40 ...[more]

PMID: 21753881

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Project description:PurposeTo evaluate the clinical effectiveness and safety of foldable capsular vitreous body (FCVB) implantation for severe retinal detachment.MethodsA retrospective analysis was performed on 26 patients with severe ocular trauma and one with recurrent retinal detachment. Clinical data-including surgery success, complications, retinal reattachment, vision, and intraocular pressure (IOP)-were analyzed for patients who underwent 23G pars plana vitrectomy and FCVB implantation combined with silicone oil tamponade.ResultsThe mean follow-up period was 10.44 ± 2.68 months. All surgeries were smooth; the FCVBs were properly positioned and supported the retina well, and the retinal reattachment rate reached 92.59%. At the six-month follow-up, preoperative (1.30 ± 1.20) and postoperative (0.63 ± 0.79) vision was significantly different (t = 3.03, P = 0.005), and the postoperative IOP (7.93 ± 3.57 mm Hg) was lower than the preoperative IOP (13.98 ± 10.72 mm Hg) (t = 2.74, P = 0.01). Among 20 patients followed up for >12 months, preoperative (1.20 ± 0.95) and postoperative (0.75 ± 0.91) visions were significantly different (t = 1.831, P = 0.005), and the postoperative IOP (9.85 ± 6.48 mm Hg) was lower than the preoperative IOP (14.85 ± 12.17 mm Hg) (t = 1.82, P = 0.01). No endophthalmitis, sympathetic ophthalmia, and rejection of FCVB occurred during follow-up.ConclusionsFCVB combined with silicone oil tamponade showed good efficacy and safety in severe retinal detachment treatment during the follow-up period.Translational relevanceVitreous substitution is deemed a highly challenging and interesting research topic in ophthalmology. Traditional method such as silicone oil tamponade often causes various complications such as silicone oil emulsification, silicone oil migration, and corneal degeneration. The foldable capsular vitreous body as a novel vitreous substitute combined silicone oil injection into it can stay in the eyeball for a long time without obvious complications.

Project description:I. Exp Design 1. Type of experiment: Comparison of native versus cultured RPE cells 2. Experimental factors: Native RPE versus ARPE-19 cells grown on different matrices 3. How many hybridizations in exp: 21 4. If a common reference used for all the hybs: no 5. Quality control steps: three independent arrays for each condition 6. Description: The expression profile of ARPE-19 cells grown on different matrices were compared to morphologically normal native macular RPE cells that were laser capture microdissected from 3 donors. II. Samples used, extract prep, and labeling 1. Biosource: Human donor globes from NDRI (63, 71, 74 years old) and ARPE-19 cells. 2. Manipulations: Human donor globes were cryopreserved, and morphologically normal RPE cells from the macula were laser capture micodissected. ARPE-19 cells were grown on different matrices (plastic, Matrigel, collagen I, collagen IV, laminin, and fibronectin). 3. Extract preparation: Total RNA from cells were extracted with the RNeasy kit (Qiagen) using the manufacturer’s instructions. 4. Labeling protocol: Total RNA from cells was reverse transcribed with 33P-dCTP and 33P-dATP, and second strand cDNA was labeled with 33P-dCTP and 33P-dATP. 5. No external controls were added. III. Hybridization procedures and parameters 1. Sample, array type, batch and serial # used 2. Hybridization protocol: Hybridization was carried out using the manufacturer’s recommendations. Arrays were prehybridized with Microhyb solution containing denatured Cot-1 DNA and poly dA at 42oC for two hours. Hybridization was carried out at 42oC overnight using a hybridization oven set at 8-10 rpm. Arrays were washed twice at 50oC for 20 minutes using 2x SSC, 1%SDS and once at room temperature for 15 minutes using 0.5x SSC, 1%SDS. IV. Measurement data and specifications of data processing 1,2. Arrays were exposed to a phosphorimaging screen for 3 days and scanned at 50 mm resolution with a BioRad FX Pro-Plus phosphorimager. TIFF images from the phosphorimager were exported into ResGen Pathways 3 software for analysis. 3. Data processing: A gene was expressed if its background subtracted intensity was greater than 1.4 fold background. The data were normalized using a simple global scaling procedure, and Cluster/Treeview and Statistical Analysis of Microarrays (SAM version 1.12) programs were used for analysis. Keywords: other

Dataset Information

Protein kinase C? downregulation via siRNA-PKC? released from foldable capsular vitreous body in cultured human retinal pigment epithelium cells.

Publications

Protein kinase Cα downregulation via siRNA-PKCα released from foldable capsular vitreous body in cultured human retinal pigment epithelium cells.

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