重组AAV2-色素上皮衍生因子对人视网膜微血管内皮细胞增殖的影响
중조AAV2-색소상피연생인자대인시망막미혈관내피세포증식적영향
The effect of rAAV2-PEDF on proliferation of human retinal capillary endothelium cells
  • 万方数据
    中华眼科杂志 중화안과잡지
    Chinese Journal of Ophthalmology
    2011年 3期 197-201 ,共5页

    李涛%马红婕%梁小玲%丁小燕%罗燕%林少芬%唐仕波

    리도%마홍첩%량소령%정소연%라연%림소분%당사파

    视网膜血管%内皮细胞%依赖病毒%细胞增殖%转染%眼蛋白质类%神经生长因子类%舍平类 視網膜血管%內皮細胞%依賴病毒%細胞增殖%轉染%眼蛋白質類%神經生長因子類%捨平類
    시망막혈관%내피세포%의뢰병독%세포증식%전염%안단백질류%신경생장인자류%사평류
    Retinal vessels%Endothelial cells%Dependovirus%Cell proliferation%Transfection%Eye proteins%Nerve growth factors%Serpins
    目的 体外培养人视网膜微血管内皮细胞,探讨重组rAAV2-色素上皮衍生因子(PEDF)对该细胞在不同氧环境下增殖的影响.方法 实验研究.2%胰蛋白酶和0.1%Ⅱ型胶原酶消化视网膜,获得视网膜微血管内皮细胞;接种于预先包被纤维连接蛋白的培养瓶内,用含10%胎牛血清、内皮细胞生长因子、胰岛素-转铁蛋白-硒添加物的内皮细胞培养基,置于5%CO237 ℃培养箱内培养.相差显微镜观察细胞形态及生长情况.抗第Ⅷ因子相关抗原抗体鉴定内皮细胞.以125 μmol/L CoCl2建立HRCECs化学低氧模型,观察低氧对内皮细胞增殖的影响.按照105病毒基因组数/细胞的剂量进行rAAV2-PEDF病毒转染,分别设空白和阴性对照,激光共焦显微镜下观察EGFP阳性细胞,免疫印迹法检测PEDF蛋白表达.MTT法测定rAAV2-PEDF对不同氧条件下对细胞增殖的影响.流式细胞仪检测rAAV2-PEDF对不同氧条件下对细胞凋亡的影响.两组间比较采用独立样本t检验,多组间比较采用方差分析.结果 原代培养的人视网膜微血管内皮细胞48～72 h贴壁,2周左右细胞融合.第Ⅷ因子相关抗原鉴定细胞呈阳性.转染病毒48 h后,激光共焦显微镜下观察可见EGFP阳性细胞,免疫印迹法检测,实验组PEDF表达明显强于其他组.MTT结果显示,单纯低氧处理,正常对照组A值为0.085±0.021,实验组A值为0.166±0.024(t=3.938,P＜0.05).rAAV2-PEDF干预正常氧条件下人视网膜微血管内皮细胞,A值分别为:正常对照组0.171±0.011,rAAV2-EGFP组0.178±0.016,rAAV2-PEDF组0.169±0.017(F=0.01,P＞0.05).rAAV2-PEDF干预低氧条件下人视网膜微血管内皮细胞,A值分别为:单纯CoCl2组0.166±0.013,CoCl2+rAAV2-EGFP组0.155±0.012,CoCl2+rAAV2-PEDF组0.116±0.015(F=6.25,3.50;P＜0.05).rAAV2-PEDF干预正常氧条件下人视网膜微血管内皮细胞,正常对照组凋亡细胞比例为2.3%,rAAV2-EGFP组为3.3%,rAAV2-PEDF组为1.7%.rAAV2-PEDF干预低氧条件下人视网膜微血管内皮细胞,单纯CoCl2组凋亡细胞比例为3.6%,CoCl2+rAAV2-EGFP组为6.7%,CoCl2+rAAV2-PEDF组为36.4%.结论 rAAV2-PEDF转染人视网膜微血管内皮细胞后PEDF可稳定表达,并能显著抑制低氧诱导下内皮细胞的增殖.
    목적 체외배양인시망막미혈관내피세포,탐토중조rAAV2-색소상피연생인자(PEDF)대해세포재불동양배경하증식적영향.방법 실험연구.2%이단백매화0.1%Ⅱ형효원매소화시망막,획득시망막미혈관내피세포;접충우예선포피섬유련접단백적배양병내,용함10%태우혈청、내피세포생장인자、이도소-전철단백-서첨가물적내피세포배양기,치우5%CO237 ℃배양상내배양.상차현미경관찰세포형태급생장정황.항제Ⅷ인자상관항원항체감정내피세포.이125 μmol/L CoCl2건립HRCECs화학저양모형,관찰저양대내피세포증식적영향.안조105병독기인조수/세포적제량진행rAAV2-PEDF병독전염,분별설공백화음성대조,격광공초현미경하관찰EGFP양성세포,면역인적법검측PEDF단백표체.MTT법측정rAAV2-PEDF대불동양조건하대세포증식적영향.류식세포의검측rAAV2-PEDF대불동양조건하대세포조망적영향.량조간비교채용독립양본t검험,다조간비교채용방차분석.결과 원대배양적인시망막미혈관내피세포48～72 h첩벽,2주좌우세포융합.제Ⅷ인자상관항원감정세포정양성.전염병독48 h후,격광공초현미경하관찰가견EGFP양성세포,면역인적법검측,실험조PEDF표체명현강우기타조.MTT결과현시,단순저양처리,정상대조조A치위0.085±0.021,실험조A치위0.166±0.024(t=3.938,P＜0.05).rAAV2-PEDF간예정상양조건하인시망막미혈관내피세포,A치분별위:정상대조조0.171±0.011,rAAV2-EGFP조0.178±0.016,rAAV2-PEDF조0.169±0.017(F=0.01,P＞0.05).rAAV2-PEDF간예저양조건하인시망막미혈관내피세포,A치분별위:단순CoCl2조0.166±0.013,CoCl2+rAAV2-EGFP조0.155±0.012,CoCl2+rAAV2-PEDF조0.116±0.015(F=6.25,3.50;P＜0.05).rAAV2-PEDF간예정상양조건하인시망막미혈관내피세포,정상대조조조망세포비례위2.3%,rAAV2-EGFP조위3.3%,rAAV2-PEDF조위1.7%.rAAV2-PEDF간예저양조건하인시망막미혈관내피세포,단순CoCl2조조망세포비례위3.6%,CoCl2+rAAV2-EGFP조위6.7%,CoCl2+rAAV2-PEDF조위36.4%.결론 rAAV2-PEDF전염인시망막미혈관내피세포후PEDF가은정표체,병능현저억제저양유도하내피세포적증식.
    Objective To culture human retinal capillary endothelium cells(HRCECs) in vitro and explore the effect of rAAV2-PEDF on proliferation of HRCEs. Methods Retinas were digested by 2. 5%trypsin and 0.1% collagenase Ⅰ in order. The isolated cells were cultured on fibronectin-coated dishes in media of human endothelial-sFM basal growth medium (HE-SFM BGM) with 10% fetal bovine serum,insulin-transferin-selenium (ITS) and endothelial cell growth factor (ECGF). The cultured cells were identified by anti-factor Ⅷ related antigen though immunohistochemistry stain. The effect of hypoxia induced by CoCl2 on proliferation of HRCECs was assessed by MTT assay. After rAAV2-PEDF were transfected into HRCECs, the EGPF positive cells were observed by laser confocal scanning microscop, the protein expression of PEDF were detected by Western blot, and the proliferation of HRCECs were checked by MTT assay. Flow cytometry was used to analyze the apoptosis of HRCECs. Results Cultured HRCECs attached in the bottom of dishes in 48 h-72 h and grew to confluence in 2 weeks after seeding. HRCECs were with a positive brown staining for factor Ⅷ. EGPF positive cells were seen under laser confocal scanning microscop after 48h of rAAV2-EGFP transfection. The expression level of PEDF protein was higher in experimental group than in control group. The results of MTT assay showed the numeric value OA was 0.085±0.021 in normal group and 0.166±0.024 in hypoxia group(t =3.938,P＜0.05). In normal oxygen condition, the numeric value OA was 0.171±0.011 in normal control group, 0.178±0.016 in rAAV2-EGFP treated group, and O. 169±0.017 in rAAV2-PEDF treated group(F=0.01 ,P＞0.05). In hypoxia condition, the numeric value OA was 0.166±0.013 in CoCl2 treated group, 0.155±0.012 in CoCl2 + rAAV2-EGFP treated group, and 0.116±0.015 in CoCl2 + rAAV2-PEDF treated group. In normal oxygen condition, the ratio of apoptosis was 2. 3% in normal contral group,and 3. 3% in rAAV2-EGFP treated group,and 1.7% in rAAV2-PEDF treated group. In hypoxia condition, the ratio of apoptosis was 3.6% in CoCl2 treated group,6.7% in CoCl2 + rAAV2-EGFP treated group, and 36. 4% in CoCl2 + rAAV2-PEDF treated group.Conclusions PEDF gene can stably express in HRCECs after rAAV2-PEDF transfection and can obviously inhibit proliferation of HRCECs in hypoxia.
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