CAJ | 학술논문

目的：观察多聚嘧啶序列结合蛋白相关剪接因子(PSF)对体外培养的视网膜色素上皮(RPE)细胞磷脂酰肌醇3激酶(PI3K)/丝氨酸-苏氨酸蛋白激酶(Akt)信号通路的调控作用。方法将体外培养的 RPE 细胞分为 PSF 高表达组、PSF 高表达对照组、PSF 低表达组、PSF 低表达对照组及假转染组。应用脂质体2000将上调 PSF 表达的真核质粒增强型绿色荧光蛋白(pEGFP)-C2-PSF、下调 PSF 表达的真核质粒 pGenesil-PSF-RNAi 以0.25、0.50、1.00μg 的递增量分别转染至 PSF 高表达组及 PSF 低表达组细胞。PSF 高表达对照组细胞转染 pEGFP-C2空载质粒。PSF 低表达对照组细胞转染 pGenesil-scramble-siRNA 质粒。假转染组仅做转染处理,不加入任何表达质粒。采用水溶性四氮唑法检测胰岛素样生长因子1(IGF-1)刺激条件下 PSF 对 RPE 细胞增生的影响。应用脂质体2000将0.50μg 真核质粒pEGFP-C2-PSF、1.00μg 真核质粒 pGenesil-PSF-RNAi 及 pEGFP-C2空载质粒转染细胞分别作为 PSF 高表达组、PSF 低表达组、对照组,采用实时定量聚合酶链反应(PCR)检测 PSF 对 IGF-1诱导的 RPE 细胞内血管内皮生长因子(VEGF)mRNA 表达的影响。采用蛋白免疫印迹法(Western blot)检测 PSF 对 IGF-1诱导的磷酸化 Akt (pAkt)蛋白表达的影响。在 IGF-1刺激后,将 RPE 细胞分为单纯渥曼青霉素(Wortmannin)处理组、单纯 PSF 高表达组、渥曼青霉素联合 PSF 高表达处理组,并以未经渥曼青霉素处理的常规体外培养的 RPE 细胞为对照组,采用实时定量 PCR 检测各组 VEGF 的表达。结果IGF-1刺激后,PSF 高表达组、PSF 高表达对照组及假转染组之间 RPE 细胞增生率比较,差异有统计学意义(F =29.728,P <0.05)；PSF 低表达组、PSF 低表达对照组及假转染组之间 RPE 细胞增生率比较,差异有统计学意义(F =14.121,P <0.05)。PSF 高表达组 RPE 细胞中 VEGF mRNA 表达较对照组明显降低,差异有统计学意义(P =0.0003)；PSF 低表达组 RPE 细胞中 VEGF mRNA 表达较对照组明显提高,差异有统计学意义(P =0.0309)。Western blot 检测结果显示,IGF-1刺激可上调 pAkt 蛋白表达水平,PSF 高表达可明显下调 pAkt 蛋白表达水平。IGF-1刺激后,渥曼青霉素联合 PSF 高表达处理组 VEGF 表达较单纯渥曼青霉素处理组明显降低,差异有统计学意义(P <0.05)。结论PSF 能抑制 IGF-1刺激后体外培养的 RPE细胞中 PI3K/Akt 信号通路活化,下调 VEGF 表达。目的：觀察多聚嘧啶序列結閤蛋白相關剪接因子(PSF)對體外培養的視網膜色素上皮(RPE)細胞燐脂酰肌醇3激酶(PI3K)/絲氨痠-囌氨痠蛋白激酶(Akt)信號通路的調控作用。方法將體外培養的 RPE 細胞分為 PSF 高錶達組、PSF 高錶達對照組、PSF 低錶達組、PSF 低錶達對照組及假轉染組。應用脂質體2000將上調 PSF 錶達的真覈質粒增彊型綠色熒光蛋白(pEGFP)-C2-PSF、下調 PSF 錶達的真覈質粒 pGenesil-PSF-RNAi 以0.25、0.50、1.00μg 的遞增量分彆轉染至 PSF 高錶達組及 PSF 低錶達組細胞。PSF 高錶達對照組細胞轉染 pEGFP-C2空載質粒。PSF 低錶達對照組細胞轉染 pGenesil-scramble-siRNA 質粒。假轉染組僅做轉染處理,不加入任何錶達質粒。採用水溶性四氮唑法檢測胰島素樣生長因子1(IGF-1)刺激條件下 PSF 對 RPE 細胞增生的影響。應用脂質體2000將0.50μg 真覈質粒pEGFP-C2-PSF、1.00μg 真覈質粒 pGenesil-PSF-RNAi 及 pEGFP-C2空載質粒轉染細胞分彆作為 PSF 高錶達組、PSF 低錶達組、對照組,採用實時定量聚閤酶鏈反應(PCR)檢測 PSF 對 IGF-1誘導的 RPE 細胞內血管內皮生長因子(VEGF)mRNA 錶達的影響。採用蛋白免疫印跡法(Western blot)檢測 PSF 對 IGF-1誘導的燐痠化 Akt (pAkt)蛋白錶達的影響。在 IGF-1刺激後,將 RPE 細胞分為單純渥曼青黴素(Wortmannin)處理組、單純 PSF 高錶達組、渥曼青黴素聯閤 PSF 高錶達處理組,併以未經渥曼青黴素處理的常規體外培養的 RPE 細胞為對照組,採用實時定量 PCR 檢測各組 VEGF 的錶達。結果IGF-1刺激後,PSF 高錶達組、PSF 高錶達對照組及假轉染組之間 RPE 細胞增生率比較,差異有統計學意義(F =29.728,P <0.05)；PSF 低錶達組、PSF 低錶達對照組及假轉染組之間 RPE 細胞增生率比較,差異有統計學意義(F =14.121,P <0.05)。PSF 高錶達組 RPE 細胞中 VEGF mRNA 錶達較對照組明顯降低,差異有統計學意義(P =0.0003)；PSF 低錶達組 RPE 細胞中 VEGF mRNA 錶達較對照組明顯提高,差異有統計學意義(P =0.0309)。Western blot 檢測結果顯示,IGF-1刺激可上調 pAkt 蛋白錶達水平,PSF 高錶達可明顯下調 pAkt 蛋白錶達水平。IGF-1刺激後,渥曼青黴素聯閤 PSF 高錶達處理組 VEGF 錶達較單純渥曼青黴素處理組明顯降低,差異有統計學意義(P <0.05)。結論PSF 能抑製 IGF-1刺激後體外培養的 RPE細胞中 PI3K/Akt 信號通路活化,下調 VEGF 錶達。
목적：관찰다취밀정서렬결합단백상관전접인자(PSF)대체외배양적시망막색소상피(RPE)세포린지선기순3격매(PI3K)/사안산-소안산단백격매(Akt)신호통로적조공작용。방법장체외배양적 RPE 세포분위 PSF 고표체조、PSF 고표체대조조、PSF 저표체조、PSF 저표체대조조급가전염조。응용지질체2000장상조 PSF 표체적진핵질립증강형록색형광단백(pEGFP)-C2-PSF、하조 PSF 표체적진핵질립 pGenesil-PSF-RNAi 이0.25、0.50、1.00μg 적체증량분별전염지 PSF 고표체조급 PSF 저표체조세포。PSF 고표체대조조세포전염 pEGFP-C2공재질립。PSF 저표체대조조세포전염 pGenesil-scramble-siRNA 질립。가전염조부주전염처리,불가입임하표체질립。채용수용성사담서법검측이도소양생장인자1(IGF-1)자격조건하 PSF 대 RPE 세포증생적영향。응용지질체2000장0.50μg 진핵질립pEGFP-C2-PSF、1.00μg 진핵질립 pGenesil-PSF-RNAi 급 pEGFP-C2공재질립전염세포분별작위 PSF 고표체조、PSF 저표체조、대조조,채용실시정량취합매련반응(PCR)검측 PSF 대 IGF-1유도적 RPE 세포내혈관내피생장인자(VEGF)mRNA 표체적영향。채용단백면역인적법(Western blot)검측 PSF 대 IGF-1유도적린산화 Akt (pAkt)단백표체적영향。재 IGF-1자격후,장 RPE 세포분위단순악만청매소(Wortmannin)처리조、단순 PSF 고표체조、악만청매소연합 PSF 고표체처리조,병이미경악만청매소처리적상규체외배양적 RPE 세포위대조조,채용실시정량 PCR 검측각조 VEGF 적표체。결과IGF-1자격후,PSF 고표체조、PSF 고표체대조조급가전염조지간 RPE 세포증생솔비교,차이유통계학의의(F =29.728,P <0.05)；PSF 저표체조、PSF 저표체대조조급가전염조지간 RPE 세포증생솔비교,차이유통계학의의(F =14.121,P <0.05)。PSF 고표체조 RPE 세포중 VEGF mRNA 표체교대조조명현강저,차이유통계학의의(P =0.0003)；PSF 저표체조 RPE 세포중 VEGF mRNA 표체교대조조명현제고,차이유통계학의의(P =0.0309)。Western blot 검측결과현시,IGF-1자격가상조 pAkt 단백표체수평,PSF 고표체가명현하조 pAkt 단백표체수평。IGF-1자격후,악만청매소연합 PSF 고표체처리조 VEGF 표체교단순악만청매소처리조명현강저,차이유통계학의의(P <0.05)。결론PSF 능억제 IGF-1자격후체외배양적 RPE세포중 PI3K/Akt 신호통로활화,하조 VEGF 표체。
Objective To observe the regulation of PTB-associated splicing factor (PSF)exerts on phosphatidylinositol 3 kinase (PI3K)/Akt signaling pathway in cultured retinal pigment epithelial (RPE) cells.Methods ARPE-1 9 RPE cells were divided into five groups including PSF overexpression (0.25,0.50,1.00 μg of pEGFP-C2-PSF plasmid DNA),PSF overexpression control (pEGFP-C2 empty vector DNA),PSF inhibition (0.25,0.50,1.00 μg of pGenesil-PSF-RNAi plasmid DNA),PSF inhibition control (pGenesil-scramble-siRNA empty vector)and sham transfected group (treated with lipofactamine 2000 reagent,but without adding plasmid DNA)groups.After transfecting with plasmid DNA,the cells were stimulated with insulin-like growth factor-1 (IGF-1 ).IGF-1-stimulated ARPE-1 9 cells were also treated with Wortmannin and/or PSF over-expression.WST-1 assay was used to detect the proliferation rates,the VEGF mRNA levels were analyzed using real time polymerase chain reaction (PCR ), the levels of phosphorylation Akt and total Akt expression were measured by western blotting.Results After IGF-1 stimulation, the difference of the cell proliferation rates between PSF overexpression group, PSF overexpression control group and sham transfected group was statistically significant (F = 29.728,P <0.05).The difference of the cell proliferation rates between PSF inhibition group,PSF inhibition control group and sham transfected was also statistically significant (F =14.121,P <0.05).Compared with control group,the VEGF mRNA levels was decreased in PSF overexpression group (P =0.000 3),but increased in PSF low expression group (P = 0.030 9).Furthermore,overexpression of PSF could down-regulate the activation of pAkt after IGF-1 stimulation.When combined with Wortmannin treatment,the VEGF mRNA levels in PSF overexpression group was significantly lower than the control group (P <0.05).Conclusions After IGF-1 treatment,PSF plays a role in suppressing the proliferation and VEGF expression in RPE cells by inactivating PI3K/Akt signaling pathway.