TGF-β1对胎儿晶状体上皮细胞增殖的作用

目的 探讨转化生长因子－β1（transforming growth factor-β1,TGF－β1)对胎儿晶状体上皮细胞增殖的作用。方法 用SP免疫对48例人胎儿不同胎龄晶状体上皮细胞进行细胞增殖核抗原（proliferating cell nuclear antigen,PCNA),TGF－β1免疫组织化学染色。结果 PCNA，TGF－β1在人胎儿不同胎龄晶状体上皮细胞均表达；免疫阳笥细胞随着胎龄的增加而减少。PCNA阳性细胞核为桔黄色或棕黄色；TGF－β1阳性细胞浆为桔黄色或棕黄色的颗粒状或斑块状；经相关分析：TGF－β1与PCNA呈正相关。结论 PCNA，TGF－β1在人胎儿不同胎龄晶状体上皮细胞均有表达；TGF－β1与PCNA呈正相关，具有促进晶状体上皮细胞增殖的作用。     

人胎儿晶状体上皮细胞增殖及相关因子的动态研究

目的：观察增殖细胞核抗原（PCNA）及转化生长因子－β1（TGF－β1），表皮生长因子（EGF）在人胎和晶状体上皮细胞的表达，并探讨其在晶状体发育中的作用。方法：用SP免疫细胞化学法对48例人胎儿不同胎龄晶状体上皮细胞进行PCNA，TGF－β1，EGF免疫细胞化学染色。结果：PCNA，TGF－β1，EGF在人胎儿不同胎龄状体上皮细胞内均有表达；免疫阳性细胞随着胎龄的增加而减少。PCNA阳性细胞核为桔黄色或棕黄色；TNF－β1，EGF阳性细胞浆为桔共色或棕黄色的颗粒状或斑块状；经相关分析，TGF－β1与EGF以及TGF－β1，EGF与PCNA均呈正相关。结论：PNCA，TGF－β1，EGF在人胎儿不同胎龄晶状体上皮细胞内均有表达，TGF－β1，EGF具有协同作用，共同促进晶状体上皮细胞的增殖。     

DispaseⅡ消化法原代培养兔结膜上皮细胞及鉴定

目的研究DispaseⅡ消化法原代培养兔结膜上皮细胞,观察其体外生长特性,并采用免疫组织化学染色鉴定培养的上皮细胞。方法 DispaseⅡ消化法原代培养兔结膜上皮细胞,观察不同时期细胞的生长情况、细胞形态,做广谱角蛋白、MUC5AC免疫荧光染色及PAS染色鉴定;并比较不同部位结膜上皮细胞增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)表达阳性情况。结果 DispaseⅡ消化法能够获得大量的较纯净的结膜上皮细胞,细胞生长增殖快,能传代4～5代;绝大多数细胞广谱角蛋白免疫荧光染色阳性,少数细胞MUC5AC表达阳性及PAS染色阳性。睑结膜、穹隆部结膜和球结膜细胞PCNA阳性率分别为:(32.14±1.69)%、(27.67±1.20)%、(22.76±1.73)%,兔睑结膜上皮细胞PCNA阳性率较穹隆部及球结膜上皮细胞的PCNA阳性率高,各部位PCNA阳性率间比较差异有统计学意义(P<0.05)。结论 DispaseⅡ消化法是进行体外结膜上皮细胞培养的理想方法,兔眼睑结膜处可能存在结膜干细胞。     

暴露性干眼病大鼠模型结膜上皮细胞凋亡相关基因的表达

目的 探讨暴露性干眼病大鼠模型结膜上皮细胞凋亡相关基因的表达与组织损伤的关系。方法 25只健康成年Wistar大鼠随机分为4组实验组和1组正常组。实验结束分别处死每组大鼠，取双眼结膜进行免疫组织化学染色观察结膜组织中凋亡相关基因bax及Bcl-2的表达情况。结果 各实验组大鼠结膜上皮细胞Bax表达阳性细胞数与正常组相比增加，而Bcl-2阳性表达细胞数较正常组减少，差异均有统计学意义（P〈0．05）；结膜上皮细胞Bax表达阳性细胞数与暴露时间呈正线性相关（r=0．76，P〈0．05），而Bcl-2表达阳性细胞数与暴露时间呈负线性相关（r=-0．78，P〈0．05）。结论 暴露性干眼病结膜组织中上皮细胞凋亡可能是导致结膜上皮破坏进而功能丧失的原因之一，bax增加及Bcl-2减少与结膜上皮细胞凋亡有关。     

结膜印迹细胞学检查在干眼病诊断中的价值

目前,干眼病成为眼科门诊的常见病和多发病,原有的三项诊断性试验(角膜荧光素染色、泪液分泌试验、泪膜破裂时间)越来越不能满足对干眼病的确切诊断,亟待开展新的方法。结膜印迹细胞学技术是近年来新开展的一项实验室检查手段,可以从形态学方面对结膜上皮细胞和杯状细胞的病理改变进行确切的描述,且不会给患者带来任何痛苦,具有良好的临床应用前景。因此,我们对2004年6月-2005年6月门诊诊断为或高度怀疑干眼病的患者共16例进行了结膜印迹细胞学检查,以期探讨结膜印迹细胞学技术在干眼病早期诊断中的价值及意义。资料和方法1.病例选择与分组:…     

泪液缺乏性干眼治疗转归的定量检测

目的：应用角膜共焦显微镜、结膜印迹细胞学在泪液缺乏性干眼治疗前后定量测定病变角膜上皮细胞、结膜细胞,以期进一步准确评价干眼的治疗效果及预后。 方法：选择临床确诊为泪液缺乏性干眼症患者21例41眼,根据患者病史、症状、角结膜体征及相关检查确诊为泪液缺乏性干眼。应用聚乙二醇滴眼液及卡波姆凝胶联合治疗所有干眼患者,治疗前后依据结膜印迹细胞检查对干眼患者结膜细胞的鳞状化生及杯状细胞数量的多少进行分级,并对治疗前后印迹细胞分级进行统计学比较。应用海德堡激光共聚焦显微镜观察干眼治疗前后角膜上皮下神经纤维的形态学改变,定量评价同级结膜细胞干燥程度下治疗前后角膜上皮细胞密度的变化。 结果：印迹细胞检查:治疗前≤Ⅱ级6眼,Ⅲ级25眼,≥Ⅳ级10眼;治疗6mo后≤Ⅱ级19眼(46.3%),Ⅲ级17眼(41.5%),≥Ⅳ级5眼（12.2%）。治疗前后不同级别印迹细胞患者比例显著改变(P＜0.05）。共焦显微镜下细胞形态学改变：治疗前明显的角膜上皮细胞坏死脱落区,细胞密度下降,角膜上皮下神经纤维出现分支杂乱、异常走行,未见上皮下神经纤维5眼。治疗6mo后区域性细胞密度增加,所有患者均可见上皮下神经纤维,少数病例仍可见分支杂乱、异常走行。共焦显微镜显示在同级结膜印迹细胞程度下,角膜上皮细胞密度治疗前后进行比较,统计学上具有显著性差异（P<0.05）。治疗后细胞密度明显增加。 结论：应用角膜激光共焦显微镜和结膜印迹细胞定量观察泪液缺乏性干眼的治疗转归,从而确定以上两种方法在干眼治疗评价中具有重要价值。     

配戴透气性硬性角膜接触镜后结膜表面细胞变化

目的研究透气性硬性角膜接触镜对结膜表面细胞的影响。方法选取验配透气性硬性角膜接触镜的屈光不正患者30例60眼,根据屈光度、角膜曲率和配适情况,选择适合的镜片。于戴镜前及戴镜后6个月、1a分别进行结膜印迹细胞学检查,显微镜下观察结膜上皮细胞、杯状细胞,并对其进行分级,计数100倍条件下,7.20mm×5.35mm范围内上皮细胞和杯状细胞。结果配戴透气性硬性角膜接触镜前下睑结膜上皮细胞(318.5±155.4)个,下睑结膜杯状细胞(13.6±6.8)个,球结膜上皮细胞(244.1±110.9)个,杯状细胞(15.2±5.7)个;6个月后下睑结膜上皮细胞(305.6±79.9)个,杯状细胞(13.2±6.6)个,球结膜上皮细胞(226.7±86.3)个,球结膜杯状细胞(15.1±5.5)个;1a后下睑结膜上皮细胞(272.9±86.4)个,下睑结膜杯状细胞(12.3±5.9)个,球结膜上皮细胞(228.5±155.4)个,球结膜杯状细胞(14.3±6.6)个。戴镜后6个月、1a,下睑结膜及球结膜上皮细胞、杯状细胞密度与配戴前相比,差异均无统计学意义(均为P>0.05);戴镜后1a,CIC分级与戴镜前相比,差异无统计...     

特发性黄斑前膜的组织病理学特征

目的：观察各期特发性黄斑前膜（IMEM）的病理结构特征，分析黄斑前膜病理组织学特点及其发展 规律。方法：前瞻性临床研究。选取2016年2月至2018年9月在台州医院路桥院区进行玻璃体切割 联合内界膜-黄斑前膜剥除术治疗的IMEM患者60例（62眼），根据OCT图像4期分期方案分为1期 IMEM组、2期IMEM组、3期IMEM组及4期IMEM组。将术中剥除的带内界膜的IMEM标本HE染 色后进行膜平铺及切片观察，并进一步行SMA、GFAP、S-100、CD34、CD68等免疫组织化学染色 及弹力纤维染色、Masson染色等特殊染色检查。记录各组膜组织类型、病理组织学结构特征、主要 细胞和纤维成分及免疫组织化学的阳性率。采用χ2 检验进行各组膜组织类型分布及免疫组织化学阳 性率比较，Kruskal-Wallis H检验进行组间细胞密度比较。结果：将膜组织类型分为致密型及稀疏型， 4组膜组织类型分布差异有统计学意义(χ2=11.44，P=0.006)。切片观察组织病理学特点发现：从1期 到4期组织的细胞聚集度降低，细胞的胞质减少，组织内胶原纤维由致密变得疏松。4组间细胞密度 总体比差异有统计学意义（H=13.73，P=0.003），多重比较分析发现2期与4期的细胞密度差异有统计 学意义（H=3.69，P=0.001）。免疫组织化学染色显示CD34均阴性表达，GFAP均阳性表达，部分膜组 织表达SMA、S-100、CD68，但各组间SMA、S-100、CD68阳性率比较差异均无统计学意义。弹力 纤维染色及Masson染色提示各组的IMEM纤维成分均为胶原纤维，从1期到4期纤维逐渐扩张增厚。 结论：IMEM属于非血管性纤维增生组织，其主要细胞成分为胶质细胞及成纤维细胞，各期细胞成 分相似，纤维成分为胶原纤维。在疾病发展后期细胞密度下降，整个过程纤维呈逐渐扩张状态。     

移植物抗宿主病相关的干眼病

目的 观察异基因造血干细胞移植术后移植物抗宿主病的眼部表现。方法 对20例异基因造血干细胞移植术后白血病患者的随访结果进行回顾性研究,随访的主要眼科检查项目包括视力、裂隙灯显微镜、荧光素染色、虎红染色、泪膜破裂时间、Schirmer Ⅰ和SchirmerⅡ试验,部分患者行共焦显微镜检查、结膜印迹细胞检查和病理活检。结果 术后14例患者出现移植物抗宿主病。8例出现干眼病,占移植物抗宿主病患者的57％。其中4例为严重干眼病,患者伴有视力明显下降,甚至角膜溃疡;病理检查结果示：结膜杯状细胞消失或明显减少,结膜上皮角化,角膜上皮鳞状化生,结膜炎性反应细胞以T淋巴细胞为主。结论 于眼病是慢性移植物抗宿主病的主要眼部合并症,严重影响患者生活质量,应引起足够重视。     

组织工程角膜上皮治疗兔角膜缘干细胞缺乏症的研究

目的 评价以纤维凝胶膜为载体,体外培养自体角膜缘干细胞移植术治疗角膜缘干细胞缺乏症的临床效果。方法 制作兔眼角膜缘干细胞完全缺乏模型,取对侧眼角膜缘组织进行干细胞培养,并以纤维凝胶膜为载体制成组织工程角膜上皮。将实验动物随机分为4组,其中Ⅰ-Ⅲ组为实验组,行组织工程角膜上皮移植术,Ⅰ组移植术后观察3个月,Ⅱ组移植术后观察1个月,Ⅲ组移植术后观察2周。Ⅳ组为对照组,移植不含干细胞的凝胶膜,术后观察3个月。以角膜上皮染色、角膜混浊及新生血管3项指标进行临床疗效评定,通过病理检查评估术后不同时期角膜上皮修复情况,印迹细胞学检查移植前后角膜上皮的细胞表型,免疫组织化学染色观察角膜上皮特异性角蛋白K3、MUCSAC及转录因子p63在移植角膜上皮的表达。结果 实验组角膜上皮逐渐愈合,透明度提高,新生血管减退或消失;对照组角膜呈持续混浊,上皮缺损迁延不愈,最终大量新生血管长入,上皮结膜化。临床指标评分比较,差异有统计学意义（P=0．021）。角膜上皮印迹细胞学检查显示对照组为PAS（＋）的结膜细胞表型,而实验组上皮细胞PAS（-）。组织病理学显示实验组为正常角膜上皮结构,对照组为结膜化生的上皮,可见血管和杯状细胞。免疫组织化学显示实验组角膜上皮表达角膜上皮特异性角蛋白l（3（AE5）,不表达结膜特异性MUCSAC,在上皮基底部表达转录因子p63。对照组上皮持续表达MUCSAC。结论 组织工程角膜上皮移植术是一种有效的治疗角膜缘干细胞缺乏症的方法,可重建眼表,修复角膜上皮的损伤,抑制角膜上皮结膜化和新生血管。纤维凝胶膜为一种新型的组织工程材料,吸收快,透明度高,具有良好的组织相容性,是一种较理想的移植载体材料。（中华眼科杂志．2006．42：679-685）     

Staging of conjunctival squamous metaplasia by impression cytology

We modified the conventional impression cytology technique for conjunctival study by designing a 24-well Teflon sample holder, using cellulose acetate paper cut in an asymmetrical shape, and introducing Gill's modified Papanicolaou stain. Using this modified technique, we studied 35 normal subjects and 67 patients with various ocular surface disorders, 42 of whom were later found to have squamous metaplasia. Six different cytological stages were defined based on changes of goblet cell density, nucleus, and cytoplasm, encompassing three major steps: (1) loss of goblet cells, (2) increase of cellular stratification or enlargement of superficial cells, and (3) keratinization. This staging system allowed us to correlate pathological changes with clinical findings, and to investigate the action mechanism of squamous metaplasia of conjunctival epithelium. This modified impression cytology technique may help increase understanding of various ocular surface disorders.     

Impression cytology of the conjunctival epithelium in patients with chronic renal failure

AIMS: To assess ocular surface changes in patients with chronic renal failure (CRF), to compare the results with the degree of corneo-conjunctival calcium deposits, and to determine whether precipitation of calcium salts predisposes ocular surface modifications. METHODS: Impression cytology from 50 CRF patients on regular haemodialysis and 22 age and sex matched control subjects were studied. Specimens were obtained from the temporal bulbar conjunctiva using cellulose acetate filter paper. The samples were fixed in 95% ethanol, stained with the periodic acid Schiff (PAS) stain, and evaluated by light microscopy and were graded by a masked observer. Corneo-conjunctival calcification was graded by the Porter and Crombie classification. RESULTS: In the study group, three patients (6%) disclosed grade 0, 14 patients (28%) grade 1, and 33 patients (66%) grade 2-3 cytological changes. There was a statistically significant difference between the patient and the control groups (p= 0.0007), but no correlation could be found between the impression cytology grades and the calcium deposit grades (p=0.62). CONCLUSION: The ocular surfaces of CRF patients differ significantly from those of normal individuals, and it can be detected using impression cytology. These data suggest that the severity of conjunctival changes are not related to the presence or extent of calcium deposition.     

干眼症和前睑缘炎及季节性过敏性结膜炎中三种检测方法结果的比较

目的:比较三组临床患者和对照组患者的泪液功能测试,眼表染色以及结膜印象细胞学检测的结果。方法:这是一个单中心,前瞻性,双盲,随机对照试验。研究包括泪液分泌分数小于10mm和泪膜破裂时间(TBUT)小于10s的20例干眼症患者,有干燥和结痂睫毛的20例前睑缘炎患者,上眼睑睑板结膜乳头肥大的20例季节性过敏性结膜炎患者和20例对照组患者。比较泪液分泌的分数,泪膜破裂时间,眼表染色(牛津分级方案),杯状细胞密度(尼尔森分级方案评估所有患者的印象细胞学和化生分数)。结果:三组患者和对照组患者在泪液功能测试结果,眼表染色评分,杯状细胞密度和化生分数上有显著差异(P<0.001)。结论:结果证明这三种疾病的炎症反应造成严重眼表损害,并在局部产生炎症。这种损伤产生非常严重的影响,尤其是对结膜杯状细胞密度和化生。杯状细胞损伤程度与临床研究结果有关联。     

长期使用青光眼药物对眼表的影响

目的 探讨长期使用青光眼药物对患者眼表的影响.方法 收集局部使用青光眼药物3个月以上的患者65例108只眼,分为单种药物使用组(A组)和多种药物使用组(B组);另收集正常对照组(c组)25例25只眼.对所有观察对象进行泪膜破裂时间测定(BUT)、基础泪液分泌试验(ST)、角膜上皮荧光素染色、结膜上皮虎红染色以及结膜印迹细胞(IC)检查.结果 正常对照组BUT(12.25±5.40)s,ST(11.65±4.66)mm,A组BUT(6.97±4.87)8 9ST(7.19±5.36)mm,明显低于正常对照组(BUT P=0.000;STP=0.001). B组BUT(4.81±2.45)s,显著短于正常对照组(P=0.000)和A组(P=0.008);ST(5.20±2.97)mm,显著少于正常对照组(P=0.000)和A组(P=0.026).A组和B组的角膜荧光素染色评分和结膜虎红染色评分显著高于正常对照组.与正常对照组相比,A组和B组IC评分2-3级所占比例明显增高,结膜上皮表现出鳞状上皮化趋势.结论 长期使用青光眼药物会导致患者泪膜稳定性下降、泪液分泌减少,角膜上皮荧光素染色增多,结膜上皮杯状细胞减少、上皮细胞呈鳞状上皮化等泪膜、角膜和结膜的损伤.     

Impression cytology

Impression cytology refers to the application of cellulose acetate filter material to the ocular surface to remove the superficial layers of the conjunctival epithelium. Impression cytology has been found to be useful in assessing the ocular surface in various dry eye disorders, such as keratoconjunctivitis sicca (KCS), cicatricial ocular pemphigoid, and vitamin A deficiency. The technique is noninvasive, is easy to perform, causes minimal discomfort to the patient, and can be used to follow changes in the conjunctival ocular surface over time. With this method, the morphology of the conjunctival ocular surface can be studied and the degree of squamous metaplasia assessed. A specific criteria based on the appearance of the epithelial cells and the density of the goblet cells can be used to assign a grade (0-3) to the ocular surface. The grade of the ocular surface is related to the degree of squamous metaplasia and usually parallels the severity of clinical disease. Impression cytology can also be used to differentiate between various dry eye disorders. Disorders that are extrinsic or environmental (such as KCS) often affect the exposed interpalpebral ocular surface before the more protected inferior palpebral ocular surface. Intrinsic surface disorders (such as ocular pemphigoid) affect the palpebral, as well as the bulbar ocular surfaces, early in the disease.     

Impression cytology of conjunctival melanosis and melanoma.

Impression cytology using cellulose acetate paper has been used in various ocular surface disorders as a simple non-invasive diagnostic test. To assess its value in differentiating melanocytic tumours, 24 patients with a range of pigmented lesions of the conjunctiva were examined using this technique. Cytological and histological diagnoses were compared in 23 cases. In 73% of cases impression cytology predicted the histological diagnosis by detection of superficial atypical melanocytes and their proportion relative to benign epithelial cells. This pilot study shows impression cytology to be a useful diagnostic aid in the differentiation of pigmented tumours of the bulbar conjunctiva.     

Impression cytology of the ocular surface

Impression cytology refers to the application of a cellulose acetate filter to the ocular surface to remove the superficial layers of the ocular surface epithelium. These cells can then be subjected to histological, immunohistological, or molecular analysis. Proper technique is essential as the number of cells sampled can vary considerably. Generally two to three layers of cells are removed in one application but deeper cells can be accessed by repeat application over the same site. Applications for impression cytology include diagnosing a wide range of ocular surface disorders, documenting sequential changes in the conjunctival and corneal surface over time, staging conjunctival squamous metaplasia, and monitoring effects of treatment. It is also a useful investigational tool for analysing ocular surface disease with immunostaining and DNA analysis. It is non-invasive, relatively easy to perform, and yields reliable information about the area sampled with minimal discomfort to the patient. Major ophthalmic centres should develop and introduce this technique into routine clinical practice. This is best achieved with a team approach including the ophthalmologist, pathologist, microbiologist, and the immunologist.     

联合片状同种异体结膜移植与羊膜移植治疗早期碱烧伤

目的：探讨联合羊膜的小片状同种异体结膜移植重建眼表的可行性。 方法：新西兰白兔双眼上方球结膜碱烧伤后24h,实验组行以羊膜为基底的小片状同种异体结膜移植,对照组行单纯小片状同种异体结膜移植,术后各周行印迹细胞的PAS染色、扫描电镜检查。 结果：实验组未见明显排斥反应,全部植片成活,对照组7眼植片排斥脱落,植片成活率81．6％,明显低干实验组（P〈0．05）。实验组术后4wk,对照组术后第5wk结膜外观、杯状细胞形态、数目、功能均恢复正常。 结论：羊膜与小片状同种异体结膜移植相结合可能成为修补早期大面积结膜碱烧伤的有效途径。     

Impression cytology of melanocytic conjunctival tumours using the Biopore membrane

PURPOSE: To compare a new Biopore membrane impression cytology method with the routinely used exfoliative cytology in patients with a melanocytic lesion of the conjunctiva. METHODS: Sixty-eight consecutive patients with a conjunctival melanocytic lesion underwent Biopore membrane impression cytology as well as exfoliative cytology. A histologic sample was also available in 26 cases. All Biopore samples were stained immediately with RAL 555. Both Biopore and exfoliative cytology samples were assessed by two cytopathologists and graded into four different categories of atypia. RESULTS: Twenty-three out of 26 Biopores and 20 out of 24 for the exfoliative smears correlated with the corresponding histologic sample. Biopore cytology resulted in higher numbers of cells with a greater density compared to exfoliative cytology. CONCLUSIONS: Biopore cytology can be used for cytologic sampling of conjunctival melanocytic lesions. Because of the larger amount and higher density of cells obtained with the Biopore membrane, interpretation by a pathologist is easier and faster. Sampling of the fornix, caruncula, and ocular material in children is difficult with the Biopore method, and exfoliative cytology seems to be the favorable test in those situations.