Effects Of RNA Interference Targeting PDGF-αreceptor On RPEcells Proliferation And Apoptosis In Vitro

Objective: To investigate the effect of RNA interference targeting PDGF-α receptor on RPE cell proliferation and apoptosis in vitro, so that experimental evidence was provided for the clinical treatment of proliferative vitreoretinopathy. Methods: Retinal pigment epithelium cell were cultured in vitro, and PDGFR-αsh RNA which chemically synthesized was transfected into human RPE cell mediated by cationic liposomes(Lipofectamine TM2000), the morphological changes of h RPE cells was observed by inverted optical microscope. According to diferent transfection composition, the cells were divided into four different groups,blank control group; Negative control group; 1.0μg/ml PDGFR-αsh RNAgroup; 2.0μg/ml PDGFR-αsh RNA group. PDGFR-αshort hairpin RNA has effect on h RPE cells 24 h 、 48 h 、 72 h, the cell proliferation ability was detected by MTT, and calculate the inhibition rate. PDGFR-αshort hairpin RNA has effect on h RPE cells 48 h, the expression of PDGFR-αm RNA of h RPE cells was detected by Reverse Transcription Polymerase Chain Response(RT-PCR). the expression of PDGFR-α protein was detected by Western blot and immunohistochemistry, cell apoptosis was analyzed by Hochest 33258,the Flow Cytometry was used to analyze the effect of Cycle and apoptosis.Results: After treated with PDGFR-αsh RNA 48 h, the cells of experimental group become shrinkage, gap widened, adherent is not strong, and many dead cells floating, compared with the low concentration of treatment group, the effect was stronger of cells becoming shrinkage fragmentation of nucleus and dead cells floating in high concentration of treatment group. PDGFR-αsh RNA has effect on h RPE cells 24-72 h, and the proliferation of h RPE cells were inhibited,and the inhibition of h RPE cells increased with increased concentration and the extension of time,After transfection 24, 48, 72 h, the inhibition rate of the experimental group was(9.4±0.03; 20.9±0.02; 3.2±0.01)%,(14.9±0.15; 31.3±0.01; 51.7±0.01)%, contrast with the blank control group(0; 0; 0)% and negative control group(1.7±0.04; 2.6±0.02;2.4±0.01)%, the experimental groups effect on inhibition were stronger,the difference was statistically significant(P<0.05). After treated with PDGFR-αsh RNA 48 h, the expression of m RNA and protein of PDGFR-α gene was significantly reduced contrast with control group, and the effect was stronger with increased drug concentration, the difference was statistically significant(P<0.05). After treated with PDGFR-αsh RNA 48 h,staining showed that: the experimental group hyperchromatic nuclei 、mitotic figures cells increased significantly compared with the control group. The results of cell apoptosis detected by Flow Cytometry showded that treatment group with PDGFR-αsh RNA was increased compared with the control group, the apoptosis rate of experimental group was(37.2±1.73)%,(51.8±1.41)%, contrast with blank control group(11.5±0.95)% and negative control group(13.8±1.61)%, the difference was statistically significant(P<0.05). The results of cell cycle detected by Flow Cytometry showded that cells witch treated with PDGFR-αsh RNA were arrested in G1 phase, the The percentage of G1 phase of experimental group was(64.44±1.13)%,(75.99±0.84)%, compared with blank control group(54.11±1.13)% and negative control group(55.53±1.81)%, the difference was statistically significant(P<0.05).Conclusions: 1. PDGFR-αsh RNA can inhibit the proliferation of h RPE cells, and induce apoptosis of cells, and make cells be arrested in G1 phases. 2. PDGFR-αsh RNA possibly through reduced expression of PDGFR-α gene to control the PVR occurrence and development.