15羟二十四碳四烯酸对缺氧视网膜微血管内皮细胞增生的抑制作用及其机制
15간이십사탄사희산대결양시망막미혈관내피세포증생적억제작용급기궤제
Inhibition and mechanism of 15 (S)-hydroxyeicosate traenoic acid on proliferation of hypoxic retinal microvascular endothelial cells in vitro
  • 万方数据
    中华实验眼科杂志 중화실험안과잡지
    CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
    2014年 12期 1067-1073 ,共7页

    胡夏云%贺涛%邢怡桥%王琼

    호하운%하도%형이교%왕경

    视网膜新生血管%视网膜微血管内皮细胞%缺氧诱导因子-1α%Bcl-2%半胱天冬酶-3 視網膜新生血管%視網膜微血管內皮細胞%缺氧誘導因子-1α%Bcl-2%半胱天鼕酶-3
    시망막신생혈관%시망막미혈관내피세포%결양유도인자-1α%Bcl-2%반광천동매-3
    Retinal neovascularization%Retinal microvascular endothelial cell%Hypoxia inducible factor1α%Bcl-2%Caspase-3
    背景 缺氧导致的视网膜新生血管性疾病已成为主要的致盲疾病,目前临床上缺乏有效的化学治疗方法,因此,研究其发病的分子机制从而指导临床用药十分关键. 目的 探讨15羟二十四碳四烯酸(15-HETE)对缺氧条件下培养的视网膜微血管内皮细胞(RMVECs)增生、凋亡的影响及其作用通路. 方法 分离C57BL/6J小鼠的RMVECs并用细胞除杂法进行培养,用间接免疫组织化学及免疫荧光检测法鉴定培养的细胞对内皮细胞的特征性标志物Ⅷ因子相关抗原的反应.将生长良好的细胞分为常氧组和缺氧组,后者以终浓度为125 μmol/L的CoCl2处理细胞建立缺氧模型.各组细胞换无血清含内皮细胞生长添加剂(ECGS)和高糖的DMEM培养基继续培养48 h,再按照培养基中加入15-HETE浓度的不同(终浓度分别为0.0、0.1、1.0、5.0μmol/L)分别将常氧组和缺氧组细胞亚分为4个组.各组细胞分别用15-HETE处理48 h,然后用MTT法检测各组细胞的增生值(吸光度,A值),并计算15-HETE的抑制率;分别采用逆转录PCR(RT-RCR)法及Western blot法检测各组RMVECs中缺氧诱导因子-1α(HIF-1α)、bcl-2和caspase-3 mRNA及其蛋白的相对表达量. 结果 间接免疫组织化学法检测结果显示,培养的细胞对Ⅷ因子相关抗原呈阳性表达,细胞阳性率为(94.38±4.25)％.常氧培养条件下不同浓度15-HETE培养组RMVECs增生值(A值)的差异无统计学意义(F=0.283,P=0.837),但缺氧培养条件下不同浓度15-HETE培养组RMVECs增生值的差异有统计学意义(F=702.582,P＜0.001),其中缺氧+1.0 μmol/L 15-HETE处理组、缺氧+5.0 μmol/L 15-HETE处理组RMVECs增生值明显低于单纯缺氧培养组,差异均有统计学意义(P＜0.05).0.1、1.0、5.0μmol/L 15-HETE处理组对RMVECs的抑制率分别为(1.09±0.31)％、(21.09±3.53)％和(49.86±4.15)％,随着15-HETE浓度的增加,抑制率逐渐升高.常氧条件下各组bcl-2、caspase-3和HIF-1α mRNA的表达差异均无统计学意义,与单纯常氧培养组比较,单纯缺氧培养组RMVECs中bcl-2和HIF-1α mRNA的相对表达量分别升高1.5倍和1.7倍,而caspasse-3 mRNA的表达降低了70％,组间差异均有统计学意义(P＜0.05);在用不同浓度15-HETE干预的缺氧细胞中,bcl-2、HIF-1α的相对表达量随着15-HETE浓度的增高呈逐渐下降的趋势,而caspasse-3mRNA的表达则逐渐上升.常氧条件下各组细胞bcl-2、pro-caspase-3蛋白相对表达量差异均无统计学意义(P＞0.05),但与单纯常氧培养组比较,单纯缺氧培养组bcl-2、pro-caspase-3蛋白相对表达量分别上升1.6倍和1.9倍,差异均有统计学意义(P＜0.05);在缺氧条件下,bcl-2和pro-caspase-3蛋白的表达量随15-HETE浓度的增高逐渐下降,5.0 μmol/L 15-HETE处理组分别是单纯缺氧培养组的40.4％和42.5％,差异均有统计学意义(P＜0.05). 结论 15-HETE可能通过下调HIF-1α、bcl-2及激活caspase-3的方式抑制缺氧诱导下RMVECs的增生,从而对缺氧导致的新生血管生成产生抑制作用,其作用呈剂量依赖的方式.
    배경 결양도치적시망막신생혈관성질병이성위주요적치맹질병,목전림상상결핍유효적화학치료방법,인차,연구기발병적분자궤제종이지도림상용약십분관건. 목적 탐토15간이십사탄사희산(15-HETE)대결양조건하배양적시망막미혈관내피세포(RMVECs)증생、조망적영향급기작용통로. 방법 분리C57BL/6J소서적RMVECs병용세포제잡법진행배양,용간접면역조직화학급면역형광검측법감정배양적세포대내피세포적특정성표지물Ⅷ인자상관항원적반응.장생장량호적세포분위상양조화결양조,후자이종농도위125 μmol/L적CoCl2처리세포건립결양모형.각조세포환무혈청함내피세포생장첨가제(ECGS)화고당적DMEM배양기계속배양48 h,재안조배양기중가입15-HETE농도적불동(종농도분별위0.0、0.1、1.0、5.0μmol/L)분별장상양조화결양조세포아분위4개조.각조세포분별용15-HETE처리48 h,연후용MTT법검측각조세포적증생치(흡광도,A치),병계산15-HETE적억제솔;분별채용역전록PCR(RT-RCR)법급Western blot법검측각조RMVECs중결양유도인자-1α(HIF-1α)、bcl-2화caspase-3 mRNA급기단백적상대표체량. 결과 간접면역조직화학법검측결과현시,배양적세포대Ⅷ인자상관항원정양성표체,세포양성솔위(94.38±4.25)％.상양배양조건하불동농도15-HETE배양조RMVECs증생치(A치)적차이무통계학의의(F=0.283,P=0.837),단결양배양조건하불동농도15-HETE배양조RMVECs증생치적차이유통계학의의(F=702.582,P＜0.001),기중결양+1.0 μmol/L 15-HETE처리조、결양+5.0 μmol/L 15-HETE처리조RMVECs증생치명현저우단순결양배양조,차이균유통계학의의(P＜0.05).0.1、1.0、5.0μmol/L 15-HETE처리조대RMVECs적억제솔분별위(1.09±0.31)％、(21.09±3.53)％화(49.86±4.15)％,수착15-HETE농도적증가,억제솔축점승고.상양조건하각조bcl-2、caspase-3화HIF-1α mRNA적표체차이균무통계학의의,여단순상양배양조비교,단순결양배양조RMVECs중bcl-2화HIF-1α mRNA적상대표체량분별승고1.5배화1.7배,이caspasse-3 mRNA적표체강저료70％,조간차이균유통계학의의(P＜0.05);재용불동농도15-HETE간예적결양세포중,bcl-2、HIF-1α적상대표체량수착15-HETE농도적증고정축점하강적추세,이caspasse-3mRNA적표체칙축점상승.상양조건하각조세포bcl-2、pro-caspase-3단백상대표체량차이균무통계학의의(P＞0.05),단여단순상양배양조비교,단순결양배양조bcl-2、pro-caspase-3단백상대표체량분별상승1.6배화1.9배,차이균유통계학의의(P＜0.05);재결양조건하,bcl-2화pro-caspase-3단백적표체량수15-HETE농도적증고축점하강,5.0 μmol/L 15-HETE처리조분별시단순결양배양조적40.4％화42.5％,차이균유통계학의의(P＜0.05). 결론 15-HETE가능통과하조HIF-1α、bcl-2급격활caspase-3적방식억제결양유도하RMVECs적증생,종이대결양도치적신생혈관생성산생억제작용,기작용정제량의뢰적방식.
    Background Retninal neovascular diseases caused by hypoxia has become a major blinding disease,which is lack of effective chemical treatment currently,it's important to study the molecuar mechanism of the disease,so as to guide the clinical medication.Objective This study was to explore the effect of 15 (S)-hydroxyeicosate traenoic acid (15-HETE) on the proliferation of hypoxic retinal microvascular endothelial cells (RMVECs) and its probable mechanism.Methods RMVECs were isolated from C57BL/6J mice and incubated and then identified with anti-Ⅷ factor antibody by immunochemistry and immunofluorescence.The cells were divided into the normoxia group and the hypoxia group.The hypoxia cell models were established by treated with 125 μmol/L CoCl2.The cells were cultured with serum-free DMEM containing endothelial cell growth supplement (ECGS)and high glucose for 48 hours,and then different concentrations of 15-HETE (0.0,0.1,1.0,5.0 μmol/L) were added in the medium for 48 hours respectively to subgroup the groups.The proliferation of the cells (absorbance,A) was detected using MTT.The relative expression levels of protein and mRNA of hypoxia inducible factor-1α (HIF-1α),bcl-2 and caspase-3 were assayed by reverse transcription PCR (RT-RCR)and Western blot.Results The cells showed the positive response for anti-Ⅷ factor antibody with the positive rate of (94.38 ±4.25)％.No significant difference was found in the cell proliferation of various groups under the normoxia condition (F =0.283,P =0.837),but under the hypoxia condition,the proliferation values were significantly different among various groups (F =702.582,P＜0.001).The cell proliferation value in the 1.0 μmol/L 15-HETE group and 5.0 μmol/L 15-HETE group was lower than that of the simple hypoxia group respectively(both at P＜0.05).The inhibitory rates in the 0.1,1.0,5.0 μ mol/L 15-HETE groups were (1.09±0.31) ％,(21.09± 3.53) ％ and (49.86 ±4.15) ％,showing a dosedependent manner.No significant difference was seen in the expression levels of bcl-2,caspase-3 and HIF-1α mRNA in various groups under the normoxia conditions.However,compared with the simple normoxia group,the relative expressions of bcl-2 mRNA and HIF-1α mRNA in the cells were increased by 1.53 folds and 1.7 folds in the simple hypoxia group respectively,and caspasse-3 mRNA expression decreased by 70％ (all at P ＜ 0.05).Under the normoxia condition,the expression of bcl-2 and pro-caspase-3 protein in the cells were not significantly different among the various groups (P＞0.05),however,the expressions of bcl-2 and pro-caspase-3 proteins were elevated by 1.6 folds and 1.9 folds in the hypoxia group in compared with the normoxia group (P＜0.05).Compared with the simple hypoxia group,the expressions of bcl-2 and pro-caspase-3 were lowed by 40.4％ and 42.5％ in the 5.0 μmol/L 15-HETE group (P＜0.05).Conclusions 15-HETE inhibits the proliferation of RMVECs and therefore suppresses neovascularization by down-regulating the expressions of HIF-1α and bcl-2 and the activation of caspase-3 in a dose-dependent manner.
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