人外伤性白内障晶状体上皮细胞的组织块培养

目的建立人外伤性白内障晶状体上皮细胞体外培养的简单有效方法，观察晶状体上皮细胞的体外生长规律和特点。方法应用改良组织块贴附培养法对儿童及成人外伤性白内障的晶状体上皮细胞进行体外培养，倒置显微镜下观察其生长规律。结果儿童和成人外伤性白内障晶状体上皮细胞都具有增生能力，晶状体上皮细胞均可传3代，晶状体上皮细胞多次传代后生长缓慢。结论儿童和成人外伤性白内障晶状体上皮细胞体外培养增生能力有限，儿童晶状体上皮细胞增生能力较强。改良组织块贴附培养法简单易行，重复性好，是晶状体上皮细胞体外培养的较好方法。     

人晶状体上皮细胞的组织块培养

目的建立人晶状体上皮细胞体外培养的简单有效方法,观察不同年龄人晶状体上皮细胞的体外生长规律和特点。方法应用改良组织块培养法对胎儿、成人和年龄相关性白内障的晶状体上皮细胞进行体外培养,在倒置显微镜下观察其生长、分化规律。结果胎儿、成人和年龄相关性白内障晶状体上皮细胞都具有增殖能力,胎儿和成人晶状体上皮细胞可传3代,年龄相关性白内障晶状体上皮细胞传代培养基本不能增殖。结论人晶状体上皮细胞体外培养困难,不同年龄人晶状体上皮细胞体外均能增殖,但增殖能力均很有限;改良组织块培养法是晶状体上皮细胞体外培养的较好方法。     

盖玻片辅助人晶状体上皮细胞原代培养法

目的：建立人晶状体上皮细胞原代培养的简便方法并比较不同来源人品状体上皮细胞的生物学特性。方法：取胎龄20周合法引产胚胎眼晶状体囊膜、中山眼科中心眼库眼晶状体囊膜和白内障患者术中撕取的前囊膜，分别在培养皿中铺平，加10乩10％DMEM培养液润湿后加盖盖玻片防止卷曲并促进粘贴．添加培养液浸没盖玻片，37℃培养。同时取相同来源的囊膜按照组织块法培养。观察细胞增殖情况并比较原代人晶状体上皮细胞与人晶状体上皮细胞系SRA01／0413晶体蛋白的表达差异。结果：在盖玻片辅助下，胚胎眼晶状体囊膜第2天即可见明显的增殖细胞由囊膜缘长出，眼库眼囊膜和白内障患者术中撕取的囊膜在3～4d的潜伏期后亦可见增殖细胞长出；组织块法培养出现部分组织块漂浮，且胚胎眼囊膜潜伏期延长至3-4d，眼库眼囊膜和白内障患者晶状体囊膜潜伏期延长至4-5d。结论：盖玻片辅助的改良组织块培养法能尽快获得体外培养的原代晶状体上皮细胞，且操作简便，值得推广应用于品状体病的研究。     

波形纤维蛋白在老年性白内障晶状体上皮细胞的表达

目的观察波形纤维蛋白在老年性白内障晶状体上皮细胞的变化.方法用卵白素-生物素过氧化物酶法对22例老年性白内障患者晶状体前囊膜上皮细胞进行波形纤维蛋白染色,采用包埋前免疫酶电镜技术处理6个晶状体前囊膜标本,并观察其超微结构;利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳及Westemnblot法分析4个晶状体表层组织(前囊膜、上皮细胞和表层皮质)中的波形纤维蛋白.结果老年性白内障晶状体上皮细胞的波形纤维蛋白表达减弱,与对照组比较差异有显著性(t=2.0948,P<     

胰蛋白酶预防后发性白内障的实验研究

目的 探讨应用晶状体上皮细胞清除的方法预防后发性白内障。方法 老年性白内障手术中前囊连续环形撕囊后取得人晶状体前囊，应用不同浓度胰蛋白酶对前囊进行消化处理。观察上皮细胞的清除情况。结果 0.25%，0.2%胰蛋白酶溶液处理的晶状体前囊，晶状体上皮细胞可以完全脱落，0.1%胰蛋白酶溶液处理晶状体前囊，上皮细胞部分脱落，结论 该结果可为临床应用胰蛋白酶清除晶状体上皮细胞防治后发生白内障提供理论依据。     

兔眼晶状体上皮细胞的体外培养

目的 建立兔晶状体上皮细胞体外培养的模型。方法 采用组织块培养法,对兔眼晶状体前囊膜进行培养,并利用形态学检查方法和免疫组化技术鉴定。结果 组织块接种２４ｈ后即可见细胞生长,且保持上皮细胞形态,１ｗｋ左右细胞融合,在体外可传至７代,５代以后细胞呈成纤维细胞状,α－晶状体蛋白间接免疫荧光呈阳性反应。结论 成功地建立晶状体上皮细胞体外培养模型,可用于后发性白内障发病机制的研究。     

MTT比色法检测苦参碱对兔晶状体上皮细胞增生的影响

目的研究苦参碱对体外培养的兔晶状体上皮细胞增生的影响,探索白内障术后晶状体后囊浑浊的治疗药物。方法取第2代对数生长期的兔晶状体上皮细胞,培养24h后,加入不同浓度的苦参碱(0.1、0.2、0.4、0.6、0.8、1.0、1.2、1.4、1.6mg/mL)作用24h后,采用MTT法观察苦参碱对兔晶状体上皮细胞增生的影响。结果苦参碱随着浓度的增加,其抑制兔晶状体上皮细胞增生的作用逐渐增强,增生抑制率的升高呈现明显的剂量依赖性。结论苦参碱能有效抑制体外培养的兔晶状体上皮细胞增生,在防止白内障术后晶状体后囊浑浊方面有一定的应用前景。     

老年性和糖尿病性白内障晶状体上皮细胞的超微结构改变

目的 观察正常人，老年性白内障、糖尿病性白内障晶状体上皮细胞的超微结构，探讨细胞凋亡在白内障发生中的作用。方法 收集正常人、老年性白内障、糖尿病性白内障晶状体前囊膜送透射电镜扫描，取老年性白内障和糖尿病性白内障前囊膜各15例，用脱氧核苷酸末端转移酶缺口标记原位细胞检测法，检测晶状体上皮细胞的凋亡细胞。结果 老年性和糖尿病性白内障晶状体上细胞的超微结构在形态上为扁平状，胞浆出现空泡变性，胞核出现固缩，染色质边聚，浓缩等改变。糖尿病性白内障晶状体上皮细胞的胞浆空泡变性更加严重。在光镜下老年性和糖尿病性白内障的晶状体上皮均有凋亡细胞。结论 老年性白内障和糖尿病性白内障的发生与晶状体上皮的细胞凋亡密切相关。     

老年性白内障晶状体上皮细胞超微结构的观察

目的 :观察老年性白内障晶状体上皮细胞的超微改变 ,探讨细胞凋亡在老年性白内障形成中的作用。方法 :老年性白内障患者 2 4例 ,行囊外或超声乳化联合后房型人工晶状体植入术 ,术中取中央部晶状体前囊膜 ,分别行光镜 ,扫描电镜 ,透射电镜观察。结果 :老年性白内障晶状体上皮细胞的超微改变有细胞形态大小不一 ,扁平细胞多无正常细胞结构 ,低柱状及柱状细胞 ,细胞结构大体正常 ,但细胞间隙增大 ,胞浆内可见空泡变性 ,部分细胞溶解坏死 ,部分细胞皱缩 ,呈早期细胞调亡改变。结论 :晶状体上皮细胞坏死与凋亡均与老年性白内障的发生有关。     

碱性成纤维细胞生长因子基因在胎儿与白内障患者晶状体上皮细胞内表达的比较

目的:研究碱性成纤维细胞生长因子基因在胎儿和白内障患者晶状体上皮细胞内表达的区别。方法:采用原位杂交方法,用cDNA探针检测胎儿培养及组织切片中的晶状体上皮细胞和白内障患者前囊中的晶状体上皮细胞的bFGF的mRNA,并用图像分析进行相对定量,比较胎儿培养细胞、组织切片细胞及患者囊膜细胞的积分光吸收度值。结果:胎儿的培养及组织切片中晶状体上皮细胞和白内障患者前囊中的晶状体上皮细胞都存在bFGF基因表达。胎儿体外培养晶状体上皮细胞、胎儿组织切片晶状体上皮细胞和白内障患者晶状体上皮细胞积分光吸收度值分别为627.1±268.7,131.5±42.8和79.2±26.3。胎儿体外培养晶状体上皮细胞积分光吸收度值显著高于胎儿组织切片晶状体上皮细胞(P<0.01);白内障患者晶状体上皮细胞积分光吸收度值显著低于胎儿晶状体上皮细胞(P<0.01)。结论:晶状体上皮细胞体外培养可增加bFGF基因表达;胎儿晶状体上皮细胞bFGF基因表达显著高于白内障患者晶状体上皮细胞。     

体外培养的晶状体上皮细胞株的确定及生长、分化规律

目的建立牛、兔、人晶状体上皮细胞的体外培养，进一步认识晶状体上皮细胞生长、分化规律。方法应用组织块培养方法进行３种晶状体上皮细胞的体外培养，经Ｇｉｍｓａ染色，在倒置显微镜下对培养的晶状体上皮细胞的生长、分化规律进行观察。结果培养至６ｗｋ的人胎儿晶状体上皮细胞中有特征性的“晶状体小体”形成；牛、兔晶状体上皮细胞去分化是发生在第３代，而人晶状体上皮细胞在第４代开始出现去分化；它们传代至第８代时生长都趋于停止，出现老化表现；来自人的晶状体上皮细胞生长增殖率与年龄呈负相关关系（ｒ＝－０．９９６）。结论“晶状体小体”的形成可作为确定晶状体上皮细胞株的一项特征性依据，而体外培养的人、牛、兔晶状体上皮细胞具有相同的有限生长潜能，在相同的条件下，牛、兔晶状体上皮细胞的生长增殖速度比人晶状体上皮细胞快，但易于发生去分化；此外，人晶状体上皮细胞的生长增殖率与年龄密切相关，年龄越小，晶状体上皮细胞的生长增殖速度越快。     

鼠晶状体上皮细胞的体外培养

目的:建立鼠晶状体上皮细胞体外培养的模型。方法:应用组织块贴片法和酶逐步消化法对10~14d的SD大鼠晶状体上皮细胞进行体外培养,在相差显微镜下观察其生长规律。结果:组织块贴片法在加入培养基4~5d后见细胞生长,2wk细胞融合。而酶逐步消化法在加入培养基后7d左右见细胞贴壁,2wk左右见细胞融合。结论:鼠晶状体上皮细胞体外培养较困难,本试验采用酶逐步消化方法和组织块贴片法。成功地建立了鼠晶状体上皮细胞体外培养的模型,为研究后发性白内障发病机制提供了基础。     

Pigment Epithelium-derived Factor in Cataractous Aqueous Humor and Lens Epithelial Cells

Purpose: To study the characteristics of PEDF in cataractous aqueous humor and its expression in human lens epithelium. Methods: The PEDF concentration in the aqueous humor was measured by enzyme -linked immunosorbent assay in senile (130cases) and congenital (18cases) cataract patients who underwent cataract phacoemulsification extraction surgery. Anterior lens capsular specimens were obtained from these patients to count lens epithelial cells (LEC) density. The Lens Opacities Classification System Ⅲ was used to classify the senile cataracts as cortical, nuclear, posterior subcapsular and mixed types of opacity, and quantitative analysis of the nuclear opacities was performed by Pentacam Scheimpflug imaging system. Anterior lens capsular specimens from another senile (10cases) and congenital (10cases) cataract were collected for immunofluorescence with polyclonal antibodies specific to human pigment epithelium -derived factor (PEDF). Results:The mean aqueous level of PEDF was(178. 9±87. 5)ng/ml, and there was negative linear correlation of PEDF level and age (r=0. 811, P<0. 001). In senile cases, the aqueous PEDF concentration decreased with increasing nuclear opacities (r=0. 447, P < 0.01) , and the mean PEDF level in nuclear cataract was significantly lower than that in posterior subcapsular opacity (P < 0.01) . PEDF immunostaining was detected in LEC of all capsular specimens. Conclusion : The PEDF level in human aqueous humor is related to age, types of cataracts and lens opacity. PEDF also express in human LEC. The study results suggest PEDF may regulate and/or protect LEC by paracrine and autocrine, and lack of PEDF may play a role in cataractogenesis.     

平阳霉素体外诱发大鼠白内障的早期生化变化

目的和方法：用器官培养的方法在培养基中加入一定剂量的平阳霉素作用于大鼠晶体而诱发形成白内障,并在白内障形成的早期过程中观察了晶体内非蛋白质巯基,蛋白质巯基和二硫键同仁以及脂类过氧化水平的动态变化。结果和结论;培养１０ｍｉｎ时,非蛋白质巯基首先下降;随后蛋白质巯基含量也开始降低;二硫键的含量至４０ｍｉｎ时才呈现升高的趋势;而ＭＤＡ在培养的３０ｍｉｎ后明显升高。     

Studies on primary cultures of adult lens cells from normal and hereditary cataractous mice.

Lens epithelial cells from normal and congenital cataractous mice strains were cultured under similar conditions. Both normal and cataractous cells actively propagated and reached confluency on the eleventh day. These cells, thereafter, underwent morphological changes characterized by cell elongation, aggregation and formation of lentoid bodies at about 15 days.Electron microscopy revealed these lentoid bodies to consist of immature lens cells. These structures derived from cataractous cells had numerous vacuoles in the cytoplasm much more so than in the normal lens cells. In addition, some lentoid bodies closely resembled mature fibers of the intact lens. It was also demonstrated that these lentoid bodies showed positive immunofluorescence when reacted with fluorescent antiserum to γ-crystallin.There were certain differences observed between the cultured cells derived from normal lens and Nakano cataract. The disappearance of organelles and denucleation process were delayed in the lentoid bodies found in cultured Nakano cells when compared to normal cell culture. In addition a second type of lentoid body, although present as a minor population, was observed in the Nakano cell culture. Other subtle differences were observed during the course of culturing normal and cataractous lens cells.     

正常和老年性白内障晶体的钙调节

测定了正常和白内障晶体上皮、皮质和核的钙-ATP酶、钙调蛋白和钙含量，分析了它们之间的相关性。发现正常晶体钙、钙调蛋白与钙-ATP酶呈正相关。老年性白内障晶体钙调蛋白与钙-ATP酶活性无相关性，钙-ATP酶活性下降。提示老年性白内障晶体钧-ATP酶活性下降是钙含量升高的重要原因，推测与氧化性损伤累及钙-ATP酶流基有关。     

晶状体上皮细胞增生与Pax-6表达的相关分析

目的检测Pax-6基因在体外培养的不同阶段小鼠晶状体上皮细胞(lens epithelial cells,LECs)中的表达,分析其与LECs增生特性的关系。方法 利用囊膜组织块贴壁法及胰酶消化法体外培养不同阶段LECs;免疫组织化学法检测LECs中Pax-6蛋白表达情况;并利用RT-PCR法检测其mRNA水平的表达情况。结果 在原代培养细胞、传第1代及传第2代的LECs中明显检测到Pax-6mRNA水平的阳性表达,LECs相应各泳道有一与227 bp相一致的扩增片段;RT-PCR法同样发现在原代培养细胞、传第1代及传第2代、传第3代的LECs中均能检测到Pax-6蛋白水平的表达,Pax-6标记指数分别为11.75%、20.00%、26.75%、5.25%,Pax-6阳性物质染色呈弥漫性、均一性分布于LECs细胞核内。阴性对照组在各阶段均未见Pax-6的表达。结论 LECs中有Pax-6基因存在,该基因的正常表达与LECs增生特性相关。     

Comparison of cell-suspension and explant culture of rabbit limbal epithelial cells

Currently, most investigators directly use limbal explants to culture corneal epithelial cells. However, it has not been identified that limbal stem cells do readily migrate from the limbal explants onto culture plate or amniotic membrane carrier. In this study a cell-suspension culture system for rabbit limbal stem cells was developed and compared with the direct explant method in the aspect of stem cells content in the culture system. Rabbit limbal epithelial cells were dissociated from rabbit eyes by dispase and single cell suspension was made for cell-suspension culture. DeltaNp63 expression of cultured rabbit limbal epithelial cells by cell-suspension technique and explant technique was detected. In cell-suspension culture, isolated cell-suspension was evaluated by flow cytometric analysis for vimentin expression and residual limbal tissue after dispase treatment was examined by scanning electron microscopy. In limbal epithelial cells suspension less than 5% cells were vimentin positive. Examination of residual limbal tissue confirmed that all the limbal epithelial cells had been removed. Histological examination revealed that with cell-suspension culture the cultured epithelial cells could differentiate better than with explant technique. In cells cultured with cell-suspension, there were much more cells expressing DeltaNp63 than in explant cultured cells. In cells cultured with explants, most of DeltaNp63 labelling cells mustered around the explants, and peripheral cells on the slides were DeltaNp63 negative. These results suggested that with pure limbal epithelial cells suspension including basal cells, which could directly enter into culture system, cell-suspension culture technique was significantly superior to explant culture technique in terms of stem cells content.