羊膜培养液抑制角膜新生血管的实验研究

目的　研究羊膜培养液对小鼠角膜新生血管的抑制作用及机制。方法　应用cornealmicropocket方法 ,将含 10 0ng碱性成纤维细胞生长因子 (bFGF)和 12 %hydronpolymer的缓释颗粒植入角膜层间 ,制备小鼠角膜新生血管模型。实验分对照组、羊膜组和去上皮羊膜组 ,每组 10只眼 ,收集各组培养液于模型制作当日起 ,每日滴眼 4次至术后 7d摄片观测角膜新生血管长入情况并处死动物。对脐静脉血管内皮细胞 (HUVEC)进行体外培养 ,并应用Boyden小房技术和荧光结合(CyQUANT)实验分别检测羊膜培养液对HUVEC细胞迁移和细胞增殖的影响。利用酶联免疫吸附(ELISA)法检测各组培养液中金属蛋白酶抑制剂 1(TIMP1)和 2 (TIMP2 )的含量。结果　羊膜培养液明显抑制由bFGF诱发的小鼠角膜新生血管的形成 ,对照组、羊膜组和去上皮羊膜组角膜新生血管面积分别是 (2 4 8± 0 76 )mm2 、(0 6 4± 0 5 2 )mm2 和 (1 96± 0 6 5 )mm2 。羊膜培养液明显抑制血管内皮细胞的迁移和增殖 ,而对照组和去上皮羊膜组对HUVEC细胞迁移无作用。羊膜培养液中TIMP2水平明显增高 ,而TIMP1的含量无变化。结论　羊膜培养液中TIMP1的含量未增加。而羊膜上皮产生或释放大量的TIMP2 ,抑制血管内皮细胞的迁移和增殖 ,其可能是羊膜培养液抑制角膜新生血管的     

羊膜上皮细胞培养液抑制角膜基质细胞凋亡的实验研究

目的 对羊膜上皮细胞培养液抑制由肿瘤坏死因子(TNF-α)诱发的角膜基质细胞凋亡进行研究。方法 体外培养兔角膜基质细胞(RCK),30ng/ml TNF-α诱发角膜基质细胞凋亡。实验分为对照组、羊膜上皮细胞培养液组(羊膜组)和阴性对照组。应用FITC-dUTP-TUNEL和DAPI染色检测RCK细胞凋亡发生率,分别测定经24h培养后各组细胞凋亡特异性DNA梯形降解。Western blot检测细胞凋亡保护性因子Bcl-2、促进因子Bax在经羊膜上皮细胞培养液处理后于细胞中表达强度及Bcl-2/Bax比值变化。结果 羊膜上皮细胞培养液明显抑制由TNF-α诱发的兔角膜基质细胞凋亡,DAPI染色显示24h对照组,羊膜培养液组和阴性对照组细胞凋亡发生率分别为:42.4％±4.3％,2.2％±0.3％和2.7％±0.4％。DNA降解实验显示羊膜上皮细胞培养液明显抑制TNF-α诱导的DNA片段降解。Western blot显示羊膜上皮细胞培养液明显刺激RCK细胞Bcl-2蛋白的表达和分泌。结论 羊膜培养液中可能释放的多种细胞因子对TNF-α诱发的角膜基质细胞调亡具有明显的抑制作用,其作用机制之一是刺激Bcl-2在RCK细胞的表达,并使Bcl-2/Bax比值上调。     

人羊膜上皮细胞培养液抑制角膜新生血管的实验研究

背景 角膜新生血管(CNV)是一种常见的眼部病变,研究其发病机制及其抑制剂是眼科研究的热点和难点. 目的 研究人羊膜上皮细胞(AECs)培养液对CNV的抑制作用及机制. 方法 消化法培养及鉴定人AECs,并收集培养液,酶联免疫吸附试验(ELISA)法检测色素上皮衍生因子(PEDF)和白细胞介素1受体拮抗剂( IL-1Ra)在培养液中的质量浓度.兔角膜上皮细胞分离后分别用无血清DMEM培养基、人AECs培养液、混合培养液(DMEM+人AECs培养液)培养48 h,采用实时定量聚合酶链反应(QRT-PCR)法检测不同培养液培养的角膜上皮细胞中血管内皮生长因子(VEGF)及碱性成纤维细胞生长因子(bFGF)的表达.用含质量分数10％胎牛血清的DMEM培养基培养人脐静脉血管内皮细胞(UVECs),并分别加入无血清DMEM、混合培养液和人AECs培养液,划痕法和细胞计数试剂盒8(CCK8)法检测各组培养液对人UVECs迁移的影响.分别在上述3种培养基中加入终质量浓度为50 μg/L的bFGF,CCK8法检测各培养液中人UVECs的增牛情况.在原子力显微镜(AFM)下观察人AECs培养液对人UVECs超微结构的影响. 结果 人羊膜培养和传代细胞角蛋白免疫组织化学染色阳性证实为人AECs.与无血清DMEM组相比,人AECs培养液组的兔角膜上皮细胞VEGF mRNA(1.00±0.22 vs.2.98±0.46)及bFGF mRNA( 1.00±0.36vs.2.55±0.48)的表达均明显下降,差异均有统计学意义(P=0.001、0.002);培养后不同时间人AECs培养液组人UVECs的增生吸光度(A)值明显下降,差异均有统计学意义(P＜0.05),人UVECs的迁移率下降,差异有统计学意义(P＜0.05).AFM观察见人AECs培养液组的血管内皮细胞膜粗糙、表面颗粒紊乱,细胞间连接及伪足减少.ELISA法检测人AECs培养液中PEDF的质量浓度为(70.41 ±0.68) μg/L,IL-1Ra的质量浓度为(153.56±0.36) ng/L,无血清DMEM组中未检出.结论 人AECs培养液可抑制角膜上皮VEGF及bFGF的表达,抑制血管内皮细胞的增生和迁移,细胞结构和功能改变,这可能是其抑制CNV的机制之一.     

抗血管内皮生长因子小干扰RNA抑制大鼠角膜新生血管形成的研究

目的 探讨抗血管内皮生长因子小干扰RNA(VEGF-siRNA)对角膜碱烧伤急性期新生血管(CNV)形成的抑制作用.方法 实验研究.体外化学合成VEGF序列特异性双链小干扰RNA(VEGF-siRNA),脂质体介导转染体外培养的大鼠角膜上皮和基质细胞,实时PCR法检测转染后各时段VEGF mRNA表达水平,酶联免疫吸附实验测定转染后各时段VEGF蛋白表达水平.制作大鼠角膜碱烧伤动物模型,采用VEGF-siRNA转染的角膜上皮移植联合前房注射脂质体包裹的VEGF-siRNA治疗急性期CNV,免疫组化和ELISA分别检测角膜VEGF表达,观察术后CNV形成.细胞实验和动物实验中siRNA处理组和对照组VEGF蛋白含量比较,采用两样本均数比较的t检验.结果 siRNA对角膜上皮细胞VEGFmRNA的抑制效率达60%～84%,角膜基质细胞VEGFmRNA的抑制效率达59%～76%,角膜上皮和基质细胞VEGF蛋白表达也有相应的下降.采用VEGF-siRNA转染的角膜上皮移植联合前房注射VEGF-siRNA治疗,实验组CNV面积显著小于对照组;实验组VEGF表达水平显著低于对照组.结论 VEGF-siRNA能显著抑制大鼠角膜碱烧伤急性期CNV形成.     

姜黄素对大鼠碱烧伤角膜新生血管的抑制作用

目的:探讨姜黄素(curcumin)对大鼠角膜新生血管(corne-al neovascularization,CNV)形成的影响及CNV形成过程中血管内皮生长因子(vascular endothelial growth factor,VEGF)和诱导型一氧化氮合酶(inducible nitric oxide syn-thesis,iNOS)的表达情况。方法:以碱烧伤建立角膜新生血管模型,采用裂隙灯照相、免疫组化和RT-PCR等方法分别检测使用姜黄素前后各时期角膜组织中VEGF和iNOS的表达情况,并进行统计学分析。结果:姜黄素结膜下注射组角膜新生血管面积明显减少(t值分别为4.453,4.440,3.887,4.718,3.585,P<0.05);免疫组化染色,VEGF和iNOS在正常角膜上皮基底细胞有弱表达,新生血管对照组表达明显增强,其表达主要分布在新生血管形成区域和上皮全层;结膜下注射组表达明显减少;VEGF mRNA和蛋白表达一致。结论:姜黄素可以有效抑制角膜新生血管的发生,其机制与降低角膜新生血管VEGF和iNOS有关。     

自体口腔黏膜上皮细胞培养眼表重建的实验研究

管计数、VEGF阳性表达量均最少.印迹细胞检杏:口腔黏膜细胞移植组角膜上皮未发现杯状细胞.结论 利用体外培养的自体口腔黏膜上皮细胞进行移植,可减少角膜新生血管的形成,其抑制新生血管的作用优于单纯羊膜移植抑制新生血管的作用.     

环氧合酶-2及其抑制剂对角膜新生血管作用的研究

目的:观察大鼠角膜新生血管(cornealneovasculariza-tion,CNV)形成过程中环氧合酶-2(COX-2)的表达情况及与CNV的关系,探讨COX-2抑制剂Celecoxib对CNV的抑制作用。方法:利用免疫组织化学方法、RT-PCR法检测碱烧伤后各个时期COX-2和VEGF蛋白、mRNA在角膜各层中的分布,并对其进行半定量。比较实验组和对照组各个时期COX-2和VEGF蛋白和mRNA表达量的差别,并对其进行统计学分析,了解COX-2与VEGF的相关性。结果:①活化的COX-2和VEGF蛋白、mRNA的表达在角膜新生血管的形成中均有一动态变化。②VEGF的表达区域和COX-2表达的部位高度一致。③实验II组和实验Ⅲ组COX-2和VEGF蛋白、mRNA表达量与对照组的差别有显著性(P<0.05),实验I组与对照组的差别无显著性(P>0.05)。实验组和对照组COX-2与VEGF的表达呈正相关。结论:①COX-2在炎症性角膜新生血管形成过程中表达上调,其调控VEGF的表达,在角膜新生血管的形成过程中起着重要作用。②Celecoxib能抑制COX-2的表达,有效的抑制角膜新生血管的形成。     

骨髓间充质干细胞移植对角膜化学伤后炎症和血管生成相关因子表达的影响

背景 已有研究证明骨髓间充质干细胞(BMSCs)移植可重建损伤的角膜上皮并促进角膜上皮的修复,但BMSCs的作用机制尚不明确. 目的 探讨BMSCs移植在大鼠角膜化学伤后抗炎和抗新生血管形成的作用.方法 从2只成年清洁级SD大鼠双侧股骨及胫骨提取骨髓并进行BMSCs培养和传代,第3代细胞种植于羊膜.18只成年清洁级SD大鼠用NaOH滤纸片贴附于角膜中央的方法制作角膜碱烧伤模型,然后按随机数字表法随机分为3组,每组6只眼,均取右眼为模型眼.造模1周后,BMSCs移植组行带有BMSCs的羊膜移植；羊膜移植组行无BMSCs的羊膜移植；模型对照组不进行移植.术后2周裂隙灯显微镜下进行角膜透明度评分并计算角膜新生血管(CNV)面积,ELISA法检测各组大鼠角膜中白细胞介素2(IL-2)、IL-10、干扰素γ(IFN-γ)和转化生长因子B(TGF-β)的含量,荧光实时定量PCR法检测各组角膜中基质金属蛋白酶2(MMP-2)、血管内皮生长因子(VEGF)、表皮生长因子(EGF)和碱性成纤维细胞生长因子(bFGF) mRNA的表达.结果 传代细胞CD90及CD29表达阳性率分别为99.78％及99.79％,CD34、CD45及CD11b的表达率分别为7.90％、1.16％和1.28％,符合间充质干细胞的特征,种植于羊膜后生长状态良好.角膜碱烧伤后1周,各组大鼠角膜水肿混浊评分和CNV面积的差异均无统计学意义(F=0.021,P=0.979；F=0.076,P=0.927).移植术后2周,羊膜移植组和BMSCs移植组大鼠角膜混浊水肿评分均明显低于模型对照组(P=0.011、0.001),CNV面积均小于模型对照组(P=0.005、0.000).与羊膜移植组比较,BMSCs移植组CNV面积较小(P=0.001).角膜中Thl细胞分泌的IL-2和IFN-γ在BMSCs移植组明显下降,与模型对照组及羊膜移植组比较差异均有统计学意义(P=0.000、0.002、0.003、0.045),而Th2细胞分泌的IL-10和TGF-β明显高于模型对照组和羊膜移植组(P=0.000、0.000、0.000、0.021).3个组间VEGF在角膜中的表达差异无统计学意义( F=4.880,P=0.056).BMSCs移植组大鼠角膜中MMP-2及bFGF的表达均明显低于模型对照组及羊膜移植组(P＜0.01).结论 BMSCs移植可调节大鼠角膜碱烧伤后炎性相关因子和新生血管相关因子的分泌,从而减轻角膜炎症反应,抑制CNV的形成.     

羊膜对兔角膜上皮细胞影响的实验研究

目的 :观察羊膜对在其上皮面培养的兔角膜上皮细胞增生的影响。方法 :将兔角膜上皮细胞原代培养后 ,接种于羊膜上皮面 ,并用MTT自动比色法分别于接种后 1、3、5、7天检测羊膜对兔角膜上皮细胞增生的影响。结果 :接种后 1 ,3,5天 ,羊膜对兔角膜上皮细胞有明显的促增生作用 (P <0 0 5)。结论 :羊膜有促进兔角膜上皮细胞增生的作用 ,可用做角膜表面结构重建的理想材料。     

兔角膜及房水中VEGF的表达与角膜新生血管形成的相关性

目的:探讨兔角膜与房水中VEGF的表达与角膜新生血管(corneal neovasucularization,CNV)形成的相关性。方法:兔33只,随机分为正常对照组、实验组和实验对照组,角膜缝线制备CNV模型,实验对照组自术后当日起隔日结膜下注射一定剂量地塞米松治疗。术后每日观察CNV生长情况并计算其面积。分别于术后1,4,7,14,21d检测角膜组织及房水中VEGF的表达情况。结果:实验对照组CNV生长情况及角膜组织炎症细胞浸润程度较实验组明显受到抑制(P<0.05)。角膜组织中VEGF主要定位于上皮细胞、基质纤维细胞、内皮细胞、浸润的炎症细胞及新生血管内皮细胞胞质中。实验组角膜组织内VEGF表达的阳性率随时间推移逐渐增高,且表达强度与CNV面积具有显著的相关性。角膜缝线后14,21d实验组VEGF表达阳性率显著高于实验对照组(P<0.05)。实验组房水中VEGF的含量随着时间的推移逐渐增长,各检测点均高于实验对照组,并且房水及角膜组织中VEGF的表达与CNV面积显著正相关。结论:CNV的形成与炎症细胞浸润密切相关,角膜及房水中的VEGF可能在CNV形成过程中发挥着重要的调控作用。地塞米松可能通过抑制炎症反应、下调VEGF等细胞因子的表达,进而抑制CNV的生长。     

人羊膜匀浆上清液对兔角膜上皮细胞中bFGF表达的影响

目的:观察不同浓度人羊膜匀浆上清液对体外培养的兔角膜上皮细胞碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)表达的影响,探讨人羊膜匀浆上清液在促进角膜上皮损伤修复中的作用。方法:将兔角膜上皮细胞离体培养并传代,然后分组。实验组分别在含100mL/L胎牛血清的DMEM培养液中分别加入终浓度为40,80,160μg/mL 的人羊膜匀浆上清液,对照组仅用100mL/L胎牛血清的DMEM培养液。分别在培养24,48和96h后,采用甲基噻唑基四唑(methyl thiazolyl tetrazolium,MTT)比色法观察人羊膜匀浆上清液对兔角膜上皮细胞增殖的影响。同时,采用RT-PCR技术检测三个不同等级浓度对兔角膜上皮细胞bFGF-mRNA表达的影响。结果:培养24h后,与对照组（0.087±0.013）比较,加入终浓度为40,80,160μg/mL人羊膜匀浆上清液的实验组角膜上皮细胞增殖数量（分别为0.185±0.010,0.318±0015,0.501±0.014）明显增加,差异具有显著性(P<0.05);同时,随着作用时间的延长,差异愈加明显;加入40μg/mL和80μg/mL浓度羊膜匀浆上清液组兔角膜的bFGF-mRNA（分别为0.750±0.007,0.785±0.006）和空白对照组（0.708±0.013）比较,差异无显著性意义（P>0.05）,但160μg/mL浓度羊膜匀浆上清液组（1.013±0.120）与其它两组及对照组（0.708±0.013）比较,差异均具有显著性（P<0.05）。结论:人羊膜匀浆上清液具有促进兔角膜上皮细胞增殖的作用,并随着人羊膜匀浆上清液所含蛋白浓度的增加,其促进作用增强;人羊膜匀浆上清液具有促进体外培养的兔角膜上皮细胞bFGF-mRNA表达的作用,但与羊膜匀浆上清液的浓度有关。     

人羊膜作为载体体外培养兔角膜上皮细胞移植治疗碱烧伤动物的实验研究

目的观察培养生长于人羊膜的兔角膜上皮细胞使其扩增并移植治疗角膜碱烧伤的效果。为培养角膜上皮细胞移植技术及应用于临床实践提供最佳的理论和实验依据。方法新西兰白兔30只（30眼）,随机分为3组（n=10）,右眼制成碱烧伤模型。A组：角膜上皮细胞羊膜移植组;B组单纯羊膜移植组;C组为对照组（碱烧伤后不作任何移植）。术后观察角膜透明度、角膜新生血管及上皮修复情况,裂隙灯显微镜照相记录。3组均于术后1周、2周、1个月及3个月时各取1眼角膜标本行病理组织检查。结果A组除1眼移植片在第7天脱落外,所有移植片在术后3d水肿消退,角膜逐渐透明。B组移植片持续水肿,眼前段炎性反应稍重,但较碱烧伤对照组轻。C组角结膜高度水肿浑浊,烧伤后观察3个月未发现角膜恢复透明现象。病理组织检查显示：A组角膜及周边上皮细胞为多层结构,角膜新生血管消失,基质的炎性细胞浸润减退;B组覆盖上皮细胞表现为完整上皮细胞型,C组角膜浑浊,新生血管及肉芽组织形成。结论人羊膜作载体体外培养兔角膜上皮细胞移植重建角膜基底膜和角膜上皮结构治疗兔碱烧伤后的角结膜损伤是一种合理有效的方法。     

Analysis of angiogenesis induced by cultured corneal and oral mucosal epithelial cell sheets in vitro

The aim of this study was to compare angiogenesis-induction capabilities of cultured corneal epithelial cells (CCE) and cultured oral mucosal epithelial cells (COE) in vitro, and identify candidate factors that induce corneal neovascularization after transplantation of COE sheets. Rabbit corneal and oral mucosal epithelial cells were co-cultured with mitomycin C-treated NIH/3T3 cells on culture plates and inserts. After CCE and COE were multilayered, culture medium was replaced by basal medium and incubated. Angiogenic potential was examined by invasion, migration and tube formation assays with human umbilical vein endothelial cells (HUVECs). Protein secretion of fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF), angiopoietin-1 and transforming growth factor beta1 was assessed in conditioned medium by ELISA. Gene expression of FGF2 and VEGF was also quantified by real-time RT-PCR and neutralizing antibodies against FGF2 and VEGF were employed for blocking assays. COE induced significantly greater invasion, migration and tube formation of HUVECs, when compared to CCE. CCE secreted a significantly lower amount of FGF2 than COE, while amounts of VEGF were approximately equal in both culture media. Similarly, significantly higher expression of FGF2 mRNA was observed with COE, while no significant difference in VEGF mRNA expression was observed between COE and CCE. Only anti-FGF2 neutralizing antibody significantly suppressed HUVEC invasion and migration induced by COE, without suppression in CCE. In conclusion, angiogenic potential of COE is greater than that of CCE and FGF2 is a candidate involved in the induction of corneal neovascularization after COE sheet transplantation.     

Suppression of corneal neovascularization by culture supernatant of human amniotic cells.

PURPOSE: To examine the applicability of culture supernatant of human amniotic cells on basic fibroblast growth factor (bFGF)-induced corneal neovascularization. METHODS: Human amniotic epithelial and mesenchymal cells (AC) were obtained from human amniotic membranes by digesting with collagenase and maintained in serum-containing medium. The AC preparations predominantly contained cytokeratin-positive cells (91.2 +/- 3.1%, n = 4). The culture supernatant was prepared by cultivating AC in serum-free medium for 24 hours. Neovascularization was obtained by a micropocket assay with Hydron pellets containing bFGF. Migration assay was carried out by a double-chamber method using human umbilical vein endothelial cells (HUVEC). Cell growth assay was done by MTT assay using HUVEC. RESULTS: Basic fibroblast growth factor-induced corneal neovascularization was significantly reduced by administration of AC culture supernatant. When either control or AC culture supernatant was administered three times per day for 10 days, the area with neovascularization was 13.2 +/- 3.2 mm2 and 4.0 +/- 1.4 mm2 for the control and AC culture supernatant-treated eyes, respectively (n = 7, p = 0.021). AC culture supernatant strongly inhibited bFGF-induced migration and cell growth of HUVEC. CONCLUSIONS: Amniotic cell culture supernatant contains potent inhibitors of neovascularization. This effect is explained in part by inhibition of migration and cell growth of vascular endothelial cells. AC culture supernatant may be applicable for treatment of corneal diseases with neovascularization.     

Corneal Neovascularization Suppressed by TIMP2 Released from Human Amniotic Membranes

Purpose: To investigate the effects of culture medium of human amniotic membrane (AM) on corneal neovascularization (CNV) induced by basic fibroblast growth factor (bFGF) in mice. Methods: Culture medium of amniotic membrane was prepared by cultivating AM (with epithelium side up) in EGM basic medium for 3 days, and was collected separately to three groups, e.g. control (EGM only), AM with epithelium (AM) and AM without epithelium (De-AM). Corneal neovascularization was induced in mice by using micropocket assay with Hydron polymer pellets containing 100 ng bFGF. Migration and proliferation of human umbilical cord vein endothelial cells (HUVEC) were performed in Boyden chambers and by using the CyQUANT fluorescence binding assay respectively. The levels of tissue inhibitors of metalloproteinase 1 and 2 (TIMP1, TIMP2) in culture medium were determined by ELISA assay. Results: CNV induced by bFGF was significantly suppressed by culture medium of amniotic membrane. When the medium was applied as an eyedrop     

低浓度地塞米松对兔角膜上皮细胞的影响

目的: 探讨地塞米松(dexam ethasone,D EX)对培养的兔角膜上皮细胞和角膜上皮伤口愈合的影响。方法: 体外实验原代培养兔角膜上皮细胞, 用四唑盐(M TT)实验和流式细胞仪检测不同浓度 D EX 对兔角膜上皮细胞的作用, 同时通过 H E 、免疫组织化学染色和扫描电镜及临床观察兔角膜上皮细胞刮除后 0.1g/L D EX 用药组和对照组伤口愈合的情况。结果: M TT 检测显示小于 0.1g/L 浓度的 D EX 对兔角膜上皮细胞的生长无明显影响。流式细胞仪检测到 0.1g/L 浓度的 D EX 对兔角膜细胞生长周期无抑制作用。动物实验: 0.1g/L 地塞米松作用后上皮愈合时间显著性缩短, 两组中新生成的上皮细胞均有较强的 PC N A 蛋白的表达。0.1g/L 地塞米松组与对照组相比, 电子显微镜检查角膜基质排列更规则, 炎症反应较轻。结论: 低于 0.1g/L 浓度的地塞米松对培养的兔角膜上皮细胞的生长无抑制作用。0.1g/L 浓度的地塞米松眼液能有效的促进角膜上皮生长并降低炎症反应, 将有利于准分子激光角膜屈光手术后早期角膜伤口愈合。     

Growth factor changes in ex vivo expansion of human limbal epithelial cells on human amniotic membrane.

PURPOSE: Human limbal epithelial cells cultured on human amniotic membrane have been used for transplantation to treat corneal surface injuries. We determined whether the amniotic basement membrane affects the growth of human limbal epithelial cells through the production of growth factors. METHODS: The epithelial cells grown out from limbal basal epithelium were placed on conventional culture plastic or on the epithelial side of denuded amniotic membrane under serum-free conditions. Culture supernatant was assayed for growth factor release at 24, 48, and 96 hours. RESULTS: The cells grown on both substrata produced similar levels of epidermal growth factor (EGF). Cells grown on amniotic membrane showed enhanced secretion of tissue inhibitor of metalloproteinase type 1 (TIMP1) and reduced production of transforming growth factor beta1 and beta2. Depletion of EGF and TIMPI in cell culture slowed down cell growth and reduced EGF receptor expression, respectively. CONCLUSION: Increased TIMPI influences the proteolytic system in the cell and extracellular matrix interaction, and decreased transforming growth factor beta1 and beta2 may stimulate corneal cell proliferation. We show that the amniotic membrane leads to differential expression of cytokines of limbal epithelial cells cultured on its surface. Such effects may be favorable to the growth and differentiation of the cells when used for ocular surface reconstruction.     

培养角膜缘干细胞羊膜移植治疗碱烧伤动物的实验研究

目的 观察培养生长于羊膜的角膜缘干细胞移植的治疗角膜缘碱烧伤伤的效果。方法 将兔角膜缘干细胞在的代培养后接种于羊膜,对新西兰大白兔角膜缘碱烧伤动物模型行角膜缘干细胞羊膜移植术,并对治疗后的角膜进行临床及病理学检查。结果 体外培养的兔角膜缘士细胞可在羊膜上继续增殖、分化为密集的角膜上皮细胞层;角膜缘干细胞移植术后兔角膜缘轻度充血、角膜上皮完整基质细胞浸润减轻、新生血管减少。组织病理学染色证实,角膜缘     

Ultraviolet transmittance of human limbal epithelial cells cultured on human amniotic membranes

PURPOSE: To evaluate ultraviolet (UV) A and B transmittance by human limbal epithelial cells cultured on human amniotic membranes. METHODS: Human limbal epithelial cells were taken from the limbus of donor corneas and were cultured on human amniotic membranes with inactivated 3T3 fibroblasts for 2 to 4 weeks. Then, the cultured cells were examined histologically. Next, cells from different culture periods were irradiated with UV-A (365 nm) or UV-B (302 nm) at energy levels ranging from 50 to 800 microW/cm2, and UV transmittance was measured with a UV light meter. RESULTS: Histological examination revealed a monolayer of corneal epithelial cells on the amniotic membrane after 2 weeks of culture, and a layer of 3-4 cells was formed after 4 weeks. Transmittance of UV-A and UV-B was highest by the amniotic membrane alone, followed in decreasing order by limbal epithelial cells cultured on amniotic membranes for 2 weeks, 3 weeks, and 4 weeks. CONCLUSIONS: These results indicate that UV absorbance increases in proportion to the number of limbal epithelial cell layers in cultures on amniotic membranes. Limbal epithelial cells may need to be cultured until 3-4 layers are formed in order to prevent ocular damage by UV light after transplantation.