背景 角膜新生血管(CNV)是导致角膜盲的主要原因之一,研究表明,ADP-核糖基化因子(ARF)对肿瘤细胞的增生有调节作用,抑制ARF能抑制血管的形成,但ARF抑制剂对CNV是否发挥作用尚不清楚. 目的 探讨ARF抑制剂在碱烧伤诱导的CNV形成过程中的作用及其机制.方法 7～8周龄清洁级雄性BABL/c小鼠60只按照随机数字表法分为PBS对照组和ARF抑制剂干预组,每组各30只.所有小鼠采用角膜碱烧伤诱导法构建CNV模型,ARF抑制剂干预组小鼠于造模后l周腹腔内注射0.5 g/L ARF抑制剂溶液0.5 ml,每周3次,共1周,PBS对照组小鼠以同样方式注射0.5 ml PBS.各组分别取24只小鼠,于造模后0、4、7、14d采用实时定量PCR(real-time PCR)和Western blot法检测小鼠角膜组织中ARF mRNA和蛋白的动态表达,并于造模后14 d,采用Western blot法检测各组小鼠角膜组织中血管内皮生长因子(VEGF)的表达.各组另取6只小鼠,于造模后2、4、7、14 d裂隙灯显微镜下观察CNV形成情况,并于造模后14d,采用全视网膜铺片免疫荧光组织化学法检测角膜组织中CD31在CNV中的表达.体外实验中应用ARF抑制剂干预人视网膜血管内皮细胞(RECs),CCK8法和细胞划痕法检测ARF对人RECs增生和迁移的影响.实验动物的使用和喂养遵循视觉及眼科学研究协会有关规定及苏州大学《实验动物管理及使用指南》. 结果 造模后PBS对照组和ARF抑制剂干预组小鼠角膜组织中ARF mRNA在各个时间点均有表达,在造模后14d达高峰,各组小鼠角膜中ARF mRNA的表达随着时间的延长而增加,差异有统计学意义(F时间=65.17,P=0.00),不同时间点的组间比较差异无统计学意义(F分组=1.98,P=0.18)；造模后14 d,ARF抑制剂干预组实验后不同时间点ARF蛋白水平相对表达值比较差异有统计学意义(F=10.77,P=0.00).裂隙灯显微镜下检查发现,ARF抑制剂干预组CNV相对面积为0.45 ±0.05,PBS对照组为0.72±0.11,2个组间差异有统计学意义(t=-3.87,P＜0.05).全角膜铺片免疫荧光组织化学法检测表明,ARF抑制剂干预组小鼠角膜组织CD31表达面积明显小于PBS对照组.造模后14 d,Western blot法检测显示,ARF抑制剂干预组VEGF蛋白表达水平为1.20±0.21,明显低于PBS对照组的2.47±0.33,差异有统计学意义(t=-5.62,P＜0.05).CCK8法检测结果显示,随着ARF抑制剂质量浓度的增加,ARF抑制剂对人RECs的抑制率增加,差异有统计学意义(F=8.47,P=0.02).细胞划痕实验后24 h,100μg/L和1 000 μg/L ARF抑制剂干预组人RECs迁移距离分别为(5.46±1.32) μm和(5.04±1.68)μm,与PBS对照组的(8.49±1.18) μm相比明显减小,差异均有统计学意义(t=-2.94、-2.91,P＜0.05). 结论 ARF抑制剂能抑制碱烧伤诱导的CNV发生,可能与其下调VEGF的表达以及抑制血管内皮细胞的增生和迁移有关.
揹景 角膜新生血管(CNV)是導緻角膜盲的主要原因之一,研究錶明,ADP-覈糖基化因子(ARF)對腫瘤細胞的增生有調節作用,抑製ARF能抑製血管的形成,但ARF抑製劑對CNV是否髮揮作用尚不清楚. 目的 探討ARF抑製劑在堿燒傷誘導的CNV形成過程中的作用及其機製.方法 7～8週齡清潔級雄性BABL/c小鼠60隻按照隨機數字錶法分為PBS對照組和ARF抑製劑榦預組,每組各30隻.所有小鼠採用角膜堿燒傷誘導法構建CNV模型,ARF抑製劑榦預組小鼠于造模後l週腹腔內註射0.5 g/L ARF抑製劑溶液0.5 ml,每週3次,共1週,PBS對照組小鼠以同樣方式註射0.5 ml PBS.各組分彆取24隻小鼠,于造模後0、4、7、14d採用實時定量PCR(real-time PCR)和Western blot法檢測小鼠角膜組織中ARF mRNA和蛋白的動態錶達,併于造模後14 d,採用Western blot法檢測各組小鼠角膜組織中血管內皮生長因子(VEGF)的錶達.各組另取6隻小鼠,于造模後2、4、7、14 d裂隙燈顯微鏡下觀察CNV形成情況,併于造模後14d,採用全視網膜鋪片免疫熒光組織化學法檢測角膜組織中CD31在CNV中的錶達.體外實驗中應用ARF抑製劑榦預人視網膜血管內皮細胞(RECs),CCK8法和細胞劃痕法檢測ARF對人RECs增生和遷移的影響.實驗動物的使用和餵養遵循視覺及眼科學研究協會有關規定及囌州大學《實驗動物管理及使用指南》. 結果 造模後PBS對照組和ARF抑製劑榦預組小鼠角膜組織中ARF mRNA在各箇時間點均有錶達,在造模後14d達高峰,各組小鼠角膜中ARF mRNA的錶達隨著時間的延長而增加,差異有統計學意義(F時間=65.17,P=0.00),不同時間點的組間比較差異無統計學意義(F分組=1.98,P=0.18)；造模後14 d,ARF抑製劑榦預組實驗後不同時間點ARF蛋白水平相對錶達值比較差異有統計學意義(F=10.77,P=0.00).裂隙燈顯微鏡下檢查髮現,ARF抑製劑榦預組CNV相對麵積為0.45 ±0.05,PBS對照組為0.72±0.11,2箇組間差異有統計學意義(t=-3.87,P＜0.05).全角膜鋪片免疫熒光組織化學法檢測錶明,ARF抑製劑榦預組小鼠角膜組織CD31錶達麵積明顯小于PBS對照組.造模後14 d,Western blot法檢測顯示,ARF抑製劑榦預組VEGF蛋白錶達水平為1.20±0.21,明顯低于PBS對照組的2.47±0.33,差異有統計學意義(t=-5.62,P＜0.05).CCK8法檢測結果顯示,隨著ARF抑製劑質量濃度的增加,ARF抑製劑對人RECs的抑製率增加,差異有統計學意義(F=8.47,P=0.02).細胞劃痕實驗後24 h,100μg/L和1 000 μg/L ARF抑製劑榦預組人RECs遷移距離分彆為(5.46±1.32) μm和(5.04±1.68)μm,與PBS對照組的(8.49±1.18) μm相比明顯減小,差異均有統計學意義(t=-2.94、-2.91,P＜0.05). 結論 ARF抑製劑能抑製堿燒傷誘導的CNV髮生,可能與其下調VEGF的錶達以及抑製血管內皮細胞的增生和遷移有關.
배경 각막신생혈관(CNV)시도치각막맹적주요원인지일,연구표명,ADP-핵당기화인자(ARF)대종류세포적증생유조절작용,억제ARF능억제혈관적형성,단ARF억제제대CNV시부발휘작용상불청초. 목적 탐토ARF억제제재감소상유도적CNV형성과정중적작용급기궤제.방법 7～8주령청길급웅성BABL/c소서60지안조수궤수자표법분위PBS대조조화ARF억제제간예조,매조각30지.소유소서채용각막감소상유도법구건CNV모형,ARF억제제간예조소서우조모후l주복강내주사0.5 g/L ARF억제제용액0.5 ml,매주3차,공1주,PBS대조조소서이동양방식주사0.5 ml PBS.각조분별취24지소서,우조모후0、4、7、14d채용실시정량PCR(real-time PCR)화Western blot법검측소서각막조직중ARF mRNA화단백적동태표체,병우조모후14 d,채용Western blot법검측각조소서각막조직중혈관내피생장인자(VEGF)적표체.각조령취6지소서,우조모후2、4、7、14 d렬극등현미경하관찰CNV형성정황,병우조모후14d,채용전시망막포편면역형광조직화학법검측각막조직중CD31재CNV중적표체.체외실험중응용ARF억제제간예인시망막혈관내피세포(RECs),CCK8법화세포화흔법검측ARF대인RECs증생화천이적영향.실험동물적사용화위양준순시각급안과학연구협회유관규정급소주대학《실험동물관리급사용지남》. 결과 조모후PBS대조조화ARF억제제간예조소서각막조직중ARF mRNA재각개시간점균유표체,재조모후14d체고봉,각조소서각막중ARF mRNA적표체수착시간적연장이증가,차이유통계학의의(F시간=65.17,P=0.00),불동시간점적조간비교차이무통계학의의(F분조=1.98,P=0.18)；조모후14 d,ARF억제제간예조실험후불동시간점ARF단백수평상대표체치비교차이유통계학의의(F=10.77,P=0.00).렬극등현미경하검사발현,ARF억제제간예조CNV상대면적위0.45 ±0.05,PBS대조조위0.72±0.11,2개조간차이유통계학의의(t=-3.87,P＜0.05).전각막포편면역형광조직화학법검측표명,ARF억제제간예조소서각막조직CD31표체면적명현소우PBS대조조.조모후14 d,Western blot법검측현시,ARF억제제간예조VEGF단백표체수평위1.20±0.21,명현저우PBS대조조적2.47±0.33,차이유통계학의의(t=-5.62,P＜0.05).CCK8법검측결과현시,수착ARF억제제질량농도적증가,ARF억제제대인RECs적억제솔증가,차이유통계학의의(F=8.47,P=0.02).세포화흔실험후24 h,100μg/L화1 000 μg/L ARF억제제간예조인RECs천이거리분별위(5.46±1.32) μm화(5.04±1.68)μm,여PBS대조조적(8.49±1.18) μm상비명현감소,차이균유통계학의의(t=-2.94、-2.91,P＜0.05). 결론 ARF억제제능억제감소상유도적CNV발생,가능여기하조VEGF적표체이급억제혈관내피세포적증생화천이유관.
Background Corneal neovascularization (CNV) is one of the causes of corneal blindness.Studies showed that ADP-ribosylation factor (ARF) can regulate the growth of tumor cells,and inhibiting ARF will decrease angiogenesis.However,whether ARF antagonist plays an action on CNV is unclear.Objective The aim of this study was to explore the effect and mechanism of ARF inhibitor on alkali-burn induced CNV.Methods Sixty clean male BABL/c mice aged 7-8 weeks were divided into PBS group and ARF antagonist group according to randomized number table.CNV models were induced by NaOH burn method in all the mice.ARF at the concentration of 0.5 g/L(0.5 ml) was intraperitoneally injected 3 times per week for 1 week followed the induction of CNV in the ARF antagonist group,and 0.5 ml PBS was used in the PBS group.CNV was examined 2,4,7,14 days after injection by the slit lamp microscope and the CNV related area in the cornea was calculated.Betore modeling(0 day) and 4,7,14 days after modeling,real-time PCR and Western blot were used to analyze the expressions of ARF mRNA and protein in the corneas.Forteen days after modeling,the expression of the CD31 in the CNV was detected using immnofluorescence of corneal whole mount;the expression of vascular endothelial growth factor(VEGF) in the cornea was assayed by Western blot.Cellular wound scratch test was employed to evaluate the effects of ARF antagonist on proliferation and migration of human retinal vascular endothelial cells (RECs).All animal experiments were done in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and Guideline for the Care and Use of Laboratory Animals on the the Soochow University Animal Care Committee.Results ARF mRNA and protein were expressed in the mice corneas in both the PBS group and the ARF antagonist group at various time points.The expression of ARF mRNA in mice corneas was enhanced with the lapse of the time (Ftime =65.17,P =0.00),but no significant difference was found among the groups (Fsroup =1.98,P=0.18).There was also significant difference in the expression of ARF protein in mice corneas at different time points in the ARF antagonist group (F =10.77,P =0.00).The related CNV area was 0.45±0.05 in the ARF antagonist group,and that in the PBS group was 0.72±0.11,with significant difference between them (t =-3.87,P ＜ 0.05).The green fluorescence area of C D31 expression in the cornea was smaller in the ARF antagonist group than that of the PBS group.Expression level of VEGF in the ARF antagonist group was 1.20±0.21,and that in the PBS group was 2.47±0.33,showing a significant difference (t =-5.62,P ＜ 0.05).As the increase of ARF antagonist concentration,the inhibiting rate of cell proliferation was reinforced among 10,100 and 1 000 μg/L ARF antagonist groups (F=8.47,P =0.02).Twenty-four hours after scratch test,the migrating distance of human RECs was (5.46±1.32) μm and (5.04±1.68) μm in the 100 μg/L and 1 000 μg/L ARF antagonist groups,respectively,which were shorter than (8.49± 1.18) μm of the PBS group (t=-2.94,-2.91,both at P＜0.05).Conclusions ARF inhibitor can reduce CNV by down-regulating the expression of VEGF in alkaline burn cornea and inhibiting the proliferation and migration of vascular endothelial cells.
