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。     

Avastin对小鼠角膜碱烧伤后新生淋巴管形成的影响

目的观察小鼠角膜碱烧伤后新生淋巴管形成情况,探讨Avastin对小鼠角膜碱烧伤后新生淋巴管形成的影响及其机制。方法制作小鼠角膜碱烧伤模型,随机分为治疗组和烧伤组,烧伤组连续两周内每日左氧氟沙星滴眼液滴眼3次,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、5d、7d、12d和18d取材。采用免疫组化法,观察VEGF-C在碱烧伤后不同时间段角膜组织内的表达;应用淋巴管内皮透明质酸受体(LYVE-1)标记淋巴管内皮,观察小鼠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。在烧伤组碱烧伤角膜内,VEGF-C主要表达于角膜上皮细胞和角膜基质内入侵的炎性细胞,于碱烧伤后3d,VEGF-C的表达达到高峰(0.05),碱烧伤后12d,VEGF-C的表达恢复至正常水平。在治疗组碱烧伤角膜内,VEGF-C的表达在碱烧伤后3d低于烧伤组。Avastin治疗组角膜内,碱烧伤后12d见LYVE-1阳性表达开放状态的新生淋巴管,淋巴管出现的时间较烧伤组明显延迟。结论 Avastin能减少小鼠碱烧伤后角膜新生淋巴管的形成,其机制可能与抑制角膜组织内淋巴管生成因子VEGF-C的表达相关。     

淋巴管内皮细胞透明质酸受体-1在发育小鼠肾内的表达

目的:探讨淋巴管内皮细胞透明质酸受体-1(LYVE-1)在发育小鼠肾内的表达.方法:胚胎期第13～18天(E13～18)的胎鼠和出生后第4、 14和21天(P4～21)及成年小鼠的肾,行LYVE-1免疫组织化学显色.结果:LYVE-1免疫阳性淋巴管丛最早在胚胎期第14天的小鼠肾检测到,主要位于肾皮质的动脉周围.在胚胎期第15天的肾小球中最早检测到LYVE-1免疫阳性细胞,但LYVE-1免疫阳性细胞在肾小球中的数量和位置不定.结论:LYVE-1免疫阳性细胞主要位于肾动脉周围淋巴管,在肾小球中也有LYVE-1表达.     

肺癌组织及淋巴管E-cadherin、β-catenin和LYVE-1的表达

目的观察肺癌组织E-cadherin和β-catenin的表达,LYVE-1特异性标记淋巴管;探讨钙黏蛋白及其受体在癌细胞淋巴道转移中的作用。方法取肺癌手术材料30例,通过免疫组化法,观察E-cadherin、β-catenin和LYVE-1在癌细胞及淋巴管的表达。结果癌细胞对E-cadherin、β-catenin呈阳性表达,低分化组、有淋巴结转移组E-cadherin表达减弱。淋巴管对LYVE-1阳性表达,E-cadherin阴性表达,β-catenin弱阳性表达。结论LYVE-1在淋巴管特异性表达;E-cadherin表达与肿瘤分化程度和有无淋巴结转移呈负相关;E-cadherin/β-catenin复合物对肿瘤细胞与淋巴管内皮细胞的黏附不起主要作用。     

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。     

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。     

吲哚类化合物MAZ51对小鼠移植瘤生长及淋巴管生成的影响

目的:观察吲哚类化合物MAZ51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,成瘤后给予MAZ51,观察其对移植瘤生长的影响.免疫组织化学标记淋巴管,分析移植瘤周边区和正常皮肤的毛细淋巴管密度(LMVD).结果:移植瘤周边区的LMVD高于正常皮肤组织.给予MAZ51后,明显减慢移植瘤的生长,但瘤周边区的LMVD并没有变化.结论:实验结果显示肿瘤周边区有淋巴管新生.MAZ51影响移植瘤的生长,但不影响淋巴管的生成.     

角膜缘干细胞移植治疗兔眼表碱烧伤的实验研究

目的 :探讨眼表碱烧伤后行角膜缘干细胞移植术的疗效 ,并与单纯行角膜移植术组作比较。方法 :在 16只兔双眼上制作碱烧伤模型 ,1d后 ,右眼行异体板层角膜缘干细胞移植术 ,左眼行异体板层角膜移植术 ,术后观察 6月 ,根据术眼的角膜新生血管分级、植片的混浊度、水肿度来计算移植排斥反应指数 (RI) ,以RI值判断植片的存活情况。结果 :行异体板层角膜缘干细胞移植术的 16眼中有 14眼RI <9,治愈率达 87.5 % ;行单纯异体板层角膜移植的 16眼中 6眼RI值 <9,有效率 3 7.5 % ;两者相比有显著性差异 (P <0 .0 0 1)。两组的新生血管数、植片的混浊度、水肿度及RI值均有显著性差异。结论 :含干细胞的异体角膜缘移植治疗眼表碱烧伤的疗效明显优于单纯异体角膜移植 ,是一种较有效的方法。     

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。     

血管内皮生长因子C及其受体3在人恶性黑色素瘤组织内的表达及意义

目的 观察人恶性黑色素瘤组织内血管内皮生长因子C(VEGF-C)及其受体3(VEGFR-3)的表达,探讨VEGF-C和VEGFR-3在恶性黑色素瘤淋巴管生成及淋巴道转移中的作用.方法 取人恶性黑色素瘤组织48例(石蜡标本30例,术后新鲜组织18例),应用免疫组织化学和RT-PCR技术,观察VEGF-C和VEGFR-3蛋白及mRNA在恶性黑色素瘤组织内的表达情况.以淋巴管内皮透明质酸受体(LYVE-1)标记淋巴管,计数恶性黑色素瘤组织淋巴管数密度.结果 VEGF-C和VEGFR-3蛋白主要表达于恶性黑色素瘤细胞胞浆内,在肿瘤周围的血管和淋巴管内皮上也可见VEGFR-3蛋白表达,VEGF-C和VEGFR-3蛋白在淋巴结转移组恶性黑色素瘤组织内的表达水平明显高于无淋巴结转移组(P<0.05).在18例新鲜恶性黑色素瘤中,淋巴结转移组VEGF-C和VEGFR-3mRNA的表达明显高于无淋巴结转移组(P<0.01).LYVE-1表达于肿瘤间质内的淋巴管内皮细胞,淋巴结转移组恶性黑色素瘤组织中的淋巴管数密度(LMVD)为9.845±2.454,无淋巴结转移组恶性黑色素瘤组织中的淋巴管数密度为6.534±2.193,淋巴结转移组恶性黑色素瘤组织内的淋巴管数密度明显高于无淋巴结转移组(P<0.01).结论恶性黑色素瘤组织内VEGF-C表达明显增高,并通过上调其受体VEGFR-3的表达促进恶性黑色素瘤组织内淋巴管的生成,从而促进恶性黑色素瘤的淋巴道转移.     

小鼠恶性黑色素移植瘤模型的建立及其淋巴管内皮细胞透明质酸受体1的表达

目的:建立小鼠皮肤恶性黑色素移植瘤模型,探讨小鼠皮肤恶性黑色素移植瘤组织内淋巴管的生成情况。方法:体外培养B16瘤细胞,接种B16瘤细胞于C57BL/6小鼠右侧背部皮内,构建C57BL/6小鼠皮肤恶性黑色素移植瘤模型;应用H-E染色、淋巴管内皮细胞透明质酸受体1(1ymphatic vessel endothelial hyaluronan receptor-1, LYVE-1)免疫组化染色确认模型和观察肿瘤组织内淋巴管的生成情况。结果:建立了恶性黑色素移植瘤模型;LY- VE-1主要着色于正常组织和移植瘤组织中淋巴管内皮细胞的细胞膜上;恶性黑色素瘤组织中淋巴管密度明显高于正常皮肤组织。结论:构建C57BL/6小鼠皮肤恶性黑色素移植瘤模型是获得恶性黑色素瘤组织和进一步研究的有效手段;LYVE-1是特异性较高的淋巴管内皮标记物;小鼠皮肤恶性黑色素移植瘤组织中可能存在淋巴管的新生。     

K14 + Compound niches are present on the mouse cornea early after birth and expand after debridement wounds

Background: We previously identified compound niches (CNs) at the limbal:corneal border of the mouse cornea that contain corneal epithelial progenitor cells, express Keratin 8 (K8), and goblet cell mucin Muc5AC. During re‐epithelialization after 2.5 mm epithelial debridement wounds, CNs migrate onto the cornea and expand in number mimicking conjunctivalization. When CNs form during development and whether they express corneal epithelial progenitor cell enriched K14 was not known. Results: To provide insight into corneal epithelial homeostasis, we quantify changes in expression of simple (K8, K18, K19) and stratified squamous epithelial keratins (K5, K12, K14, and K15) during postnatal development and in response to 2.5 mm wounds using quantitative polymerase chain reaction (Q‐PCR), confocal imaging and immunoblots. K14 + CNs are present 7 days after birth. By 21 days, when the eyelids are open, K8, K19, and Muc5AC are also expressed in CNs. By 28 days after wounding, the corneal epithelium shows enhanced mRNA and protein expression for K14 and retains mRNA and protein for corneal epithelial specific K12. Conclusions: The keratin phenotype observed in corneal epithelial cells before eyelid opening is similar to that seen during wound healing. Data show K14 + corneal epithelial progenitor cells expand in number after 2.5 mm wounds. Developmental Dynamics 245:132–143, 2016. © 2015 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc.     

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.     

直肠腺癌VEGF-C及VEGFR-3的表达与淋巴转移的关系

目的为探讨癌细胞淋巴管转移机理,观察血管内皮生长因子-C(VEGF-C)和血管内皮生长因子受体-3(VEGFR-3)在直肠腺癌组织及淋巴管的表达。方法取人直肠腺癌手术材料30例,用免疫组化方法检测癌区和癌周正常区VEGF-C和VEGFR-3的表达情况。结果VEGF-C主要表达在直肠腺癌的癌细胞胞浆,VEGFR-3主要在淋巴管内皮细胞有阳性表达,两者在癌区的表达率均高于正常区直肠组织;癌区淋巴管的平均面密度(33.81±5.67)高于正常区平均面密度(20.13±3.27)。结论VEGF-C和VEGFR-3在人直肠腺癌中的过表达,可能与淋巴管增生和扩张,促进癌细胞的淋巴转移有关。     

Investigation of intratumoural and peritumoural lymphatics expressed by podoplanin and LYVE-1 in the hybridoma-induced tumours

Tumour-associated lymphatics contribute to a key component of metastatic spread, however, the biological interaction of tumour cells with intratumoural and peritumoural lymphatics (ITLs and PTLs) has remained unclear. To address this important issue, we have focused on the morphological and molecular aspects of newly formed lymphatics (lymphangiogenesis) and pre-existing lymphatics in the intratumoural and peritumoural tissues by using a hybridoma-induced tumour model. In the present study, ITLs with very high vessel density within the tumour mass showed small and flattened contours that varied from non-solid-to-solid tumours, whereas PTLs were relatively disorganized and tortuous, and packed with a cluster of tumour cells at the tumour periphery. Lymphatic endothelial cells (LECs) both in ITLs and PTLs were expressed with LYVE-1 and podoplanin in various tumour tissues, in which initial lymphatics were extremely extended and dilated. The tumour cells were frequently detected adhering to or penetrating lymphatic walls, especially near the open junctions. In the metastatic tissues, lymphangiogenic vasculatures occurred within the tumour matrix, and collecting PTLs represented abnormal twisty valve leaflets. The Western blot and RT-PCR analysis showed local variations of LEC proliferating potentials and lymphatic involvement in metastasis by a distinct profile of the protein and mRNA expression by LYVE-1, podoplanin, Prox-1 and vascular endothelial growth factor-3 (VEGFR-3). These findings indicated that both ITLs and PTLs, including enlarged pre-existing and newly formed lymphatics, may play a crucial role in metastasis with an active tumour cell adhesion, invasion, migration and implantation.