【作者】 唐琼燕；

【导师】 胡义珍；

【作者基本信息】 华中科技大学 ， 眼科学， 2009， 博士

【摘要】 第一部分应用活体共聚焦显微镜进行角膜缘干细胞缺失的临床分级研究【目的】观察角膜缘干细胞缺失中央角膜上皮和角膜缘上皮在活体共聚焦显微镜下的改变，从细胞水平建立一套角膜缘干细胞缺失的临床分级系统。【方法】根据裂隙灯显微镜下荧光素染色的不同阳性表现，将临床诊断为角膜缘干细胞缺失的18例24眼初步分成4个临床阶段组，各组均利用海德堡视网膜断层扫描仪3代Rostock角膜模块(HRT3 RCM)进行活体眼表组织成像。图像信息集中阅读分析。【结果】5个形态学指标被发现有助于分析中央角膜区和角膜缘区的上皮层改变：角膜上皮细胞层数的改变；角膜上皮细胞大小和活动性改变；外侵细胞：如炎症细胞、树突状细胞；角膜上皮层内和基底膜下神经丛的改变；角膜上皮层内血管的出现。每个临床阶段组都有其对应的共聚焦显微镜下细胞水平的改变，且组与组之间中央角膜区和角膜缘区的上皮层有明显的形态学和结构学差异。角膜缘干细胞缺失临床分级系统首次从活体眼表单个细胞水平创立。【结论】角膜缘干细胞缺失中央角膜上皮和角膜缘上皮在细胞水平的形态学和结构学差异与其临床体征有较高一致性，活体共聚焦显微镜可作为诊断极早期角膜缘干细胞缺失和辅助其分级的有用工具。第二部分人胚胎干细胞标记物在角膜缘上皮和角膜上皮的表达研究【目的】研究5个典型人胚胎干细胞标记物碱性磷酸酶(ALP)、Nanog、Oct-4、阶段特异性胚胎抗原(SSEA)-1和SSEA-4在角膜缘上皮和角膜上皮的表达。【方法】可移植级别人角巩膜组织进行冷冻切片，免疫组织化学方法从蛋白水平检测ALP、Nanog、Oct-4、SSEA-1和SSEA-4在角膜缘上皮和角膜上皮的表达，RT-PCR法从mRNA水平进一步确认SSEA-4、Nanog和Oct-4在角膜和角膜缘的表达。【结果】免疫组织化学法研究显示SSEA-4广泛表达于角膜缘上皮和角膜上皮的各层，ALP散在表达于少量角膜缘前基质层细胞中。SSEA-4的表达经RT-PCR得到确认。免疫组织化学法和RT-PCR均显示SSEA-1和Oct-4不表达与角膜缘上皮和角膜上皮。RT-PCR在mRNA水平检测到Nanog在角膜缘和角膜的表达，但免疫组织化学法在蛋白水平未检测到Nanog在角膜缘和角膜的表达。【结论】SSEA-4广泛表达于成年角膜缘上皮和角膜上皮的各层中，提示SSEA-4不能作为一个理想的角膜缘干细胞标记物。Nanog是否表达于角膜和角膜缘仍需进一步确证。第三部分应用可诱导转基因大鼠模型进行角膜缘干细胞研究的初步探索【目的】探讨一种可直接药物诱导转基因大鼠模型用于角膜缘干细胞研究的可行性。【方法】可Doxycycline直接诱导型GFP转基因大鼠(ROSA26-rtTA-Collal-tetOP-H2BGFP)模型被引入。GFP基因的表达通过日常喂给水中添加Doxycycline直接激活，Doxycycline持续添加21天后停止。大鼠于1月和4月大被处以安乐死，这里的1月和4月指从开始喂给Doxycycline的日期到大鼠安乐死之间的间隔时间，安乐死大鼠在立体生物显微镜下行立体荧光照相，然后分别剜出左右眼球，左眼球进行冰冻切片，免疫组织化学染色研究；右眼球进行角巩膜铺片，荧光显微镜照相和激光共聚焦显微镜照相分析。【结果】GFP绿色可见荧光广泛表达于Doxycycline诱导的转基因1月大鼠的全身多个组织，眼表表达显著，且可见明显的高表达角膜缘条带。经Doxycycline诱导的转基因4月大鼠眼表GFP表达减退，但可见角膜缘表达强度仍高于周边角膜和中央角膜，漩涡状高表达条带连接角膜缘和角膜中央，经激光共聚焦显微镜分析，这些高表达条带内的细胞形状特殊，走行于角膜缘和角膜区翼状上皮细胞和基底上皮细胞之间。【结论】经Doxycycline诱导的4月转基因大鼠角膜缘区的GFP高阳性表达提示眼球表面分化缓慢的细胞群位于角膜缘，连接角膜缘和角膜中央的漩涡状GFP高表达条带中提示分化缓慢的细胞从角膜缘漩涡状移行入中央角膜，补给脱落的角膜上皮细胞。可Doxycycline直接诱导型转基因大鼠是研究角膜缘干细胞的有效模型。更多还原

【Abstract】 PartⅠStaging System for Limbal Stem Cells DeficiencyUsing in Vivo Confocal MicroscopyObjective To describe the corneal and limbal epithelial changes in patients with imbalstem cells deficiency (LSCD) using in vivo laser scanning confocal microscopy (LSCM),and to classify the stages of LSCD at the cellular level.Method This is a prospective, noncomparative clinical study. Patients were selectedaccording to their clinical presentations. Twenty four eyes of 18 subjects with LSCD wereclassified into 4 stages groups initially according to their cornea status. In vivo imagings ofthe corneal and limbal epithelium in all the 24 eyes were obtained with HRT 3 Rostockcornea module LSCM. The LSCM images were reviewed.Results Five useful parameters were found to evaluate all the LSCM images: theepithelium layers; epithelial cells size and activity; invading cells; epithelial andsubepithelial nerve, intraepithelial vessels. Both the central cornea and limbus showed 4different stages changes which correlated with the initial clinical 4 stages group. Thestaging system for LSCD at the cellular level was established.Conclusion The morphological and structural changes of corneal and limbal epithelium at the cellular level correlated with clinical presentations at different stages in LSCD.Confocal microscopy might be a useful tool to detect very early stage of LSCD and couldaid in the staging of LSCD.PartⅡExpression of Human Embryonic Stem Cells Markers in theLimbal and Corneal Epithelial CellsObjective To investigate the expression of five human embryonic stem cells (hESCs)markers, Alkaline phosphatase (ALP), Stage-specific embryonic antigen (SSEA)-1,SSEA-4, Nanog and Oct-4, in the limbal and corneal epithelial cells.Method Frozen sections were obtained from human corneoscleral tissues.Immunohistochemistry study for five hESCs markers, ALP, SSEA-1, SSEA-4, Nanog andOct-4, was performed to evaluate their expression at the protein level in the human limbusand cornea. The mRNA expression of SSEA-4, Nanog and Oct-4 was further confirmed byreverse transcription-PCR (RT-PCR).Results Immunohistochemistry study showed that SSEA-4 was present in all layers ofthe limbal and corneal epithelial cells and ALP was detected in a small subgroup of stromalcells in the anterior limbus. The expression of SSEA-4 was confirmed by RT-PCR. SSEA-1and Oct-4 were not detected at the protein nor the mRNA level in the limbus and cornea.Nanog mRNA transcript was detected in the cornea and limbus, but Nanog protein was notdetected using immunostaining.Conclusion The expression of SSEA-4 in mature corneal epithelial cells indicates thatit is not a marker for corneal epithelial progenitor/stem cells. Whether Nanog is expressedon the ocular surface needs to be further confirmed at the protein level. PartⅢUsing an Inducible Transgenic Mice Model toStudy Limbal Stem CellsObjective To establish an inducible GFP transgenic mice model to study the limbalstem cells.Method An inducible GFP transgenic mice (ROSA26-rtTA-Col1a1-tetOP-H2BGFP)model was selected and the expression of GFP was turned on for 21 days starting atdifferent periods and followed by 1 month and 4 months, which was counted from the datethe GFP was turned on. After the mice were euthanized, the left eyes were frozen with OCTand immunohistochemistry study for GFP expression pattern was performed on the frozensections. The sclerocornea tissues from the right eyes were isolated, mounted and observedunder confocal and fluorescence microscopy. Images were processed.Results Expression of GFP was present on the whole ocular surface in both 1 monthand 4 months mice and stronger GFP expression band was seen in the limbus. In 4 monthsmice, the expression of GFP was weaker than 1 month and parts of higher GFP expressionlimbal cells were organized in a radial stripe fashion towards the center of the corneaforming a vortex pattern. Three-dimensional reconstructions of the vortex pattern confocalimages revealed that the stripe cells were seen in the basal and suprabasal corneal epitheliallayers and different from the other cells in the peripheral and central cornea.Conclusion The epithelial cells in the limbus retained the higher GFP expression in 4months after the GFP was turned on indicating that they are the slowest cycling cells. Theradial vortex pattern suggests that these epithelial cells might migrate from the limbustowards the center of the cornea. This mouse model is a useful tool to study the in vivohomeostasis of corneal epithelial cells.更多还原