血管内皮生长因子C在小鼠碱烧伤角膜内的表达及意义

目的观察血管内皮生长因子C(VEGF-C)在小鼠角膜碱烧伤后不同时间段角膜组织内的表达情况,探讨VEGF-C在小鼠角膜碱烧伤后新生淋巴管形成过程中的作用。方法制作小鼠角膜碱烧伤模型,分别于碱烧伤后1d、3d、5d、7d、12d和18d取材。采用免疫组化法(SP),观察VEGF-C在正常角膜和碱烧伤后不同时间段角膜组织内的表达情况。应用淋巴管内皮透明质酸受体(LYVE-1)标记淋巴管,观察小鼠碱烧伤角膜内新生淋巴管的形成情况。结果在正常小鼠角膜组织内,VEGF-C表达于角膜上皮层和内皮层。在碱烧伤角膜内,VEGF-C主要表达于角膜基质内入侵的炎性细胞和角膜上皮层细胞,并且表达增高,于碱烧伤后3d,VEGF-C的表达达到高峰(P<0.01)。碱烧伤后7d,VEGF-C的表达下降至正常水平,可见阳性表达LYVE-1的处于开放状态的新生淋巴管。结论VEGF-C可能参与小鼠角膜碱烧伤后角膜新生淋巴管形成过程。     

血管内皮生长因子D在小鼠碱烧伤后角膜的表达及其作用

目的观察血管内皮生长因子D(VEGF-D)在小鼠角膜碱烧伤后不同时间角膜组织内的表达,探讨VEGF-D在小鼠角膜碱烧伤后新生淋巴管形成过程中的作用。方法制作小鼠角膜碱烧伤模型,分别于碱烧伤后1d、3d、5d、7d、12d和18d取材。应用免疫组化SP法观察VEGF-D在正常角膜和碱烧伤后不同时间角膜内的表达,应用淋巴管内皮透明质酸受体1(LYVE-1)标记淋巴管,观察小鼠碱烧伤角膜内新生淋巴管的形成情况。结果碱烧伤后1d、3d、5d,角膜内VEGF-D表达水平明显高于正常角膜(P<0.01),于碱烧伤后3d,表达达到高峰。碱烧伤后7d,VEGF-D的表达下降至正常水平。在碱烧伤角膜内,可见阳性表达LYVE-1的新生淋巴管。结论 VEGF-D过表达可能参与小鼠角膜碱烧伤后新生淋巴管形成过程。     

LYVE-1蛋白在小鼠碱烧伤角膜的表达及意义

目的观察淋巴管内皮透明质酸受体(LYVE-1)在小鼠碱烧伤角膜内的表达情况,探讨角膜新生淋巴管形成的时间过程及角膜疾病后新生淋巴管形成的作用。方法应用NaOH溶液制作小鼠角膜碱烧伤模型,分别于角膜碱烧伤后第1d、3d、5d、7d和12d取材。采用免疫组化法,观察正常角膜和碱烧伤后不同时间段角膜内LYVE-1的表达情况。结果在正常角膜组织中,LYVE-1表达于角膜上皮细胞和内皮细胞内。在角膜碱烧伤后1d、3d和5d,LYVE-1主要表达于角膜上皮内及入侵角膜基质的炎性细胞内;碱烧伤后7d,可见大量LYVE-1呈条索样表达于角膜基质中,并可见少量LYVE-1阳性表达于开放状态的淋巴管;碱烧伤后12d,角膜基质内新生淋巴管数量增多。结论正常角膜组织储备LYVE-1生物因子,在炎性角膜中,LYVE-1可能在角膜新生淋巴管运输透明质酸(HA)的过程中发挥重要作用。     

Avastin对小鼠角膜新生血管形成和TGF-β1表达的影响

目的探讨Avastin对小鼠角膜新生血管形成和转化生长因子-β1(transforming growth factor-beta1,TGF-β1)表达的影响。方法制作小鼠角膜碱烧伤模型,随机分为碱烧伤组和Avastin治疗组,碱烧伤组连续两周内每日左氧氟沙星滴眼液滴眼3次,Avastin治疗组连续两周内应用25mg/ml Avastin结膜下隔日注射5μl+每日左氧氟沙星滴眼液滴眼3次,分别于碱烧伤后3d、7d和12d取材。体视显微镜观察角膜新生血管形成情况,应用HE染色法观察角膜组织的损伤情况,采用免疫组化法观察TGF-β1在碱烧伤后不同时间段角膜组织内的表达情况。结果在不同时间段,Avastin治疗组角膜新生血管的形成均少于碱烧伤组。在正常小鼠角膜组织内,TGF-β1表达于角膜上皮层和内皮层。在碱烧伤对照组角膜内,TGF-β1主要表达于角膜上皮细胞和角膜基质内入侵的炎性细胞,在碱烧伤后3d,TGF-β1的表达达到高峰(P0.05),在碱烧伤后12d,TGF-β1的表达恢复至正常水平。在Avastin治疗组角膜内,各时间段TGF-β1的表达均低于对照组。结论 Avastin能够抑制小鼠碱烧伤后角膜新生血管的形成,其机制可能与下调碱烧伤角膜组织TGF-β1的表达相关。     

血管抑素抑制兔眼表碱烧伤角膜缘移植术后的角膜血管新生

目的:检测眼表碱烧伤角膜缘移植术后血管抑素抑制角膜新生血管的作用。方法:16只新西兰大白兔双眼制作碱烧伤模型1d后,双眼行角膜缘移植术,术后左眼局部应用血管抑素治疗2周,右眼作对照;观测4周,根据新生血管侵入角膜缘内的范围、角膜混浊与水肿程度进行分级并作统计学处理;同时测量术后7、14、21及28d的眼压。结果:术后4周时,应用血管抑素的左眼的新生血管的评分为1.19±0.10,而对照组为1.63±0.72,统计学处理显示左眼的新生血管较右眼的明显减少(P<0.05),角膜混浊与水肿程度亦明显下降。各时间点各术眼的眼压均在正常范围,无统计学差异。结论:局部应用血管抑素能有效抑制眼表碱烧伤角膜缘移植术后的新生血管增生。     

诺帝滴眼液抑制碱烧伤诱导大鼠角膜新生血管分子机制的初步研究

目的 初步探索0.1%诺帝滴眼液对角膜新生血管(corneal neovascularization,CRNV)抑制作用的分子机制.方法 建立大鼠碱烧伤角膜新生血管模型,分空白溶液组、0.1%诺帝滴眼液组、0.1%地塞米松滴眼液组.采用免疫组化及RT-PCR法检测VEGF、flk-1蛋白及mRNA表达.结果 治疗组及地塞米松组VEGF蛋白表达少于空白溶液组(P<0.05).7 d、14 d VEGF mRNA抑制率分别为:治疗组58.60%,74.60%;地塞米松组76.88%,80.95%.flk-1 mRNA抑制率:治疗组66.27%,69.63%;地塞米松组72.16%,74.81%.结论 0.1%诺帝滴眼液明显降低角膜VEGF和其受体flk-1 mRNA蛋白表达,通过下调VEGF受体量,间接减少VEGF受体后信号转导及效应蛋白合成,抑制了CRNV的发生.     

色素上皮衍生因子对角膜碱烧伤后新生血管形成的抑制

背景：角膜新生血管导致角膜透明性降低,造成严重的视觉障碍。色素上皮衍生因子是一种内源性血管生成抑制剂,其对于角膜新生血管是否具有抑制作用尚不清楚。 目的：探索局部运用色素上皮衍生因子对大鼠角膜碱烧伤后角膜新生血管的抑制作用。 方法：将20只大鼠随机分为生理盐水组与色素上皮衍生因子组,每组10只。用NaOH溶液将大鼠右眼角膜烧伤诱导产生新生血管。碱烧伤后2组每日分别给予生理盐水和色素上皮衍生因子点眼,并采用裂隙灯显微镜观察和测量各组角膜新生血管生长情况。碱烧伤后12 d处死大鼠,将角膜组织固定切片,行苏木精-伊红染色观察,并进行免疫组织化学染色检测各组大鼠角膜血管内皮生长因子和CD31的表达。 结果与结论：大鼠角膜碱烧伤后3,7,12 d,色素上皮衍生因子组大鼠角膜新生血管面积均小于生理盐水组(P < 0.05)。角膜碱烧伤后12 d,苏木精-伊红染色显示生理盐水组大鼠角膜产生大量新生血管,角膜组织结构紊乱;色素上皮衍生因子组新生血管较少,角膜组织结构趋于整齐。角膜碱烧伤后12 d,免疫组织化学染色示生理盐水组大鼠角膜上皮和基质层可见血管内皮生长因子大量表达,角膜基质层可见血管内皮生长因子和CD31大量表达;色素上皮衍生因子组新生血管稀少,CD31表达较弱。证实局部应用色素上皮衍生因子可有效抑制大鼠角膜化学伤后的血管新生。     

重组IFN-α蛋白联合endostatin基因对碱烧伤诱导角膜新生血管的抑制作用

目的探讨重组IFN-α蛋白联合endostatin基因对碱烧伤诱导角膜新生血管的抑制作用。方法兔眼球结膜下注射包有绿色荧光蛋白表达载体的脂质体，3d后用激光共聚焦显微镜观察角膜绿色荧光蛋白表达。利用碱烧伤法制备兔眼角膜新生血管模型，球结膜下联合注射重组IFN-α蛋白及包有endostatin真核表达载体的脂质体，用裂隙灯显微镜观察对新生血管的抑制作用。结果实验组角膜有很强的绿色荧光，而在对照组则无荧光。联合应用重组IFN-α蛋白和endostatin基因治疗，第7、10、13天角膜新生血管长度、面积明显小于重组IFN-α蛋白和endostatin基因的单独治疗组（P〈0．05）。结论重组IFN-α蛋白联合endostatin基因可有效抑制碱烧伤诱导角膜新生血管的生长。     

塞来昔布对小鼠移植瘤生长及淋巴管生成的影响

目的观察塞来昔布(Celecoxib)对小鼠移植瘤生长、COX-2、VEGF-C表达和淋巴管生成影响,探讨抑制肿瘤淋巴转移的机制。方法构建S180小鼠移植瘤模型,应用大体观察、HE染色、免疫组化和Western blot,比较对照组和Celecoxib给药组荷瘤鼠肿瘤生长、COX-2、VEGF-C表达和淋巴管分布。结果 Celecoxib组与对照组相比瘤体生长缓慢,第8周时肿瘤体积明显比对照组小,COX-2和VEGF-C的表达均下调,淋巴管密度降低。结论 Celecoxib抑制肿瘤的转移和生长可能与下调COX-2表达,减少VEGF-C的产生和淋巴管生成有关。     

人参皂甙Rh2对小鼠移植瘤血管内皮生长因子C及淋巴管生成的影响

目的 观察人参皂甙Rh2对小鼠移植瘤生长,肿瘤细胞血管内皮生长因子c(VEGF-C)表达和淋巴管密度的影响,探讨其作用机制.方法 用S180瘤株构建55只小鼠移植瘤模型,成瘤后灌服人参皂甙Rh2,观察用药组与对照组移植瘤的生长情况,免疫组织化学染色,比较用药2周、3周后癌细胞VEGF-C的表达及LYVE-1标记的淋巴管密度与对照组的差异.结果接种约第3周开始,对照组移植瘤生长速度明显快于用药组.用药第2周癌细胞VEGF-C表达及淋巴管密度与对照组无差异;第3周VEGF-C表达较对照组弱,淋巴管密度也较对照组低,有差异(P<0.05). 结论 人参皂甙Rh2能抑制肿瘤生长,降低淋巴管密度,其机制可能是通过降低VEGF-C在癌细胞的表达,干扰淋巴管的生成.     

Transient downregulation of microRNA-206 protects alkali burn injury in mouse cornea by regulating connexin 43

Purpose: Chemical burn in cornea may cause permanent visual problem or complete blindness. In the present study, we investigated the role of microRNA 206 (miR-206) in relieving chemical burn in mouse cornea. Method: An alkali burn model was established in C57BL/6 mice to induce chemical corneal injury. Within 72 hours, the transient inflammatory responses in alkali-treated corneas were measured by opacity and corneal neovascularization (CNV) levels, and the gene expression profile of miR-206 was measured by quantitative real-time PCR (qPCR). Inhibitory oligonucleotides of miR-206, miR-206-I, were intrastromally injected into alkali-burned corneas. The possible protective effects of down-regulating miR-206 were assessed by both in vivo measurements of inflammatory responses and in vitro histochemical examinations of corneal epithelium sections. The possible binding of miR-206 on its molecular target, connexin43 (Cx43), was assessed by luciferase reporter (LR) and western blot (WB) assays. Cx43 was silenced by siRNA to examine its effect on regulating miR-206 modulation in alkali-burned cornea. Results: Opacity and CNV levels, along with gene expression of miR-206, were all transiently elevated within 72 hours of alkali-burned mouse cornea. Intrastromal injection of miR-206-I into alkali-burned cornea down-regulated miR-206 and ameliorated inflammatory responses both in vivo and in vitro. LR and WB assays confirmed that Cx43 was directly targeted by miR-206 in mouse cornea. Genetic silencing of Cx43 reversed the protective effect of miR-206 down-regulation in alkali-burned cornea. Conclusion: miR-206, associated with Cx43, is a novel molecular modulator in alkali burn in mouse cornea.     

大鼠角膜碱烧伤后热休克蛋白70的表达

背景：角膜碱烧伤后损伤修复受许多因素的影响,热休克蛋白可促进变性、损伤蛋白质的迅速恢复或清除。 目的：观察大鼠角膜碱烧伤后热休克蛋白70的表达及其与角膜损伤修复的关系。 方法：检查大鼠眼无炎症及其他病变后,奥布卡因滴眼液点眼2次,棉签吸除结膜囊液体,将统一规格直径5 mm的滤纸片浸泡于1 mol/L NaOH溶液中10 s,然后置于大鼠角膜中央30 s制作大鼠角膜碱烧伤模型。分别于碱烧伤后6 h,1,3,7,14,21 d取材。 结果与结论：RT-PCR、免疫组织化学染色、Western blot结果均显示热休克蛋白70 mRNA和蛋白在角膜碱烧伤后1 d即开始升高,7 d时达高峰,14 d后开始下降。苏木精-伊红染色及电镜观察显示角膜损伤在烧伤后6 h即较明显,烧伤后7 d逐渐恢复。提示大鼠角膜碱烧伤后热休克蛋白70的表达与碱烧伤后角膜损伤修复过程一致,参与了大鼠角膜碱烧伤后细胞的自我保护及修复过程。     

The effect of sub-conjunctival platelet-rich plasma in combination with topical acetylcysteine on corneal alkali burn ulcer in rabbits

An experimental study examined the effect of sub-conjunctival platelet-rich plasma (sPRP) in combination with topical acetylcysteine on corneal alkali burn ulcers in rabbits. A total of 20 rabbits were used in this study. After collecting intracardiac blood samples from ten rabbits, platelet-rich plasma was obtained by centrifugation. Alkali wounds were inflicted on the central corneas of rabbits by applying a round filter paper, 6.0 mm in diameter, soaked in 1 M NaOH for 60 s. Only one eye in each rabbit was used. A total of 20 rabbits were allocated into four groups of five animals each. Group 1 served as the control group. Group 2 received 3% N-acetylcysteine (NAC) topically three times daily for 2 weeks. The third group received only sPRP whereas group 4 received sPRP with topical 3% NAC three times daily for 2 weeks. Clinical outcome was monitored by evaluation of epithelial defects, corneal opacity, duration of blepharospasm, corneal vascularisation, duration of ocular discharge and wound area diameter measurement. After 3 weeks eyes were enucleated and corneas were excised for histopathological analysis. Samples were assessed by evaluating the number of epithelial rows, stromal vascularisation and inflammation and stromal collagen arrangement. Comparison between groups showed that group 3 had significantly shorter duration of blepharospasm than the control group. Additionally, group 3 had smaller mean defect area and greater wound healing. Histopathlogical investigation revealed significantly less inflammation and vascularisation in the corneas in group 3; this group also had the best stromal collagen arrangement. In conclusion sPRP seems to improve corneal epithelial burn healing. However, acetylcysteine and sPRP combination may have a retarded healing effect as compared with platelet-rich plasma alone.     

辐照灭菌冷冻干燥羊膜修复碱烧伤

背景：经辐照灭菌的冷冻干燥羊膜便于保存和使用,但羊膜经这种方式处理后其活性是否会降低、其临床疗效是否会受到影响至今仍鲜有报道。 目的：观察辐照灭菌后的冷冻干燥羊膜对兔角膜碱烧伤的修复作用。 方法：取健康成年白兔9只,均用1 mol/L 氢氧化钠烧伤角膜后,左侧眼设为羊膜组植入辐照灭菌后的冷冻干燥的羊膜,右侧眼设为对照组不植入羊膜。植入后30 d内裂隙灯显微镜每日观察角膜透明度、角膜新生血管及角膜上皮修复情况。 结果与结论：羊膜植入后30 d,羊膜组角膜透明度、新生血管及角膜上皮修复情况明显优于对照组(P < 0.05),角膜上皮愈合好,基质纤维排列更为整齐,炎细胞浸润程度轻,新生血管更少。结果证实,兔角膜碱烧伤后植入辐照灭菌后的冷冻干燥羊膜可减轻角膜炎性反应,促进角膜上皮修复,减少角膜新生血管形成。     

The mitotic activity of the cornea in mice after infliction of a burn at different times of day

Summary The 24-hour rhythm of cellular division is preserved in symmetrical cornea by inflicting corneal burns in the morning. However, the average values of mitotic coefficients during the whole experiment appear to be lower than in the cornea of normal animals.The infliction of burn in the evening has no effect on the character of the 24 hour rhythm. However the general level of mitotic activity is much higher. A stable depression of the tempo of cellular division was noted in the injured cornea.Presented by Active Member AMN SSSR N. N. Zhukov-Verezhnikov     

D-Limonene对小鼠移植瘤生长及淋巴管生成的影响

目的:观察右旋柠烯对小鼠移植瘤生长及淋巴管生成的影响,并探讨其作用机制。方法:皮下注射肉瘤(S180)腹水型瘤株构建小鼠移植瘤模型,给予D-Lim onene干预,免疫组化染色观察瘤细胞V EG F-C表达,LY V E-1标记淋巴管,观察其分布。结果:对照组瘤细胞V EG F-C表达较强,瘤周边部淋巴管较多,有淋巴结、肺转移。用药组瘤细胞V EG F-C表达较弱;瘤周边部淋巴管较少,未见淋巴结、肺转移。结论:D-Lim onene有抑制移植瘤内瘤细胞V EG F-C表达和淋巴管生成的作用,有可能降低肿瘤的淋巴道转移。