中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2011年
8期
707-712
,共6页
包永琴%马景学%叶存喜%吕兰存%董白霞%翟英
包永琴%馬景學%葉存喜%呂蘭存%董白霞%翟英
포영금%마경학%협존희%려란존%동백하%적영
角膜新生血管%二十碳五烯酸%白细胞介素-1α%白细胞介素-6%核转录因子κB
角膜新生血管%二十碳五烯痠%白細胞介素-1α%白細胞介素-6%覈轉錄因子κB
각막신생혈관%이십탄오희산%백세포개소-1α%백세포개소-6%핵전록인자κB
Corneal neovascularization%Eicosapentaenoic acid%Interleukin-1α%Interleukin-6%Nuclear factor kappa B
背景 角膜新生血管(CNV)是炎症性角膜病变导致视力下降和角膜移植后发生排斥反应的重要原因,其发生机制和治疗一直是国内外角膜病研究的热点.目的 探讨二十碳五烯酸(EPA)对碱烧伤大鼠CNV核转录因子κB(NF-κB)、白细胞介素1α(IL-1α)、IL-6表达的影响及作用机制.方法 用浸有1 mol/L NaOH的3 mm圆形滤纸贴附于72只SD大鼠的右眼角膜中央30 s制作角膜碱烧伤模型,用随机数字表法将大鼠随机分为3个组,另6只匹配的正常大鼠作为正常对照组.碱烧伤0.02mg EPA治疗组和碱烧伤0.03mg EPA治疗组大鼠分别于碱烧伤后即刻结膜下注射0.04 ml剂量为0.02 mg或0.03mg EPA,碱烧伤模型组结膜下注射0.04ml生理盐水,正常对照组大鼠未行结膜下注射.大鼠干预后每天裂隙灯下观察CNV的生长面积和角膜水肿.分别于造模后24 h,4、7、14 d过量麻醉处死动物后制备角膜标本,采用苏木精-伊红染色法检测各组大鼠角膜的组织病理学变化,免疫组织化学方法检测各组大鼠角膜组织中CD34的表达以计数血管内皮细胞,用逆转录聚合酶链反应(RT-PCR)及蛋白印迹法分别检测各组大鼠角膜组织中IL-1α mRNA和IL-6 mRNA的表达及NF-κBp65的蛋白表达.结果 裂隙灯下见造模后2～4 d,CNV缓慢生长,角膜水肿,上皮缺损;造模后7～10 d,CNV面积增加,角膜水肿减轻;造模后14 d,CNV面积最大,血管变细.苏木精-伊红染色显示,碱烧伤后7 d碱烧伤组角膜上皮部分缺损,基质层水肿明显,可见较多炎性细胞及大量CNV;碱烧伤0.03 mg EPA治疗组与碱烧伤0.02 mg EPA治疗组均可见角膜上皮修复,基质层水肿减轻,少量炎性细胞,未见CNV.碱烧伤后7 d和14 d,碱烧伤0.02 mg EPA治疗组CNV相对面积分别为(15.80±6.43)%、(11.06±2.14)%,碱烧伤0.03 mg EPA治疗组为(16.10±7.41)%、(11.06±2.51)%,均明显小于碱烧伤组的(84.74±7.77)%、(89.63±7.50)%,差异均有统计学意义(P＜0.05).碱烧伤后7 d,碱烧伤组CD34在CNV的内皮细胞中呈强阳性表达,而在碱烧伤0.02 mg EPA治疗组、0.03mg EPA治疗组角膜中未见表达.RT-PCR检测结果表明,碱烧伤后4 d,碱烧伤0.02 mg EPA治疗组、0.03 mg EPA治疗组IL-1α、IL-6mRNA在角膜组织中的表达量明显低于碱烧伤组,蛋白印迹检测证实NF-κB p65蛋白在角膜组织中的表达低于碱烧伤组,差异均有统计学意义(P＜0.05).结论 EPA能够抑制NF-κB-IL-1α、IL-6的表达,从而抑制碱烧伤后CNV的生长.
揹景 角膜新生血管(CNV)是炎癥性角膜病變導緻視力下降和角膜移植後髮生排斥反應的重要原因,其髮生機製和治療一直是國內外角膜病研究的熱點.目的 探討二十碳五烯痠(EPA)對堿燒傷大鼠CNV覈轉錄因子κB(NF-κB)、白細胞介素1α(IL-1α)、IL-6錶達的影響及作用機製.方法 用浸有1 mol/L NaOH的3 mm圓形濾紙貼附于72隻SD大鼠的右眼角膜中央30 s製作角膜堿燒傷模型,用隨機數字錶法將大鼠隨機分為3箇組,另6隻匹配的正常大鼠作為正常對照組.堿燒傷0.02mg EPA治療組和堿燒傷0.03mg EPA治療組大鼠分彆于堿燒傷後即刻結膜下註射0.04 ml劑量為0.02 mg或0.03mg EPA,堿燒傷模型組結膜下註射0.04ml生理鹽水,正常對照組大鼠未行結膜下註射.大鼠榦預後每天裂隙燈下觀察CNV的生長麵積和角膜水腫.分彆于造模後24 h,4、7、14 d過量痳醉處死動物後製備角膜標本,採用囌木精-伊紅染色法檢測各組大鼠角膜的組織病理學變化,免疫組織化學方法檢測各組大鼠角膜組織中CD34的錶達以計數血管內皮細胞,用逆轉錄聚閤酶鏈反應(RT-PCR)及蛋白印跡法分彆檢測各組大鼠角膜組織中IL-1α mRNA和IL-6 mRNA的錶達及NF-κBp65的蛋白錶達.結果 裂隙燈下見造模後2～4 d,CNV緩慢生長,角膜水腫,上皮缺損;造模後7～10 d,CNV麵積增加,角膜水腫減輕;造模後14 d,CNV麵積最大,血管變細.囌木精-伊紅染色顯示,堿燒傷後7 d堿燒傷組角膜上皮部分缺損,基質層水腫明顯,可見較多炎性細胞及大量CNV;堿燒傷0.03 mg EPA治療組與堿燒傷0.02 mg EPA治療組均可見角膜上皮脩複,基質層水腫減輕,少量炎性細胞,未見CNV.堿燒傷後7 d和14 d,堿燒傷0.02 mg EPA治療組CNV相對麵積分彆為(15.80±6.43)%、(11.06±2.14)%,堿燒傷0.03 mg EPA治療組為(16.10±7.41)%、(11.06±2.51)%,均明顯小于堿燒傷組的(84.74±7.77)%、(89.63±7.50)%,差異均有統計學意義(P＜0.05).堿燒傷後7 d,堿燒傷組CD34在CNV的內皮細胞中呈彊暘性錶達,而在堿燒傷0.02 mg EPA治療組、0.03mg EPA治療組角膜中未見錶達.RT-PCR檢測結果錶明,堿燒傷後4 d,堿燒傷0.02 mg EPA治療組、0.03 mg EPA治療組IL-1α、IL-6mRNA在角膜組織中的錶達量明顯低于堿燒傷組,蛋白印跡檢測證實NF-κB p65蛋白在角膜組織中的錶達低于堿燒傷組,差異均有統計學意義(P＜0.05).結論 EPA能夠抑製NF-κB-IL-1α、IL-6的錶達,從而抑製堿燒傷後CNV的生長.
배경 각막신생혈관(CNV)시염증성각막병변도치시력하강화각막이식후발생배척반응적중요원인,기발생궤제화치료일직시국내외각막병연구적열점.목적 탐토이십탄오희산(EPA)대감소상대서CNV핵전록인자κB(NF-κB)、백세포개소1α(IL-1α)、IL-6표체적영향급작용궤제.방법 용침유1 mol/L NaOH적3 mm원형려지첩부우72지SD대서적우안각막중앙30 s제작각막감소상모형,용수궤수자표법장대서수궤분위3개조,령6지필배적정상대서작위정상대조조.감소상0.02mg EPA치료조화감소상0.03mg EPA치료조대서분별우감소상후즉각결막하주사0.04 ml제량위0.02 mg혹0.03mg EPA,감소상모형조결막하주사0.04ml생리염수,정상대조조대서미행결막하주사.대서간예후매천렬극등하관찰CNV적생장면적화각막수종.분별우조모후24 h,4、7、14 d과량마취처사동물후제비각막표본,채용소목정-이홍염색법검측각조대서각막적조직병이학변화,면역조직화학방법검측각조대서각막조직중CD34적표체이계수혈관내피세포,용역전록취합매련반응(RT-PCR)급단백인적법분별검측각조대서각막조직중IL-1α mRNA화IL-6 mRNA적표체급NF-κBp65적단백표체.결과 렬극등하견조모후2～4 d,CNV완만생장,각막수종,상피결손;조모후7～10 d,CNV면적증가,각막수종감경;조모후14 d,CNV면적최대,혈관변세.소목정-이홍염색현시,감소상후7 d감소상조각막상피부분결손,기질층수종명현,가견교다염성세포급대량CNV;감소상0.03 mg EPA치료조여감소상0.02 mg EPA치료조균가견각막상피수복,기질층수종감경,소량염성세포,미견CNV.감소상후7 d화14 d,감소상0.02 mg EPA치료조CNV상대면적분별위(15.80±6.43)%、(11.06±2.14)%,감소상0.03 mg EPA치료조위(16.10±7.41)%、(11.06±2.51)%,균명현소우감소상조적(84.74±7.77)%、(89.63±7.50)%,차이균유통계학의의(P＜0.05).감소상후7 d,감소상조CD34재CNV적내피세포중정강양성표체,이재감소상0.02 mg EPA치료조、0.03mg EPA치료조각막중미견표체.RT-PCR검측결과표명,감소상후4 d,감소상0.02 mg EPA치료조、0.03 mg EPA치료조IL-1α、IL-6mRNA재각막조직중적표체량명현저우감소상조,단백인적검측증실NF-κB p65단백재각막조직중적표체저우감소상조,차이균유통계학의의(P＜0.05).결론 EPA능구억제NF-κB-IL-1α、IL-6적표체,종이억제감소상후CNV적생장.
Background Corneal neovascularization (CNV) is an important cause of visual impairment and graft rejection after allograft corneal transplantation in inflammatory corneal diseases. The mechanisms and therapy relating to CNV are intensely investigated at all times. Objective This study was to evaluate the effect of eicosapentaenoic acid (EPA) on CNV induced by alkali cauterization and its mechanism. Methods The animal models of corneal neovasculation were induced in the right eyes in 72 Sprayue-Dawley rats by putting a piece of 3 mmfilter paper with 1 mol/L NaOH at the center of the cornea for 30 seconds. The rats were then divided randomly into the 0.02 mg EPA treatment group (24 rats) ,0.03 mg EPA treatment group (24 rats) ,model group (24 rats) and normal group (6 rats). EPA of 0.04 ml with doses of 0.02 mg or 0. 03 mg or saline solution of 0. 04 ml was injected subconjunctivally in model rats and immediately after cauterization. The presence of CNV and corneal edema were observed daily by slit lamp biomicroscope. 1,4,7 and 14 days after operation, corneal histopathological examination was performed by hematoxylin and eosin staining. The vascular endothelial cells were stained with CD34 by immunohistochemistry,and the expression of IL-1α,IL-6 mRNA and the nuclear factor-κBp65 ( NF-κBp65 ) proteins was measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. The use of animals complied with the Regulations for the Administration of Affair Concerning Experimental Animals by Hebei Province( version 1998 ). Results Under the slit lamp, CNV grew slowly from days 2-4 with obvious corneal edema and defect of epithelium. Larger CNV area and less edema were seen from days 7-10. Maximal vessel growth was observed 14 days after injury with thinner vessels in the model group. Histological examination showed that part of the corneal epithelium was damaged;serious corneal edema, more inflammatory cells and a lot of CNV in the stroma were presented in the model group. However, repairing of the corneal epithelium without CNV ,light corneal edema and less inflammatory cells were found in both the 0. 02 mg EPA and 0. 03 mg EPA treatment groups 7 days after alkali cauterization. The relative area of CNV in the 0. 02 mg EPA treatment group was ( 15.80±6.43 )% and ( 11.06±2. 14)% ,and that in the 0. 03 mg EPA treatment group was (16. 10±7.41 )% and (11.06±2. 51 )%, showing significant reduction in comparison with the model group [ (84. 74±7.77)% and (89.63±7.50) % ] 7 days and 14 days after operation ( P＜0. 05 ). Stronger expression of CD34 in the vascular endothelial cells of the cornea stroma was observed in the model group and an absence of CD34 was observed in the EPA-treated groups on the 7th day. RT-PCR revealed that the expression of IL-1α mRNA and IL-6 mRNA was lower in the EPA treatment groups than the model group ( P＜0. 05 ), and Western blot analysis showed that the expression of NF-κB/p65 in the corneas in the EPA treatment groups was significantly lower than that in the model group on the 4th day after operation (P＜0.05).Conclusion Topical application of EPA suppresses CNV induced by alkali burn possibly by inhibiting the expression of NF-κB,IL-1α and IL-6.
