应用反义寡核苷酸沉默PDGFR-α表达对RPE细胞增生和凋亡的影响

Effect of suppression of platelet-derived growth factor-a receptor expression with antisense oligonucleotide on proliferation and apoptosis of retinal pigment epithelium cell

背景视网膜色素上皮（RPE）细胞能分泌血小板源性生长因子（PDGF），又具有PDGF受体（PDGFR），已有研究表明PDGF对增生性玻璃体视网膜病变（PVR）的形成起着关键性作用。目的应用反义寡核苷酸（ASODN）技术抑制血小板源性生长因子受体-α（PDGFR-α）基因的表达，观察其对RPE细胞增生和凋亡的影响。方法将人RPE细胞株在含质量分数10％胎牛血清的低糖DMEM培养基中进行培养，取对数生长期细胞调节细胞密度为5×10^5个／孔，接种于96孔板，待细胞生长至80％～90％融合时进行转染。空白对照组不加PDGFR—dASODN及阳离子脂质体Lipofectamine 2000，1．0μmol／LLipo—ASODN组、2．0μmol／LLipo—ASODN组使用阳离子脂质体Lipofectamine2000将靶向PDGFR—a基因的ASODN转染至RPE细胞株。细胞转染48h后，MTT法检测各组细胞的吸光度（4）值并以A490表示，逆转录聚合酶链反应（RT—PCR）法检测PDGFR—dmRNA在RPE细胞中表达的变化；hoechst33258荧光染色观察培养细胞的凋亡情况；流式细胞仪检测RPE细胞的细胞周期和凋亡率。结果空白对照组、1．0μmol／LLipo—ASODN组及2．0μmol／LLipo—ASODN组RPE细胞的A490值分别为1．45±0．12、1．07±0．06、0．65±0．05，差异有统计学意义（F=97．72，P=0．00），1．0μmol／LLipo—ASODN组和2．0μmol／LLipo—ASODN组RPE细胞A490值明显低于空白对照组，差异均有统计学意义（P=0．00、0．00）；2．0μmol／L Lipo—ASODN组RPE细胞A490值明显低于1．0μmol／LLipo—ASODN组，差异有统计学意义（P=0．00）。Hoechst33258荧光染色显示，转染PDGFR—αASODN后RPE细胞凋亡数量多于空白对照组。流式细胞仪检测表明，转染PDGFR—αASODN后RPE细胞阻滞于G0／G1期（F=206．70。P=0．00），1．0μmol／LLipo—ASODN组和2．0μmol／LLipo—ASODN组RPE细胞凋亡率均明显高于空白对照组（37．8±1．3vs10．5±0．1，61．2±1．9∥s10．5±0．1）（F=1808．90，P=0．00）。PDGFR—α在RPE细胞中的表达随PDGFR—dASODN浓度的升高有降低的趋势。结论利用ASODN技术沉默PDGFR—α基因表达可以显著抑制PVR过程中RPE细胞的增生，并能诱导其凋亡，不同浓度PDGFR—OLASODN转染后能下调PDGFR—dmRNA的表达，PDGFR-α基因的ASODN靶向技术为PVR的基因治疗提供了实验依据。

Background Retinal pigment epithelial（RPE） cells can secrete platelet-derived growth factor （PDGF） and PDGF receptor （PDGFR）. Studies have shown that PDGF plays a key role in the formation of proliferative vitreous retinopathy（PVR）. Objective This study was to investigate the proliferation and apoptosis changes of RPE after blockage of the PDGFR-α expression by antisense oligonueleotide （ASODN） in vitro. Methods Human RPE cells strain was cultured in low glucose DMEM with 10% fetal bovine serum. Logarithmic phase cells were collected and incubated in 96-well plate at the density of 5 x 105 cells/hole. PDGFR-α ASODN was transfeeted into RPE cells at different concentrations for 48 hours. The cells of the blank control group were regularly cultured without any transfection. The changes of PDGFR-α expression were detected by reverse transeription-polymerase chain reaction（RT-PCR） ,and the proliferation of RPE was detected by MTT as the A490 value. Hoechst 33258 fluorescence staining was used to determine the apoptosis of RPE. Flow cytometry method（FCM） was applied to detect the change of cell cycle and apoptosis rate of RPE ceils. Results The A490 values of RPE cells were 1.45±0. 12,1.07±0. 06, 0. 65±0. 05 in blank control grouμ 1.0 μmol/L Lipo-ASODN group and 2.0 μ mol/L Lipo-ASODN group with the significant difference（ P = 0. 00） , and that of 1.0 μmol/L Lipo-ASODN group and 2. 0 μ mol/L Lipo-ASODN group were significantly lower than the blank control group （ P = 0. 00, 0. 00）. Hoechst 33258 staining showed that the apoptosis cells were obviously more in Lipo-ASODN group compared with blank control group. PDGFR-α ASODN transfection induced an increase of percentage of RPE cells in G0/G1 phase （ F = 206.70, P = 0.00） , and the apoptosis rates in 1.0 μ mol/L Lipo-ASODN group and 2.0 μ mol/L Lipo-ASODN group were significantly enhanced in comparison with blank control group （37.8±1.3 vs 10. 5 ± 0. 1,61.2 ± 1.9 vs 10. 5 ± 0. 1 ） （ F = 1808.90, P = 0. 00 ）. Expression intensity of PDGFR-α mRNA in RPE cells in Lipo-ASODN groups was lower. Conclusions Blocking the PDGFR-α expression with ASODN technology can suppress proliferation and induce apoptosis of RPE cells. Intensity of PDGFR-α mRNA expression in RPE cells is ASODN dose-dependent. ASODN targeted to PDGFR-α offers an experimental basis of the gene therapy for PVR.