羊膜对免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免角膜上皮细胞增生的影响。方法：将免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天检测羊膜对免角膜上皮细胞增生的影响。结果：接种后１,３,５天,羊膜对免角以细胞有显示的促增生作用（Ｐ〈０．０５）。结论：羊膜有保进免角膜上皮细胞增生的作用,可用做角膜表结构重建的理想材料。     

人羊膜上皮细胞混悬液治疗兔急性角膜碱烧伤的实验研究

目的:结合体外细胞实验和在体动物实验研究羊膜上皮细胞(amnion epithelial cell,AEC)混悬液对兔角膜上皮细胞(cornea epithelial cell,CEC)生物学行为的影响及对急性角膜碱烧伤的治疗作用.方法:细胞实验:实验组将AEC及CEC于Transwell小室中培养,上室为AEC混悬液,下室接种CEC,对照组上室仅为培养基,于不同时间点行CCK-8法检测CEC的增殖活性;免疫细胞化学法检测PCNA表达;细胞划痕法观察细胞迁移能力.动物实验:采用改良的兔角膜碱烧伤模型制备方法:将10 mm直径环钻轻置于角膜中央,向环钻中央加入NaOH溶液,1 min后去环钻迅速冲洗兔眼,随机分三组(空白对照、AEC混悬液滴眼液治疗组、结膜下注射治疗组),于术后每周行裂隙灯显微镜及荧光素钠染色观察角膜情况.第28 d摘除眼球并固定行HE染色,免疫组织化学法检测角膜VEGF、mcp-1的表达.结果:CCK-8法示AEC混悬液作用后的CEC增殖活性增强(P<0.05);免疫细胞化学法显示PCNA阳性表达增强(P<0.05);划痕试验示AEC干预后CEC迁移加快.动物实验表明,滴眼液组和结膜下注射组的角膜恢复时间明显缩短,CNV面积均少于对照组(P<0.05),滴眼液组和结膜下注射组之间差异无统计学意义(P>0.05);HE染色示治疗组角膜组织炎症细胞浸润更少,组织排列更规则;免疫组化显示组织中VEGF、mcp-1表达更低(P<0.05),而滴眼液组和结膜下注射组之间差异无统计学意义(P>0.05).结论:AEC混悬液促进CEC增殖和迁移,影响CEC生物学行为变化.经AEC混悬液点眼及结膜下注射治疗兔眼碱烧伤后角膜修复加快,且两种方法抑制炎症反应及CNV形成,有望成为治疗急性角膜碱烧伤的新方法.     

羊膜匀浆上清液对兔角膜上皮细胞增殖的影响

目的　为角膜上皮损伤修复寻找新的有效方法。方法　运用MTT比色法 ,观察羊膜匀浆上清液在不同浓度及不同时相点 ,对兔角膜上皮细胞增殖的影响。运用活细胞计数方法检测兔角膜上皮细胞在不同羊膜匀浆上清液及不同时相点时的细胞活力。结果　羊膜匀浆上清液对兔角膜上皮细胞生长有促进作用 ,并呈剂量依赖性。结论　羊膜匀浆上清液可能能够应用于临床 ,促角膜上皮细胞修复。     

羊膜培养兔角膜缘上皮细胞及自体移植--角膜缘干细胞缺损的治疗

目的　探讨以人羊膜为载体培养兔角膜缘上皮细胞及其自体移植治疗全角膜缘干细胞缺损。方法　在8只兔右眼用正庚醇脱上皮和角膜缘环切的方法构建全角膜缘干细胞缺损模型2月。其中6只兔为实验组,活体取左眼角膜缘浅层小块,置羊膜上常规和气_液培养42天后进行自体移植治疗右眼角膜缘干细胞缺损;2只兔为对照组,直接用解冻无细胞人羊膜移植治疗右眼角膜缘干细胞缺损。进行细胞和术眼活体观察、组织学观察和电镜观察。结果　角膜缘上皮细胞在羊膜上生长良好,形成复层,细胞间的联结结构存在,细胞与羊膜组织粘附牢固。实验组移植手术后角膜迅速上皮化,恢复角膜表面光滑和透明,组织学观察和电镜观察呈现生理角膜上皮层的结构特点。但眼睑闭合不全可导致手术失败。对照组术后出现角膜缘干细胞缺损导致的角膜病变。结论　以羊膜为载体培养角膜缘上皮细胞后自体移植可有效地治疗角膜缘干细胞缺损导致的角膜病变。     

羊膜移植联合鸡角膜缘上皮移植治疗兔全角膜表层损伤的组织学改变

目的：研究羊膜移植术和联合鸡角膜缘上皮移植（联合移植）术治疗兔全角膜表层损伤的组织学改变。方法：兔39只分为3组，每组13只。对照组，将角膜及角膜缘外3mm的表层组织去除；羊膜移植组，在去除表层组织的创面上移植羊膜；联合移植组，在羊膜移植片表面角膜缘区移植鸡角膜缘上皮。手术当天各组处死1只兔，7、28、90、105天各组处死3只兔，摘除眼球分别进行光镜、免疫组化，透射电镜及扫描电镜检查。结果：对照组：角膜紊乱，表面不平，上皮表面微绒毛稀少，基质纤维血管化。羊膜移植组及联合移植组：羊膜结构逐渐被基质胶元纤维取代和改建，基质纤维平行排列，角膜表面光滑，上皮表面微绒毛丰富，细胞之间可见桥粒连接。结论：羊膜移植术和联合鸡角膜缘移植术治疗兔角膜广泛损伤，能抑制新生血管和纤维组织向角膜生长，较快促进眼表重建。     

新鲜羊膜移植术对角膜新生血管抑制作用的实验研究

目的探讨新鲜羊膜移植术对角膜碱烧伤后角膜新生血管和上皮愈合作用的影响,并与保存羊膜作比较.方法用48只新西兰大白兔制作角膜碱烧伤模型,烧伤后48h设立新鲜羊膜移植组(fAMT)、保存羊膜移植组(pAMT)和对照组(C).术后每日观察角膜新生血管和上皮愈合情况.并于术后14、30d以计算机图像分析系统定量测定新生血管和上皮愈合面积.结果术后30dfAMT组、pAMT组、C组角膜新生血管面积分别为87.87mm2±3.57mm2、96.66mm2±12.00mm2、105.13mm2±6.70mm2.统计分析显示,fAMT组角膜新生血管面积小于pAMT组(P＜0.05),上述2组均小于C组(P＜0.05).结论新鲜羊膜移植术可抑制碱烧伤后角膜新生血管生长,其作用优于保存羊膜移植术.     

羊膜移植抑制兔角膜碱烧伤后新生血管增殖的研究

目的　比较采用保存羊膜和新鲜羊膜移植抑制角膜碱烧伤后新生血管增殖的效果 ;探讨应用保存人羊膜移植防治角膜碱烧伤后新生血管的手术时机。方法　制备角膜碱烧伤后新生血管增殖的动物模型 ;2 2只家兔 ( 2 2眼 )随机分为4组 :A组 ( 4眼 )作为对照组 ;B组 ( 6眼 )在碱烧伤的急性期行新鲜羊膜移植 ;C组 ( 6眼 )在急性期行保存人羊膜移植 ;D组 ( 6眼 )在瘢痕期行保存人羊膜移植。应用计算机彩色图像处理系统测定角膜新生血管面积。结果　 3个移植组和对照组比较 ,角膜新生血管面积的差异均有显著性意义 (P <0 0 1) ;B组与C组比较无显著性差异 (P >0 0 5 ) ;C组的新生血管面积明显少于D组 (P <0 0 1)。结论　保存羊膜和新鲜羊膜移植均能有效地抑制角膜碱烧伤后新生血管的增殖 ,治疗效果无显著性差异 ;在角膜碱烧伤的急性期施行羊膜移植防治新生血管增殖的效果要优于在瘢痕期手术。     

兔角膜缘上皮细胞培养后自体移植修复

目的：运用培养角膜缘上皮细胞联合人羊膜行自体移植的方法，观察植片修复兔眼角膜上皮的疗效。方法：选用健康新西兰白兔20只，制成右眼角膜缘干细胞缺乏的兔眼模型，其中12只兔行角膜缘上皮细胞培养联合羊膜自体移植，另外8只兔只进行单纯羊膜移植。术后每周对眼表情况进行评分，术后1mo眼角膜进行苏木素-伊红(HE)染色和透射电镜观察。结果：移植了含有自体角膜上皮细胞的兔眼，术后早期都形成了角膜上皮化并明显抑制了新生血管的再生，HE染色和电镜观察表明培养并移植的角膜上皮与正常的角膜上皮无明显差异；而只接受羊膜移植的兔眼，术后又出现角膜混浊和明显的新生血管，表明角膜表面被结膜上皮覆盖。结论：该方法术后早期可以恢复角膜上皮化，重建正常眼表，疗效明显优于单纯羊膜移植。     

羊膜对氧氟沙星滴眼液兔角膜通透性的影响

目的探讨羊膜对氧氟沙星滴眼液兔角膜通透性的影响。方法先给108只新西兰大白兔进行样本编号,再按号码单纯随机抽样并分为6组,每组18只,分别为正常角膜组、正常角膜羊膜移植组、机械刮除角膜上皮组、机械刮除上皮联合羊膜移植组、角膜碱烧伤组、角膜碱烧伤联合羊膜移植组。0．3％氧氟沙星眼液滴眼,每15min滴眼1次,共4次,最后1次滴眼后5、30min,1、2、4、6h,于每个时间点每组18只大白兔中任意处死3只兔抽取房水。高效液相法检测房水中氧氟沙星的浓度。结果刮除角膜上皮组与正常角膜上皮组比较,药物的前房浓度均明显增高（均P〈0．05）。正常角膜羊膜移植组5、30min、1h房水中氧氟沙星浓度与正常角膜组无明显差异（均P〉0．05）,而2、4、6h则明显高于正常角膜组（均P〈0．05）;机械刮除上皮联合羊膜移植组2、4、6h房水中氧氟沙星浓度亦明显高于机械刮除角膜上皮组（均P〈0．05）,而两组间5、30min、1h房水中氧氟沙星浓度比较无明显差异（均P〉0．05）;角膜碱烧伤联合羊膜移植组6h以内房水中氧氟沙星浓度均高于碱烧伤组（均P〈0．05）。结论羊膜具有一定药物蓄积和缓释作用,增加了氧氟沙星在角膜表面的停留时间,使药物的通透性增高。     

幼兔角膜上皮细胞损害对角膜形态的影响

角膜上皮的损害可引起角膜细胞凋亡，甚至角膜基质层变薄。我们通过损伤幼兔角膜上皮．以观察角膜厚度和角膜曲率的变化。     

A comparison of the effectiveness between amniotic membrane homogenate and transplanted amniotic membrane in healing corneal damage in a rabbit model

Purpose: To determine whether amniotic membrane homogenate is as effective in healing corneal damage as amniotic membrane transplantation in a rabbit model. Method: The rabbits were divided into three groups. The cornea of one eye in each rabbit was incised resulting in a defect of approximately 8 mm in diameter. Amniotic membrane transplantation was performed in the first group, and amnion homogenate was administered four times a day in the second group. The third group was the control group. Results: There were no differences in the diameter of the defects and the rate of corneal growth between the amnion transplant group and the amnion homogenate group. Conclusion: Amnion homogenate is as effective as transplanted amniotic membrane in promoting corneal healing in a rabbit model.     

构建组织工程化羊膜角膜上皮植片的研究进展

虽然组织工程化角膜组织已成功构建,并为体外进行角膜生理、病理、药理和角膜移植等方面的研究提供了与活体角膜很接近的模型,但其距离临床应用还有一定距离。近年来,采用组织工程技术构建的羊膜角膜上皮植片已率先应用于临床,为成千上万的由于角膜上皮缺损所致的角膜疾病患者带来希望,本文对有关羊膜的基础研究、羊膜负载角膜缘干细胞植片的构建方法及其功能的评价、角膜缘干细胞在羊膜上的生物学特性、植片移植及术后护理等方面的研究进展和存在的问题进行综述。     

改良遮盖法羊膜移植在预防和治疗外伤性角膜穿孔中的运用

目的 探讨生物胶粘贴缝合双层羊膜遮盖防治外伤性角膜穿孔的可行性。方法 15例（18眼）角膜损伤患者实施双层羊膜移植，其中角膜穿孔者14眼，深层溃疡达角膜1／3基质以上者4眼。小片羊膜覆盖在溃疡或穿孔处，将生物胶滴于羊膜、角膜表面，并将大片羊膜迅速贴于角膜表面。10／0尼龙线间断缝合大片羊膜于结膜创缘及浅层巩膜上。结果 所有患者视力均优于术前。术中及术后未见特殊并发症。结论 双层羊膜粘贴缝合治疗和预防外伤性角膜穿孔，操作简单，效果显著。     

改良羊膜移植在预防和治疗外伤性角膜穿孔中的应用

目的:探讨生物胶粘贴缝合双层羊膜遮盖防治外伤性角膜穿孔的可行性。方法:15例18眼角膜损伤患者实施双层羊膜移植,其中角膜穿孔者14眼,深层溃疡达角膜1/3基质以上者4眼。小片羊膜覆盖在溃疡或穿孔处,将生物胶滴于羊膜、角膜表面,并将大片羊膜迅速贴于角膜表面。10/0尼龙线间断缝合大片羊膜于结膜创缘及浅层巩膜上。结果:所有患者视力均优于术前。术中及术后未见特殊并发症。结论:双层羊膜粘贴缝合治疗和预防外伤性角膜穿孔,操作简单,效果显著。     

Fluorescein enhanced confocal microscopy in vivo for the evaluation of corneal epithelium

BACKGROUND: In vivo confocal microscopy is being increasingly used to evaluate corneal disorders. This study aimed to evaluate whether topical fluorescein application prior to in vivo confocal microscopy had any effect on the imaging characteristics of the corneal epithelium. METHODS: Confocal microscopy in vivo with Confoscan 3.0 (Vigonza, Italy) was performed on 22 corneas of 22 study patients before and 1 min after application of unpreserved 2% fluorescein solution on the cornea. Ten patients with normal corneal findings served as control. The quality of superficial epithelial (SE) visualization, the SE density, the image intensity and the presence of hyperreflective SE cells were evaluated. RESULTS: Eleven patients had normal corneal epithelium (group 1), six had keratoconus (group 2) and five had overt epitheliopathy (group 3). The visibility of SE layers of all subjects appeared to be enhanced in the post-fluorescein images. There was a significant difference between the mean pre-fluorescein (857.2 + 319.4 cells/mm(2)) and post-fluorescein (1378.0 +/- 292.1 cells/mm(2)) SE densities of all study subjects (paired t-test, P < 0.001). Hyperreflective SE cells were more common in the post-fluorescein images of groups 2 (83.3%), and group 3 (80%) compared with group 1 patients (36.4%). These differences were not significant between the two consecutive corneal images of control patients who received unpreserved artificial tears instead of fluorescein. CONCLUSIONS: Topical fluorescein application appears to enhance the visualization of the corneal epithelium. This technique may enable better evaluation of the corneal epithelium quantitatively and qualitatively in both normal subjects and patients with epithelial involvement.     

Intracellular potentials of isolated rabbit and human corneal epithelium

Cell membrane potentials (p.d.) of corneal epithelium were studied with 3 m-KCl-filled microelectrodes using the glass fibre method of filling. In the isolated rabbit cornea a staircase-like potential profile with five to seven single steps was obtained and a mean intracellular p.d. of 68·5±0·6 mV was recorded in basal epithelial cells. This p.d. was stimulated to 78·2±0·7 mV by 3·7 mm ascorbic acid and inhibited by KCl and NaCN. Epithelial p.d. in isolated human corneas of enucleated eyes (with normal corneal function) was 57·4± 0·4 mV. Cadaver corneas removed 2–5 hr after death or additionally stored for 12–18 hr in a moist chamber at 4°C exhibited, at least for 2 hr of incubation in modified Ringer solution, an adequate epithelial cell potential.     

羊膜移植对兔角膜碱烧伤后局部白介素-6分泌的影响

目的观察羊膜移植对兔角膜碱烧伤后泪液中白介素-6（interieukin-6，IL-6）分泌的影响。方法制作30只新西兰白兔角膜碱烧伤模型，分为羊膜移植组（20只）与对照组（10只）。术后3周内每3日1次分别检测两组兔泪液中IL-6的含量。结果①烧伤后24h内，两组兔泪液中IL-6的水平相似。②此后羊膜移植组泪液中IL-6水平略有升高，1周后开始降低，第21天时基本测不出；而对照组持续升高，第21天时仍保持较高的水平。结论羊膜移植可有效抑制局部炎症细胞IL-6的释放，在角膜碱烧伤后的抗炎治疗方面具有重要的临床意义。     

Intracellular potassium activity of isolated human and rabbit corneal epithelium

Single-barrelled open tip microelectrodes containing a potassium-selective liquid ion exchanger (Corning 477 317) were used to determine intracellular K-activity a_Kⁱ in squamous and basal cells of isolated rabbit and human corneal epithelium. The intracellular potassium activity a_Kⁱ during the five hours of incubation in Ringer solution was 114·3±23·6 mmol/l and 107·6±26·2 mmol/l for the basal cell layer of rabbit and human epithelia respectively. The corresponding values for the squamous cells of the superficial cell layer were 41·2±14·2 mmol/l and 50·2±11·1 mmol/l. Based on intracellular chemical concentrations of potassium c_Kⁱ an apparent activity coefficient could be calculated that is close to that of a dilute solution of the same ionic strength. This indicates that intracellular potassium is essentially free in both epithelia. Ouabain (5 × 10^?5 mol/l) added to the bathing solution of the rabbit cornea lowered the transmembranal voltage Ψ_M as well as the intracellular K-activities of squamous and basal cells. Three hours after application of ouabain a_Kⁱ of the basal cells had decreased to about one third of its initial value. Under all experimental conditions the potassium equilibrium potential E_K considerably exceeded Ψ_M.Our data indicate: (1) The high absolute values for a_Kⁱ imply that almost no potassium is bound or sequestered within the epithelial cells. (2) An active potassium uptake is necessary to explain the high K-activity in relation to the transmembranal voltage Ψ_M. (3) There is an intraepithelial gradient of a_Kⁱ within the corneal epithelia.