中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2012年
4期
316-319
,共4页
胰岛素样生长因子-1%缺氧诱导因子-1α%血管内皮生长因子%视网膜色素上皮细胞%增生性玻璃体视网膜疾病
胰島素樣生長因子-1%缺氧誘導因子-1α%血管內皮生長因子%視網膜色素上皮細胞%增生性玻璃體視網膜疾病
이도소양생장인자-1%결양유도인자-1α%혈관내피생장인자%시망막색소상피세포%증생성파리체시망막질병
Insulin-like growth factor-1%Hypoxia inducible factor-1α%Vascular endothelial growth factor%Retinal pigment epithelial cell%Proliferative vitreo-retinal disease
背景 增生性玻璃体视网膜疾病(PVD)是一组眼底视网膜血管性疾病,主要由视网膜色素上皮( RPE)细胞增生所致,胰岛素样生长因子-1(IGF-1)和血管内皮生长因子(VEGF)与RPE细胞的异常增生和病理性新生血管生成有关,但其信号机制及功能尚不完全明了. 目的 探讨利用小发卡环核糖核酸( shRNA)使人RPE细胞缺氧诱导因子1α(HIF-1α)基因沉默后,IGF-1对VEGF表达的影响. 方法 收集健康男性供体眼球4只,分离、收集、培养RPE细胞,用SABC法行抗人角蛋白免疫细胞化学染色进行鉴定.用体外转录法合成针对HIF-1α mRNA序列靶点的shRNA,对3～5代RPE细胞的HIF-1α进行干扰后再经50 μg/L IGF-1处理24 h,采用逆转录聚合酶链反应(RT-PCR)法检测人RPE细胞中HIF-1α及VEGF mRNA的表达,采用Western blot法检测人RPE细胞中HIF-1α及VEGF蛋白的水平.结果 分离培养的细胞呈扁平不规则多角形,97％的细胞对人角蛋白呈阳性反应.50 μg/L IGF-1作用后,人RPE细胞HIF-1α mRNA表达量(1.49±0.18)与0 μg/L IGF-1组(1.46±0.17)比较差异无统计学意义(t=0.335,P=0.743),而HIF-1α蛋白表达量(1049.86±172.54 vs 0.00±0.00)、VEGF mRNA(0.95±0.15 vs 0.35±0.07)及VEGF蛋白(391.98±56.77 vs 214.36±37.15)表达量均明显增高,差异均有统计学意义(t=16.098、9.935、6.928,P＜0.05).shRNA干扰HIF-1α mRNA表达后,RNAi转染组HIF-1α、VEGF mRNA及其蛋白水平较RNAi空白对照组及RNAi-C转染组明显下降,3个组间各指标的总体比较差异均有统计学意义(F=68.679、89.904、21.770、6.205,P＜0.05). 结论 IGF-1可通过促进人RPE细胞中HIF-1α蛋白的累积诱导VEGF的表达,是导致PVD重要的细胞因子之一.
揹景 增生性玻璃體視網膜疾病(PVD)是一組眼底視網膜血管性疾病,主要由視網膜色素上皮( RPE)細胞增生所緻,胰島素樣生長因子-1(IGF-1)和血管內皮生長因子(VEGF)與RPE細胞的異常增生和病理性新生血管生成有關,但其信號機製及功能尚不完全明瞭. 目的 探討利用小髮卡環覈糖覈痠( shRNA)使人RPE細胞缺氧誘導因子1α(HIF-1α)基因沉默後,IGF-1對VEGF錶達的影響. 方法 收集健康男性供體眼毬4隻,分離、收集、培養RPE細胞,用SABC法行抗人角蛋白免疫細胞化學染色進行鑒定.用體外轉錄法閤成針對HIF-1α mRNA序列靶點的shRNA,對3～5代RPE細胞的HIF-1α進行榦擾後再經50 μg/L IGF-1處理24 h,採用逆轉錄聚閤酶鏈反應(RT-PCR)法檢測人RPE細胞中HIF-1α及VEGF mRNA的錶達,採用Western blot法檢測人RPE細胞中HIF-1α及VEGF蛋白的水平.結果 分離培養的細胞呈扁平不規則多角形,97％的細胞對人角蛋白呈暘性反應.50 μg/L IGF-1作用後,人RPE細胞HIF-1α mRNA錶達量(1.49±0.18)與0 μg/L IGF-1組(1.46±0.17)比較差異無統計學意義(t=0.335,P=0.743),而HIF-1α蛋白錶達量(1049.86±172.54 vs 0.00±0.00)、VEGF mRNA(0.95±0.15 vs 0.35±0.07)及VEGF蛋白(391.98±56.77 vs 214.36±37.15)錶達量均明顯增高,差異均有統計學意義(t=16.098、9.935、6.928,P＜0.05).shRNA榦擾HIF-1α mRNA錶達後,RNAi轉染組HIF-1α、VEGF mRNA及其蛋白水平較RNAi空白對照組及RNAi-C轉染組明顯下降,3箇組間各指標的總體比較差異均有統計學意義(F=68.679、89.904、21.770、6.205,P＜0.05). 結論 IGF-1可通過促進人RPE細胞中HIF-1α蛋白的纍積誘導VEGF的錶達,是導緻PVD重要的細胞因子之一.
배경 증생성파리체시망막질병(PVD)시일조안저시망막혈관성질병,주요유시망막색소상피( RPE)세포증생소치,이도소양생장인자-1(IGF-1)화혈관내피생장인자(VEGF)여RPE세포적이상증생화병이성신생혈관생성유관,단기신호궤제급공능상불완전명료. 목적 탐토이용소발잡배핵당핵산( shRNA)사인RPE세포결양유도인자1α(HIF-1α)기인침묵후,IGF-1대VEGF표체적영향. 방법 수집건강남성공체안구4지,분리、수집、배양RPE세포,용SABC법행항인각단백면역세포화학염색진행감정.용체외전록법합성침대HIF-1α mRNA서렬파점적shRNA,대3～5대RPE세포적HIF-1α진행간우후재경50 μg/L IGF-1처리24 h,채용역전록취합매련반응(RT-PCR)법검측인RPE세포중HIF-1α급VEGF mRNA적표체,채용Western blot법검측인RPE세포중HIF-1α급VEGF단백적수평.결과 분리배양적세포정편평불규칙다각형,97％적세포대인각단백정양성반응.50 μg/L IGF-1작용후,인RPE세포HIF-1α mRNA표체량(1.49±0.18)여0 μg/L IGF-1조(1.46±0.17)비교차이무통계학의의(t=0.335,P=0.743),이HIF-1α단백표체량(1049.86±172.54 vs 0.00±0.00)、VEGF mRNA(0.95±0.15 vs 0.35±0.07)급VEGF단백(391.98±56.77 vs 214.36±37.15)표체량균명현증고,차이균유통계학의의(t=16.098、9.935、6.928,P＜0.05).shRNA간우HIF-1α mRNA표체후,RNAi전염조HIF-1α、VEGF mRNA급기단백수평교RNAi공백대조조급RNAi-C전염조명현하강,3개조간각지표적총체비교차이균유통계학의의(F=68.679、89.904、21.770、6.205,P＜0.05). 결론 IGF-1가통과촉진인RPE세포중HIF-1α단백적루적유도VEGF적표체,시도치PVD중요적세포인자지일.
Background Proliferative vitreo-retinal disease (PVD)is one group of ocular complications marked by the enhanced proliferation of various cells included retinal pigment epithelial (RPE) cells.Insulin-like growth factor-1 (IGF-1)and vascular endothelial growth factor(VEGF) are implicated in the aberrant cell proliferation and pathological neovascularization that characterizes PVD,but the signaling mechanism is unclear now. Objective This study was to explore the effect of IGF-1 on VEGF in cultured human RPE cells under the small hairpin loop RNA (shRNA) keeping hypoxia-inducible factor-1 α ( HIF-1 α) silencing. Methods Human retinas were isolated from 4 healthy male donors,and the RPE cells were harvested and cultured.The ceils were identified using anti-human keratin antibody.The third to fifth generation of human RPE cells were used in the experiment.One target site of HIF-1α mRNA was chosen by certain design principle,and shRNA was designed and synthesized by the target site and transferred into the cells in vitro,and then the cells were cultivated with 50 μg/L IGF-1 for 24 hours.The mRNA and protein expressions of HIF-1α and VEGF were detected by RT-PCR and Western blot respectively. Results Cultured human RPE cells showed the flat irregularly multangular shape,and 97％ cells appeared the positive response for keratin.HIF-1α mRNA expression in human RPE cells was significantly lower in 50 μg/L IGF-1 group than the 0 pg/L IGF-1 group ( 1.49±0.18 vs 1.46±0.17 ) ( t =0.335,P =0.743 ),however,the expressing levels of HIF-1α protein( 1049.86±172.54 vs 0.00±0.00) and VEGF mRNA(0.95±0.15 vs 0.35±0.07) and VEGF protein (391.98±56.77 vs 214.36±37.15)were raised in the 50 μg/L IGF-I group compared with 0 μg/L IGF-1 group (t=16.098,9.935,6.928,P＜0.05).After the HIF-1α-specific shRNA was transferred into cultured RPE cells,the expressions of both HIF-1α mRNA and its protein significantly decreased in RPE cells under 50 μg/L IGF-1 concentration condition( F=68.679,89.904,P=0.000),moreover,the expression of VEGF mRNA and its protein were significantly lowed(F=21.770,6.205,P＜0.05). Conclusions IGF-1 promotes the accumulation of HIF-1α protein and induce the expression of VEGF in human RPE cells,which probably play a pivotal role in the development of PVD.
