中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2014年
9期
773-779
,共7页
紧密连接蛋白%新生血管%碱烧伤%角膜%动物模型%BALB/c小鼠
緊密連接蛋白%新生血管%堿燒傷%角膜%動物模型%BALB/c小鼠
긴밀련접단백%신생혈관%감소상%각막%동물모형%BALB/c소서
Tight junction protein%Neovascularization%Alkali injury%Cornea%Disease model/animal%BALB/c mouse
背景 角膜新生血管(CNV)是导致角膜盲的主要原因之一,研究表明紧密连接蛋白中的闭锁小带蛋白1(ZO-1)对病理性新生血管的发生有调节作用,能通过紧密连接结构构成有效的生理屏障,抑制病理性新生血管的生长,但ZO-1对CNV是否发挥作用尚不清楚. 目的 探讨ZO-1在实验性CNV发生及发展中的作用及其机制.方法 将清洁级7～8周龄雄性BALB/c小鼠24只按随机数字表法随机分为碱烧伤15s组和碱烧伤40 s组,用NaOH滤纸贴附小鼠左眼中央角膜的方法构建CNV模型,于碱烧伤后2周在裂隙灯显微镜下观察并比较两组小鼠CNV生长情况,采用逆转录PCR(RT-PCR)法检测并比较两组小鼠角膜组织中ZO-1 mRNA的表达.另取鼠龄和性别相匹配同种小鼠54只随机分为3个组,均用NaOH滤纸贴附小鼠左眼中央角膜40 s的方法构建CNV模型,造模后分别用质量分数0.2％透明质酸钠(HA)配制的10 mg/L的ZO-1抗体和0.2％ HA配制的5 mg/L缺氧诱导因子-1α(HIF-1α)重组蛋白点眼1周,每日3次.于实验后2周摘除大鼠左眼角膜,用免疫组织化学法检测CD31在CNV中的表达,鉴定CNV的形成数目和面积;采用RT-PCR法检测3个组小鼠角膜组织中血管内皮生长因子(VEGF) mRNA的表达;采用流式细胞术检测碱烧伤角膜中巨噬细胞特异性标志物F4/80、中性粒细胞特异性标志物Ly-6G的阳性细胞比例,评价炎性细胞的浸润情况.结果 裂隙灯显微镜下可见造模后2周小鼠CNV达高峰;碱烧伤15 s组小鼠轻度、中度、重度CNV的眼数分布明显少于碱烧伤40 s组,差异有统计学意义(x2=6.032,P=O.049);碱烧伤15s组和碱烧伤40 s组小鼠角膜中ZO-1 mRNA的相对表达量分别为1.53±0.04和1.15±0.08,差异有统计学意义(t=4.157,P=0.014).免疫组织化学法检测显示,ZO-1抗体干预组和HIF-1α阳性对照组小鼠角膜组织中CD31阳性细胞数多于0.2％ HA组,差异均有统计学意义(t=-129.590、-226.820,均P=0.000),且染色面积明显大于0.2％ HA组,差异均有统计学意义(t=-5.310、-8.840,均P=0.000).ZO-1抗体干预组角膜组织内VEGFmRNA相对表达量为1.33±0.10,HIF-1α阳性对照组为1.46±0.11,均明显高于0.2％ HA组的0.93±0.06,差异均有统计学意义(t=-5.820、-7.284,均P=0.000).ZO-1抗体干预组和HIF-1α阳性对照组小鼠角膜组织中F4/80阳性细胞比例明显多于0.2％ HA组,差异均有统计学意义(t=-16.750、-17.480,均P=0.000);ZO-1抗体干预组和HIF-1α阳性对照组小鼠角膜组织中Ly-6G阳性细胞比例明显多于0.2％ HA组,差异均有统计学意义(t=-21.450、-27.680,均P=0.000). 结论 碱烧伤诱导的CNV中ZO-1表达明显下降,用抗ZO-1蛋白干预后CNV组织中VEGF mRNA表达量升高,炎性细胞浸润更明显,提示ZO-1对CNV影响的作用机制与VEGF的表达量有关.
揹景 角膜新生血管(CNV)是導緻角膜盲的主要原因之一,研究錶明緊密連接蛋白中的閉鎖小帶蛋白1(ZO-1)對病理性新生血管的髮生有調節作用,能通過緊密連接結構構成有效的生理屏障,抑製病理性新生血管的生長,但ZO-1對CNV是否髮揮作用尚不清楚. 目的 探討ZO-1在實驗性CNV髮生及髮展中的作用及其機製.方法 將清潔級7～8週齡雄性BALB/c小鼠24隻按隨機數字錶法隨機分為堿燒傷15s組和堿燒傷40 s組,用NaOH濾紙貼附小鼠左眼中央角膜的方法構建CNV模型,于堿燒傷後2週在裂隙燈顯微鏡下觀察併比較兩組小鼠CNV生長情況,採用逆轉錄PCR(RT-PCR)法檢測併比較兩組小鼠角膜組織中ZO-1 mRNA的錶達.另取鼠齡和性彆相匹配同種小鼠54隻隨機分為3箇組,均用NaOH濾紙貼附小鼠左眼中央角膜40 s的方法構建CNV模型,造模後分彆用質量分數0.2％透明質痠鈉(HA)配製的10 mg/L的ZO-1抗體和0.2％ HA配製的5 mg/L缺氧誘導因子-1α(HIF-1α)重組蛋白點眼1週,每日3次.于實驗後2週摘除大鼠左眼角膜,用免疫組織化學法檢測CD31在CNV中的錶達,鑒定CNV的形成數目和麵積;採用RT-PCR法檢測3箇組小鼠角膜組織中血管內皮生長因子(VEGF) mRNA的錶達;採用流式細胞術檢測堿燒傷角膜中巨噬細胞特異性標誌物F4/80、中性粒細胞特異性標誌物Ly-6G的暘性細胞比例,評價炎性細胞的浸潤情況.結果 裂隙燈顯微鏡下可見造模後2週小鼠CNV達高峰;堿燒傷15 s組小鼠輕度、中度、重度CNV的眼數分佈明顯少于堿燒傷40 s組,差異有統計學意義(x2=6.032,P=O.049);堿燒傷15s組和堿燒傷40 s組小鼠角膜中ZO-1 mRNA的相對錶達量分彆為1.53±0.04和1.15±0.08,差異有統計學意義(t=4.157,P=0.014).免疫組織化學法檢測顯示,ZO-1抗體榦預組和HIF-1α暘性對照組小鼠角膜組織中CD31暘性細胞數多于0.2％ HA組,差異均有統計學意義(t=-129.590、-226.820,均P=0.000),且染色麵積明顯大于0.2％ HA組,差異均有統計學意義(t=-5.310、-8.840,均P=0.000).ZO-1抗體榦預組角膜組織內VEGFmRNA相對錶達量為1.33±0.10,HIF-1α暘性對照組為1.46±0.11,均明顯高于0.2％ HA組的0.93±0.06,差異均有統計學意義(t=-5.820、-7.284,均P=0.000).ZO-1抗體榦預組和HIF-1α暘性對照組小鼠角膜組織中F4/80暘性細胞比例明顯多于0.2％ HA組,差異均有統計學意義(t=-16.750、-17.480,均P=0.000);ZO-1抗體榦預組和HIF-1α暘性對照組小鼠角膜組織中Ly-6G暘性細胞比例明顯多于0.2％ HA組,差異均有統計學意義(t=-21.450、-27.680,均P=0.000). 結論 堿燒傷誘導的CNV中ZO-1錶達明顯下降,用抗ZO-1蛋白榦預後CNV組織中VEGF mRNA錶達量升高,炎性細胞浸潤更明顯,提示ZO-1對CNV影響的作用機製與VEGF的錶達量有關.
배경 각막신생혈관(CNV)시도치각막맹적주요원인지일,연구표명긴밀련접단백중적폐쇄소대단백1(ZO-1)대병이성신생혈관적발생유조절작용,능통과긴밀련접결구구성유효적생리병장,억제병이성신생혈관적생장,단ZO-1대CNV시부발휘작용상불청초. 목적 탐토ZO-1재실험성CNV발생급발전중적작용급기궤제.방법 장청길급7～8주령웅성BALB/c소서24지안수궤수자표법수궤분위감소상15s조화감소상40 s조,용NaOH려지첩부소서좌안중앙각막적방법구건CNV모형,우감소상후2주재렬극등현미경하관찰병비교량조소서CNV생장정황,채용역전록PCR(RT-PCR)법검측병비교량조소서각막조직중ZO-1 mRNA적표체.령취서령화성별상필배동충소서54지수궤분위3개조,균용NaOH려지첩부소서좌안중앙각막40 s적방법구건CNV모형,조모후분별용질량분수0.2％투명질산납(HA)배제적10 mg/L적ZO-1항체화0.2％ HA배제적5 mg/L결양유도인자-1α(HIF-1α)중조단백점안1주,매일3차.우실험후2주적제대서좌안각막,용면역조직화학법검측CD31재CNV중적표체,감정CNV적형성수목화면적;채용RT-PCR법검측3개조소서각막조직중혈관내피생장인자(VEGF) mRNA적표체;채용류식세포술검측감소상각막중거서세포특이성표지물F4/80、중성립세포특이성표지물Ly-6G적양성세포비례,평개염성세포적침윤정황.결과 렬극등현미경하가견조모후2주소서CNV체고봉;감소상15 s조소서경도、중도、중도CNV적안수분포명현소우감소상40 s조,차이유통계학의의(x2=6.032,P=O.049);감소상15s조화감소상40 s조소서각막중ZO-1 mRNA적상대표체량분별위1.53±0.04화1.15±0.08,차이유통계학의의(t=4.157,P=0.014).면역조직화학법검측현시,ZO-1항체간예조화HIF-1α양성대조조소서각막조직중CD31양성세포수다우0.2％ HA조,차이균유통계학의의(t=-129.590、-226.820,균P=0.000),차염색면적명현대우0.2％ HA조,차이균유통계학의의(t=-5.310、-8.840,균P=0.000).ZO-1항체간예조각막조직내VEGFmRNA상대표체량위1.33±0.10,HIF-1α양성대조조위1.46±0.11,균명현고우0.2％ HA조적0.93±0.06,차이균유통계학의의(t=-5.820、-7.284,균P=0.000).ZO-1항체간예조화HIF-1α양성대조조소서각막조직중F4/80양성세포비례명현다우0.2％ HA조,차이균유통계학의의(t=-16.750、-17.480,균P=0.000);ZO-1항체간예조화HIF-1α양성대조조소서각막조직중Ly-6G양성세포비례명현다우0.2％ HA조,차이균유통계학의의(t=-21.450、-27.680,균P=0.000). 결론 감소상유도적CNV중ZO-1표체명현하강,용항ZO-1단백간예후CNV조직중VEGF mRNA표체량승고,염성세포침윤경명현,제시ZO-1대CNV영향적작용궤제여VEGF적표체량유관.
Background Corneal neovascularization (CNV) is one of the causes of corneal blindness.Studies showed that zonula occludens-1 (ZO-1) can inhibit pathological angiogenesis through physical barrier formed by tight junction structure.However,whether ZO-1 plays a role in CNV is unclear.Objective The aim of this study was to explore the effect of ZO-1,a tight junction protein on experimental CNV.Methods The CNV models were established in the left eyes of 24 clear male BALB/c mice aged 7-8 weeks by putting NaOH filter paper in the center of corneas for 15 seconds (15 s group) or 40 seconds (40 s group).CNV was examined and evaluated under the slit lamp microscope,and the expression of ZO-1 mRNA in the corneas were detected and compared by reverse transcription PCR (RT-PCR) between the two groups 2 weeks after modeling.In addition,54 models created by the same method were assigned to 3 groups according to randomized number table,0.2％ hyaluronic acid (HA),antiZO-1 neutralizing antibody (10 mg/L) +0.2％ HA and mouse hypoxia inducible factor-1α (HIF-1α) recombinant protein (5 mg/L)+0.2％ HA were topically administrated in the mice three times a day for 1 week after modeling respectively.The corneas were extracted 2 weeks after application of the drugs.Expression of CD31 in the CNV was assayed to calculate the number and the area of CNV by immunohistochemistry.The expression of VEGF mRNA in the corneas was detected by RT-PCR.The percentages of macrophage-specific F4/80 positive cells and neutrophilsspecific Ly-6G positive cells were calculated to evaluate the infiltrations of inflammatory cells in the corneas by flow cytometry.Results In 2 weeks after alkali burn of corneas,the number of severe CNV was more in the 40 s group than that in the 15 s group (x2 =6.032,P=0.049),and the expression level of ZO-1 mRNA was lower in the 40 s group than that in the 15 s group (1.15±0.08 versus 1.53±0.04) (t=4.157,P=0.014).CD31 positive cell number was more and the staining area was larger in the ZO-1 antibody group and HIF-1α positive control group than those in the 0.2％ HA group (cells:t=-129.590,-226.820,both at P=0.000;area:t =-5.310,-8.840,both at P=0.000).The relative expressions level of vascular endothelial growth factor (VEGF) mRNA was 1.33±0.10 and 1.46±0.11 in the ZO-1 antibody group and HIF-1 α positive control group respectively,which were significantly higher than 0.93±0.06 of the 0.2％ HA group (t =-5.820,-7.284,both at P =0.000).The percentages of positive cells in the ZO-1 antibody group and HIF-1α positive control group were significantly increased in comparison with the 0.2％ HA group for F4/80 (t =-16.750,-17.480,both at P =0.000) and for Ly-6G (t =-21.450,-27.680,both at P=0.000).Conclusions Alkali burn induced CNV downregulates the expression of ZO-1 mRNA.Administration of ZO-1 antibody causes the rise of VEGF mRNA in CNV and the infiltration inflammation cells,which suggests that the influence of ZO-1 on CNV is associated with the expression of VEGF.
