中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2013年
10期
908-913
,共6页
刘高勤%陈源%陈磊%肖艳辉%陈志刚%陆培荣
劉高勤%陳源%陳磊%肖豔輝%陳誌剛%陸培榮
류고근%진원%진뢰%초염휘%진지강%륙배영
角膜%新生血管%一氧化氮合成酶%血管内皮生长因子%碱烧伤
角膜%新生血管%一氧化氮閤成酶%血管內皮生長因子%堿燒傷
각막%신생혈관%일양화담합성매%혈관내피생장인자%감소상
Cornea%Neovascularization%Nitric oxide synthase%Vascular endothelial growth factor%Alkali burn
背景 研究发现一氧化氮(NO)及一氧化氮合成酶(NOS)参与新生血管的发生、发展,但其在角膜新生血管(CNV)发生过程中的具体作用机制是眼科界值得关注的问题. 目的 研究NOS及其抑制剂NW-硝基-L-精氨酸甲酯(L-NAME)在实验性CNV发生过程中的作用,并通过体外研究验证NOS及L-NAME对人血管内皮细胞(RECs)血管管腔形成的作用,为眼科新生血管性疾病的防治提供实验依据.方法7～8周龄雄性BALB/c小鼠36只用NaOH滤纸贴附角膜中央的方法构建小鼠左眼CNV模型,然后用随机数字表法将小鼠分为L-NAME干预组和PBS对照组,L-NAME干预组小鼠于造模前1周开始腹腔内注射10 g/LL-NAME 0.5 ml,每周3次,持续3周,而PBS对照组小鼠以同样的方法注射PBS.分别于造模后2、4、7、14 d在裂隙灯显微镜下观察CNV形成情况,用全角膜铺片法检测CD31在CNV中的表达以鉴定CNV面积；采用逆转录聚合酶链反应(RT-PCR)法检测2个组小鼠角膜组织中NOSmRNA的表达；采用Western blot法检测人RECs中血管内皮生长因子(VEGF)蛋白的表达；利用体外实验,观察人RECs的管腔形成能力.采用独立样本t检验对2个组小鼠上述各检测指标的结果进行差异比较,分类资料采用X2检验. 结果裂隙灯显微镜下可见小鼠造模后2周CNV生长达高峰,而L-NAME干预组小鼠CNV明显少于PBS对照组.造模后2、4、7 d,L-NAME干预组小鼠角膜中NOS mRNA的相对表达量均明显低于PBS对照组,差异均有统计学意义(t=19.481、22.059、10.961,P＜0.01).全角膜铺片法检测显示,L-NAME干预组被CD31标记的RECs面积与总面积的比值为0.59 ±0.01,明显小于PBS对照组的0.78±0.10,差异有统计学意义(t=3.078,P＜0.05).Western blot法检测表明,造模0、2、4、7d,L-NAME干预组人RECs中VEGF蛋白的相对表达量均明显降低,造模后4d、7d两组比较差异均有统计学意义(-7.696、17.953,P＜0.01).管腔形成实验中,L-NAME干预组血管网形成数目为(46.33±1.86)个,明显少于PBS对照组的(64.00±4.51)个,差异有统计学意义(t=3.623,P＜0.05). 结论 NOS参与CNV的形成和发展,L-NAME通过下调VEGF的表达量抑制碱烧伤诱导的CNV以及人RECs血管网的形成,进而阻断NOS活性,为治疗CNV性眼病的研究提供实验依据.
揹景 研究髮現一氧化氮(NO)及一氧化氮閤成酶(NOS)參與新生血管的髮生、髮展,但其在角膜新生血管(CNV)髮生過程中的具體作用機製是眼科界值得關註的問題. 目的 研究NOS及其抑製劑NW-硝基-L-精氨痠甲酯(L-NAME)在實驗性CNV髮生過程中的作用,併通過體外研究驗證NOS及L-NAME對人血管內皮細胞(RECs)血管管腔形成的作用,為眼科新生血管性疾病的防治提供實驗依據.方法7～8週齡雄性BALB/c小鼠36隻用NaOH濾紙貼附角膜中央的方法構建小鼠左眼CNV模型,然後用隨機數字錶法將小鼠分為L-NAME榦預組和PBS對照組,L-NAME榦預組小鼠于造模前1週開始腹腔內註射10 g/LL-NAME 0.5 ml,每週3次,持續3週,而PBS對照組小鼠以同樣的方法註射PBS.分彆于造模後2、4、7、14 d在裂隙燈顯微鏡下觀察CNV形成情況,用全角膜鋪片法檢測CD31在CNV中的錶達以鑒定CNV麵積；採用逆轉錄聚閤酶鏈反應(RT-PCR)法檢測2箇組小鼠角膜組織中NOSmRNA的錶達；採用Western blot法檢測人RECs中血管內皮生長因子(VEGF)蛋白的錶達；利用體外實驗,觀察人RECs的管腔形成能力.採用獨立樣本t檢驗對2箇組小鼠上述各檢測指標的結果進行差異比較,分類資料採用X2檢驗. 結果裂隙燈顯微鏡下可見小鼠造模後2週CNV生長達高峰,而L-NAME榦預組小鼠CNV明顯少于PBS對照組.造模後2、4、7 d,L-NAME榦預組小鼠角膜中NOS mRNA的相對錶達量均明顯低于PBS對照組,差異均有統計學意義(t=19.481、22.059、10.961,P＜0.01).全角膜鋪片法檢測顯示,L-NAME榦預組被CD31標記的RECs麵積與總麵積的比值為0.59 ±0.01,明顯小于PBS對照組的0.78±0.10,差異有統計學意義(t=3.078,P＜0.05).Western blot法檢測錶明,造模0、2、4、7d,L-NAME榦預組人RECs中VEGF蛋白的相對錶達量均明顯降低,造模後4d、7d兩組比較差異均有統計學意義(-7.696、17.953,P＜0.01).管腔形成實驗中,L-NAME榦預組血管網形成數目為(46.33±1.86)箇,明顯少于PBS對照組的(64.00±4.51)箇,差異有統計學意義(t=3.623,P＜0.05). 結論 NOS參與CNV的形成和髮展,L-NAME通過下調VEGF的錶達量抑製堿燒傷誘導的CNV以及人RECs血管網的形成,進而阻斷NOS活性,為治療CNV性眼病的研究提供實驗依據.
배경 연구발현일양화담(NO)급일양화담합성매(NOS)삼여신생혈관적발생、발전,단기재각막신생혈관(CNV)발생과정중적구체작용궤제시안과계치득관주적문제. 목적 연구NOS급기억제제NW-초기-L-정안산갑지(L-NAME)재실험성CNV발생과정중적작용,병통과체외연구험증NOS급L-NAME대인혈관내피세포(RECs)혈관관강형성적작용,위안과신생혈관성질병적방치제공실험의거.방법7～8주령웅성BALB/c소서36지용NaOH려지첩부각막중앙적방법구건소서좌안CNV모형,연후용수궤수자표법장소서분위L-NAME간예조화PBS대조조,L-NAME간예조소서우조모전1주개시복강내주사10 g/LL-NAME 0.5 ml,매주3차,지속3주,이PBS대조조소서이동양적방법주사PBS.분별우조모후2、4、7、14 d재렬극등현미경하관찰CNV형성정황,용전각막포편법검측CD31재CNV중적표체이감정CNV면적；채용역전록취합매련반응(RT-PCR)법검측2개조소서각막조직중NOSmRNA적표체；채용Western blot법검측인RECs중혈관내피생장인자(VEGF)단백적표체；이용체외실험,관찰인RECs적관강형성능력.채용독립양본t검험대2개조소서상술각검측지표적결과진행차이비교,분류자료채용X2검험. 결과렬극등현미경하가견소서조모후2주CNV생장체고봉,이L-NAME간예조소서CNV명현소우PBS대조조.조모후2、4、7 d,L-NAME간예조소서각막중NOS mRNA적상대표체량균명현저우PBS대조조,차이균유통계학의의(t=19.481、22.059、10.961,P＜0.01).전각막포편법검측현시,L-NAME간예조피CD31표기적RECs면적여총면적적비치위0.59 ±0.01,명현소우PBS대조조적0.78±0.10,차이유통계학의의(t=3.078,P＜0.05).Western blot법검측표명,조모0、2、4、7d,L-NAME간예조인RECs중VEGF단백적상대표체량균명현강저,조모후4d、7d량조비교차이균유통계학의의(-7.696、17.953,P＜0.01).관강형성실험중,L-NAME간예조혈관망형성수목위(46.33±1.86)개,명현소우PBS대조조적(64.00±4.51)개,차이유통계학의의(t=3.623,P＜0.05). 결론 NOS삼여CNV적형성화발전,L-NAME통과하조VEGF적표체량억제감소상유도적CNV이급인RECs혈관망적형성,진이조단NOS활성,위치료CNV성안병적연구제공실험의거.
Background Though nitric oxide (NO) and NO synthase (NOS) have a critical role in angiogenesis,their effects on corneal neovascularization (CNV) and mechanism need to be further explored.Objective The aim of this study was to explore the effects of NOS and its antagonist,Nw-nitro-L-arginine methyl ester (L-NAME) on experimental CNV in mice,and investigate the influence of NOS and L-NAME on the tube formation of human retinal endothelial cells (RECs) in vitro.Methods The CNV models were established in the left eyes of 36 male BALB/c mice aged 7-8 weeks by application of the filter paper with NaOH in the center of corneas.The mice were randomized into two groups.L-NAME of 10 g/L (0.5 ml) was intraperitoneally injected 1 week before induction of CNV three times a week for three weeks in the mice of the L-NAME injection,and PBS was used in the same way in the control group.CNV was examined under the slit lamp biomicroscope 2,4,7,14 days after NaOH burn.The expression of CD31 in the CNV was assayed to calculate the ratio of CNV area and total corneal area using whole mount technique.The expression of NOS mRNA in the corneal tissue was detected by reverse transcriptase polymerase chain reaction (PCR),and VEGF expression in the human RECs was assayed by Western blot.The vessel formation number of cultured human RECs with or without L-NAME was performed by matrigel in vitro.Grouped t test was used to compare the differences of the parameters between the two groups.Results CNV developed and peaked 2 weeks after the application of NaOH on the mice corneas,and the CNV was obviously less in the L-NAME group compared with the control group.The expression of NOS mRNA in the corneas (NOS mRNA/ GAPDH mRNA)was significantly lower in the L-NAME group than that of the control group 2,4,7 days after CNV induction (t =19.481,t=22.059,t=10.961,all at P＜0.01).The ratio of the CD31 positive area in whole corneal area was 0.59± 0.01 in the L-NAME group,and that of the control group was 0.78±0.10,showing a significant difference between the two groups (t =3.078,P＜0.05).Western blot assay showed that the relative expression of VEGF protein in human RECs was declined in the L-NAME group compared with the control group 0,2,4,7 days,with statistically significant differences in 4 days and 7 days after NaOH burn(t=7.696,t=17.953,both at P＜0.01).The number of vessel network was 46.33±1.86 in the L-NAME group and 64.00±4.51 in the control group,with a significant difference between them (t =3.623,P＜0.05).Conclusions NOS participated in the pathogenesis and aggravation of CNV induced by NaOH.L-NAME arrests CNV formation and human RECs tube formation through down regulating the VEGF expression and NOS activity.
