【作者】 罗琳；

【导师】 王传富；

【作者基本信息】 青岛大学 ， 眼科学， 2003， 硕士

【摘要】 目的 评价深低温保存兔眼及人眼角膜缘组织活性，以探讨深低温保存角膜缘组织应用于临床的可行性。 方法 3只健康新西兰大耳白兔(6只眼)，制备角膜缘组织片(约带2mm宽的周边角膜和2mm宽巩膜)，采用简化二步深低温保存法保存90天；取2只人尸眼角膜缘组织片(约带有2-3mm宽周边角膜及2-3mm宽巩膜)，来源于简化二步深低温保存法保存92天(1只眼)及726天(1只眼)的带巩膜环的角膜组织片。分别行光镜(HE染色、PAS染色)、透射电镜观察其组织结构及超微结构，酶组织化学染色[乳酸脱氢酶(LDH)、琥珀酸脱氢酶(SDH)]测定酶活性，并与新鲜眼角膜缘组织对比。 结果 ①光镜下，简化二步深低温保存兔及人角膜缘组织上皮层形态完好，细胞排列紧密，深层柱状上皮细胞胞浆少、核小、染色深。部分上皮从上皮下乳头间向下突出，每隔1-2mm横过角膜缘，呈放射状排列，形成“Vogt栅栏”。有色素颗粒沉着。未见细胞膜破裂，细胞浆均匀，未见空泡形成，细胞核未见脱失，基底膜完整，呈波浪状。基质层为疏松的纤维组织，胶原纤维排列不规则，直径粗细不均。可见血管。兔及人角膜缘上皮层PAS染色阴性；与其毗邻的角膜侧上皮层PAS染色阴性；巩膜侧上皮层偶见PAS染色阳性。②电镜下，简化二步深低温保存兔及人角膜缘组织上皮层细胞胞膜完整，上皮层表面有微绒毛和微皱褶，上皮细胞之间可见多数桥粒及半桥粒连接，细胞连接完好，底部柱状细胞为单层细胞，排列不规则，其基底连同上皮基底膜呈波浪状。胞质致密，细胞内器丰富，含有高浓度的线粒体、高尔基复合体、内质网及细丝状结构。部分线粒体肿胀，少数嵴断裂，内质网池轻度扩张。胞质内可见色素颗粒。核完整，形状不规则，核膜清晰，染色质分布均匀，中文摘要变化不明显。③简化二步深低温保存兔及人角膜缘组织上皮层LDH、SDH的酶组织化学测定可见上皮细胞内LDH为蓝色反应颗粒，分布于细胞质中;SDH为紫蓝色反应颗粒，分布于细胞质中。与新鲜角膜缘组织相比其活性无显著性差异(P>0.05)。 结论深低温保存法能保存角膜缘组织活性;能随时按需要为临床提供活性角膜缘组织材料;为深低温保存角膜缘干细胞移植治疗临床眼表疾病提供了实验依据。更多还原

【Abstract】 Objective To evaluate the vitality of limbal tissues with Cryopreservation.Methods Limbal grafts include 2mm of peripheralcornea and 2mm of sclera of 3 rabbits were preserved by simplified two-step Cryopreservation method for 90 days. The human corneal tissues were cryopreserved by the same method for 92 days and 726 days, and were made into limbal graft include 2-3mm of peripheralcornea and 2-3mm of sclera. Light and transmission electron microscopies were used to study the structure of limbal tissue cells. Enzyme histochemistry methods with image analysis system were used to evaluate the metabolic enzyme activity, lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH).Results (1) From light microscopic study, the cryopreserved limbal epithelial cells remained intact and lined up tightly. Partial epithelium arrayed radiately and shaped into Vogt bars, which projected downward over the limbus every 1 -2mm from the subepithelial papilla. And a lot of pigment granule was deposited. There were blood vessel tissue in Hypothallus. (2) From transmission electron microscopic study, the cryopreserved limbal epithelial cytomembrane remained intact and zonula occludens was as excellent as before. Partial mitochondria were swollen with no nuclei change in limbal tissue cells preserved by simplified two-step Cryopreservation method. (3) The activity of LDH and SDH in the epithelial cells of limbal tissue remained the same as in the fresh control group, which preserved by simplified two-step Cryopreservation method (P>0.05).Conclusions Simplified two-step Cryopreservation method can maintain the vitality of limbal tissue cells and provide viable limbal grafts at any time. And can provide an experimental evidence for cryopreserved limbal stem cells to treat ocular surface disorders.Postgraduated: Luo Lin(Ophthalmology) Directed by: Professor Wang Chuanfu更多还原