年龄相关性白内障晶状体上皮细胞的Smac、caspase-3表达及死亡方式

目的探讨年龄相关性白内障晶状体上皮细胞（LECs）中Smac、caspase-3的表达和LECs超微结构变化及死亡方式。方法收集皮质性（30例）、核性（24例）、后囊下性（28例）年龄相关性白内障患者晶状体前囊片,以12例高度近视摘出的透明晶状体的晶状体前囊片为对照,应用免疫组织化学链酶菌抗生素蛋白-过氧化物酶法检测Smac、caspase-3蛋白在LECs中的表达,并采用透射电镜观察LECs超微结构的改变及死亡方式。结果Smac、caspase-3蛋白在年龄相关性白内障组LECs中的阳性表达率分别为81.71%、78.05%,与透明晶状体组比较染色阳性率及染色强度差异均有统计学意义（P〈0.05）。3种类型年龄相关性白内障透射电镜下均可见LECs多表现为肿胀,线粒体嵴缺失、空泡变、髓样化,粗面内质网扩张、颗粒融合和脱颗粒现象,仅1例核性白内障中可见到核膜皱缩向外呈锐角凸起,部分双层核膜融合,模糊不清,细胞核内染色质浓缩,未见凋亡小体。结论Smac、caspase-3蛋白可能参与了年龄相关性白内障的形成。年龄相关性白内障LECs存在凋亡、胀亡等多种细胞死亡方式,细胞凋亡与细胞胀亡的发生可能存在部分分子机制的重叠。LECs超微结构的改变表明线粒体形态改变和功能异常在年龄相关性白内障发生中起重要作用。     

年龄相关性白内障基因异常表达的研究

目的比较年龄相关性白内障晶状体上皮细胞(LEC)基因表达与正常人的差异,探讨白内障发病的分子机制。方法术中撕取晶状体前囊36眼,其中单纯核性年龄相关性白内障10眼,成熟期年龄相关性白内障19眼,角膜移植供体眼及外伤性晶状体脱位7眼(正常对照)。3组晶状体囊膜分别提取总RNA,将合格的各组RNA样品做差异显示(DD-RT-PCR),寻找差异表达基因,再将其分离、扩增、测序,最后选取与白内障形成密切相关的胰岛素样生长因子结合蛋白(IGFBP-5)及溶酶体相关膜蛋白2(LAMP-2)基因进行验证。结果各组RNA纯度达到要求,总RNA完整,经差异显示,年龄相关性白内障组比正常人组低表达的条带有5条,高表达条带15条。对6条明亮条带测序后,其中3个片段分别与IGFBP-5、LAMP-2及小核糖体蛋白10(rps10)有很高的同源性,其他3个片段均为新近克隆未知功能的基因或基因组序列。验证表明,年龄相关性白内障IGFBP-5表达水平低于正常对照(P<0.01),而年龄相关性核性白内障LAMP-2表达水平高于正常对照。结论IGFBP-5和LAMP-2是年龄相关性白内障相关基因,差异显示技术在研究年龄相关性白内障发生中有重要意义。     

白内障患者晶状体上皮细胞中Fas蛋白的表达

目的:了解不同类型白内障患者晶状体上皮细胞(lens epi-thelial cell,LECs)的凋亡情况及其与Fas蛋白(CD95)的关系。方法:取年龄相关性白内障、糖尿病性白内障及高度近视并发性白内障患者超声乳化手术中取下的前囊膜标本共73例。应用光镜、透射电镜及TUNEL标记法观察LECs的凋亡情况。同时应用免疫组化和流式细胞分别定性、定量检测Fas蛋白(CD95)在LECs中的表达。结果:光镜下3种白内障患者的LECs都呈透明样变性,细胞内有空泡。电镜下可见正常以及变性、坏死的LECs。部分LECs细胞内染色质凝集并边缘化,呈早期细胞凋亡改变。TUNEL检测发现3种白内障患者的LECs中均有TUNEL阳性细胞存在,LECs的凋亡率分别为(32.20±7.91)%、(31.00±9.43)%、(28.20±8.04)%,3种白内障间未见统计学差异。免疫组化结果表明在3种白内障患者的LECs中均有Fas蛋白(CD95)表达。流式细胞定量检测年龄相关性、糖尿病性及高度近视并发性白内障患者LECs中Fas蛋白(CD95)的表达量分别为(65.72±9.95)%、(63.46±13.30)%、(31.46±17.25)%,高度近视并发性白内障LECs中Fas蛋白(CD95)的表达与其他两种白内障间存在统计学差异(t检验,P<0.05)。结论:年龄相关性白内障、糖尿病性白内障及高度近视并发性白内障患者的LECs均出现凋亡,LECs的凋亡很可能是非先天性白内障发病的细胞病理学基础。Fas蛋白(CD95)介导的LECs的凋亡在年龄相关性白内障及糖尿病性白内障的形成中起重要的作用,而在高度近视并发性白内障LECs凋亡中可能存在其他通路。     

不同类型年龄相关性白内障晶状体上皮细胞衰老标记蛋白30的表达及与细胞凋亡的关系

背景 随着人口的老龄化,白内障的发病率逐年上升,研究表明,衰老标记蛋白30( SMP-30)与白内障的发生发展关系密切. 目的 了解不同类型白内障晶状体上皮细胞( LECs)中SMP-30的表达及LECs的凋亡情况,探讨不同类型年龄相关性白内障的发病机制. 方法 收集2010年3-10月在武汉大学中南医院眼科行白内障超声乳化摘出术的年龄相关性皮质性白内障59例80眼和核性白内障53例70眼,2个组患者年龄匹配(P＞0.05).白内障手术中环形撕取晶状体前囊膜,应用免疫组织化学法和实时荧光定量聚合酶链反应( real-time PCR)技术分别定性、定量检测SMP-30蛋白及其mRNA在2个组白内障LECs中的表达.用TUNEL标记法观察LECs的凋亡情况,对比分析两种类型白内障晶状体前囊膜中SMP-30的表达及细胞凋亡的差异.结果 免疫组织化学法检测表明,SMP-30主要表达于晶状体囊膜的细胞质中,在晶状体囊膜中央部表达较弱,越近周边表达越强,差异均有统计学意义(核性:45.21±2.79 vs 76.42±11.21,P=0.042;皮质性:108.32±4.32 vs 206.34±15.67,P=0.037).核性白内障LECs中SMP-30 mRNA的表达少于皮质性,差异有统计学意义(60.02±9.08 vs 157.33±13.01,P=0.034).TUNEL染色显示,2个组白内障LECs凋亡百分率中央部明显高于周边部,差异均有统计学意义(核性:19.34％±0.11％vs 8.32％±0.57％,P=0.025;皮质性:42.07％±0.86％vs 13.55％±0.64％,P=0.010),细胞凋亡百分率低于皮质性,差异有统计学意义(14.05％±0.22％vs 27.70％±0.81％,P=0.007). 结论 两种类型年龄相关性白内障的发生均与LECs凋亡有关,SMP-30的表达与其密切相关.     

年龄相关性白内障晶状体前囊膜中核因子κB的免疫组化研究

目的:研究核因子κB蛋白在正常人、年龄相关性白内障的晶状体前囊膜上皮细胞中表达的差异,探讨NF-kB在年龄相关性白内障发病机制中的作用.方法:显微镜下随机收集年龄相关性白内障前囊膜组织30例为病例组及透明晶状体前囊膜组织5例为正常对照组,采用免疫组织化学方法检测NF-κB P65蛋白的表达.结果:正常晶状体前囊膜上皮细胞胞质和胞核中可见NF-κBP65蛋白阳性表达,以胞质为主;年龄相关性白内障晶状体前囊膜上皮细胞胞质和胞核染色呈阳性且以胞核为主.图像分析表明病例组NF-κB P65平均吸光度值高于对照组,差异有统计学意义(t=6.2109,P<0.05).结论:NF-κB表达水平升高与年龄相关性白内障发病密切相关,其表达异常可能参与年龄相关性白内障的发生与发展.     

SIRT1基因在糖尿病性白内障患者晶状体上皮细胞表达的初步研究

目的:探讨SIRT1基因在糖尿病性白内障晶状体上皮细胞的表达.方法:选取2012-01/2014-10来我院治疗的糖尿病性白内障患者、年龄相关性白内障患者和外伤性白内障患者各20例,采用RT-PCR法检测各组患者晶状体上皮细胞中SIRT1基因含量,Western blot法检测晶状体上皮细胞中SIRT1蛋白含量,TUNEL法检测晶状体上皮细胞的凋亡率.结果:RT-PCR检测结果显示,外伤性白内障患者组SIRT1 mRNA相对含量最高为1.000±0.078,其次为年龄相关性白内障患者组为0.427±0.067,糖尿病性白内障组为0.389±0.112,与外伤性白内障组比较,差异有统计学意义(P＜0.05);Western blot检测显示,外伤性白内障患者晶状体上皮细胞中SIRT1蛋白的表达量最高,其次是年龄相关性白内障组,糖尿病性白内障组SIRT1蛋白的表达量最低;TUNEL法检测结果显示,外伤性白内障组和年龄相关性白内障组患者LECs细胞凋亡率分别为(4.5±2.3)％和(8.7±4.1)％,差异无统计学意义;而糖尿病性白内障组LECs凋亡率为(24.3±6.1)％,与外伤性白内障组及年龄相关性白内障组比较差异有统计学意义(P＜0.05).结论:糖尿病性白内障患者晶体上皮细胞中SIRT1基因及蛋白表达下降,提示该基因参与了糖尿病性白内障的发生,这为我们今后进一步的研究提供了可靠的理论依据.探索调节SIRT1基因在晶状体上皮细胞中表达的有效途径,将为糖尿病性白内障的早期干预治疗提供新的思路.     

泛素羧基末端水解酶L1在年龄相关性白内障晶状体上皮细胞中的表达及其抗氧化作用

背景 氧化损伤是年龄相关性白内障发生的主要原因,而泛素蛋白酶系统参与晶状体的分化发育,研究发现其关键酶泛素羧基末端水解酶L1( UCHL1)参与帕金森病和阿尔茨海默病等年龄相关性疾病的发生发展,且与氧化应激有关. 目的 研究UCHL1在年龄相关性白内障发病过程中的作用.方法 收集24例单纯年龄相关性白内障患者术后获得的晶状体囊膜(皮质性白内障12例、核性白内障12例)、5例正常人晶状体前囊膜上皮和人晶状体上皮细胞(LECs)系SRA01/04细胞,采用免疫荧光法检测UCHL1在各组人晶状体前囊膜上皮细胞中的表达情况.构建UCHL1真核表达质粒,鉴定后采用脂质体转染法转染SRA01/04细胞作为UCHL1过表达组,同时采用绿色荧光蛋白(GFP)真核表达质粒转染SRA01/04细胞作为GFP过表达组,使用梯度过氧化氢叔丁醇(TBHB)处理24h后,采用MTT法检测各组人LECs的活性变化.结果 免疫荧光检测表明,UCHL1在各组人LECs中均有表达,但在正常晶状体囊膜、皮质性白内障以及核性白内障晶状体囊膜上皮细胞表达量的总体差异有统计学意义(F=13.441,P=0.000).皮质性白内障组以及核性白内障组晶状体囊膜上皮细胞中UCHL1的表达量均低于正常晶状体组(P=0.000、0.000),但皮质性白内障组和核性白内障组之间UCHL1的表达量差异无统计学意义(P=0.164).Western blot鉴定结果表明,UCHL1真核表达质粒转染后可见SRA01/04细胞中UCHL1的强表达.MTT检测结果显示,0.3 mol/L TBHB处理24 h后,UCHL1过表达组细胞活性吸光度(A570/630)值与GFP过表达组比较有增高的趋势,而0.2、0.4、0.5 mol/LTBHP均导致SRA01/04细胞的耐受或者大量凋亡. 结论 UCHL1具有抗氧化作用,且可能在年龄相关性白内障的发生发展过程中起抑制作用.     

白内障手术摘出前囊下晶状体上皮细胞中β1整联蛋白、Fas蛋白的表达

目的了解糖尿病性白内障及并发性(高度近视)白内障晶状体上皮细胞(LECs)中β1整联蛋白与Fas蛋白的表达情况,以探讨其与LECs凋亡的关系。方法应用免疫组化、流式细胞仪和共聚焦显微镜对52例白内障手术患者术中取出的前囊下LECs分别定性、定量及定位检测β1整联蛋白和Fas蛋白的表达。结果糖尿病性及并发性(高度近视)白内障患者免疫组化结果表明在其LECs中均有β1整联蛋白、Fas蛋白表达;流式细胞定量检测其LECs中β1整联蛋白的表达量分别为68.41±16.98%、39.22±21.18%,Fas蛋白的表达量分别为63.46±13.30%、31.46±17.25%。LECs中β1整联蛋白、Fas蛋白的表达在两种白内障间存在统计学差异(P<0.05)。β1整联蛋白与Fas蛋白在LECs中的表达具有正相关性。共聚焦显微镜观察发现β1整联蛋白与Fas蛋白大部分共同定位于LECs的细胞膜上。结论Fas蛋白介导的LECs的凋亡在糖尿病性白内障的形成中起重要的作用,而在并发性(高度近视)白内障LECs凋亡中可能存在其他通路。β1整联蛋白的作用很可能是一种促进LECs凋亡的蛋白,它与Fas蛋白一起促进LECs凋亡。     

新基因c24的克隆及其在正常及白内障晶状体上皮细胞中的表达

目的克隆新基因c24并研究其在正常晶状体和年龄相关性白内障晶状体上皮细胞(LECs)中的表达,并比较二者表达的差异。方法用改良的消减杂交法克隆新基因c24,地高辛标记c24cDNA片断制作探针,用原位杂交法研究其在晶状体前囊膜切片上的表达,并进行统计学处理。结果克隆到了新基因c24的cDNA片断,长约532bp,c24在正常晶状体和年龄相关性白内障LECs中均有表达;其在年龄相关性白内障LECs中的表达高于其在正常LECs的表达。结论c24在正常晶状体和年龄相关性白内障LECs中均有表达;在年龄相关性白内障LECs中,其表达较正常晶状体增高,提示其可能在白内障的发生中起一定的作用。     

HSP70在STZ-糖性白内障发病机制中的研究

目的:研究HSP70在链脲佐菌素—糖性白内障发生发展中的作用。方法:将66只SD大鼠随机分为2组,正常对照组与白内障组。用链脲佐菌素(STZ)诱发糖性白内障,每周观察晶状体的变化,在实验开始后2,4,8周末,分别摘取眼球,采用免疫组化技术检测热休克蛋白-70(HSP70)在晶状体上皮细胞(LECs)中的表达。结果:对照组晶状体一直保持透明,白内障组晶状体在2周末出现空泡,8周末全部混浊。HSP70在对照组中未见表达,在白内障组中表达明显,并随着白内障的发展而表达增加。结论:HSP70可能通过调节晶状体上皮细胞的生长在糖性白内障的发生、发展中起重要作用。     

hTERT在年龄相关性白内障晶状体上皮细胞中的表达及意义

目的探讨端粒酶逆转录酶（hTERT）在年龄相关性白内障患者晶状体上皮细胞（LEc）中的表达及在白内障形成中的作用。方法应用免疫组织化学及核酸原位杂交技术检测hTERT在100例年龄相关性白内障患者和30例正常LEC中的表达，并进行比较。结果年龄相关性白内障LEC中hTERT的阳性表达率明显增高（P〈0．05），染色强度以中等以上强度为主，与正常晶状体组相比，差异具有显著统计学意义（P〈0．01）。三种不同类型的年龄相关性白内障LEC中hTERT的阳性表达率及染色强度均无显著统计学差异（P〉0．05）。结论年龄相关性自内障LEC中端粒酶的激活可能是对紫外线辐射及氧化应激诱发的DNA损伤、端粒缩短的一种防御反应。但当损伤积累到一定程度时，端粒酶的激活也不足以阻止端粒缩短，LEC即发生衰老或凋亡，形成白内障。     

波形蛋白在老年性白内障晶状体上皮细胞中的表达及其意义

目的 :进一步明确波形蛋白的异常表达在老年性白内障形成中所起的作用。方法 :用链酶菌抗生物素蛋白 碱性磷酸酶 (streptavidin alkalinephosphatase,S P)免疫组化方法检测波形蛋白在 31例皮质性白内障、2 0例核性白内障及5例正常透明晶体上皮中的表达情况。结果 :波形蛋白在皮质性白内障晶体上皮中的表达比在核性白内障及正常透明晶状体中高。结论 :老年性皮质性白内障与老年性核性白内障的发生有不同的发病机制 ,皮质性白内障的发生与波形蛋白的过度表达有关     

老年性白内障晶状体上皮细胞凋亡及相关基因蛋白的表达

目的 探讨老年性白内障与晶状体上皮细胞凋亡的关系。方法透射电镜下观察老年性白内障晶状体上皮细胞的超微结构；Tunel法检测凋亡细胞百分率；并对其晶状体上皮细胞DNA进行琼脂糖凝胶电泳；免疫组化法检测P53、bcl-2在老年性白内障晶状体上皮细胞中的蛋白表达。结果 透射电镜下发现老年性白内障晶状体上皮细胞中有凋亡细胞；凋亡细胞百分率为8．4％～37．8％，琼脂糖凝胶电泳出现梯状条带；P53在老年白内障晶状体上皮细胞中蛋白表达率为16．9％～19．1％，bcl-2无蛋白表达。结论 老年性白内障的发生可能与其晶状体上皮细胞凋亡有关。     

Distribution, spherical structure and predicted Mie scattering of multilamellar bodies in human age-related nuclear cataracts

PURPOSE: To characterize multilamellar bodies (MLBs), determine their distribution along the optic axis and predict their potential Mie scattering within human age-related nuclear cataracts. Previous studies restricted to the equatorial plane have shown that MLBs are rare spherical objects that are 1-4 microm in diameter and covered by multiple layers of thin lipid-rich membranes. METHODS: Eight human aged transparent lenses were obtained from eye bank donors and eight human age-related nuclear cataracts were obtained immediately after extracapsular extraction. Each sample was Vibratome sectioned fresh into 200 microm thick sections that were fixed and embedded for light or electron microscopy. Light micrograph montages of the optic axis containing the juvenile, fetal and embryonic nuclei were examined. Mie scattering for random coated spherical particles was calculated based on assumed and measured particle parameters. RESULTS: Cells along the optic axis of the cataract contained approximately 7.5 times more MLBs as similar regions of the aged transparent lens, although these MLBs occurred with extremely low frequency. Cells of the aged transparent lens contained 1.3 MLBs mm(-2), while those of the cataract contained 9.6 MLBs mm(-2), which are equivalent to calculated densities of 5.6 x 10(2) and 4.1 x 10(3)mm(-3), respectively. While some MLBs were located within the cytoplasm near cell membranes, others were found away from membranes. The MLBs are distinct from circular profiles resulting from finger-like projections between adjacent cells. MLBs displayed varying geometries and cytoplasmic textures, although predominately spherical with interiors similar to adjacent fiber cell cytoplasm. These results are in agreement with previous theoretical analysis of light scattering from human lenses and with previous morphological studies examining the equatorial plane of the lens. Potential Mie scattering of spherical particles with the average properties of the observed MLBs and assumed refractive index properties was calculated to be forward scattering of as much as 20% of the incident light. CONCLUSIONS: The observed low frequency and absence of clustering of MLBs in the equatorial plane and along the optic axis suggests that MLBs are most likely uniformly distributed throughout the embryonic, fetal and juvenile nuclei of age-related cataracts. Because of their size, distribution, textured cytoplasm and calculated Mie scattering, MLBs probably cause local fluctuations in refractive index in human lens nuclei and, therefore, are potential sources of low-angle, forward light scattering that could impair image formation.     

Lens epithelial cell density and histomorphological study in cataractous lenses.

Human epithelial cell density was determined from flat preparation of 195 cataractous lenses from 108 males and 87 females between 30 and 80 years of age. The mature cataracts had significantly lower cell counts than the other cataracts. Cell density was significantly higher in the females than in the males. Morphohistological study of the epithelia was focused on the following cataract types: (1) nuclear, (2) posterior subcapsular, (3) mature, (4) mixed, (5) hypermature, and (6) black. The major cataractous changes in all types involved vacuolization of the cytoplasm. The mature types of cataractous epithelia showed 56% superimposed cells; the epithelia in nuclear, posterior subcapsular, and black cataracts showed between 6% and 16%. In the hypermature cataracts, four of five tissues analyzed showed superimposed cells. The superimposed areas are probably the source of increased and altered cell activity. We propose that the metaplastic processes leading to posterior capsular opacification originate from these areas. The majority of nuclear and black cataracts were almost similar to the normal human lens epithelium with more or less uniform distribution of cells. Nucleus shrinkage (5 microns) was more evident in nuclear cataracts; in subcapsular cataracts most of the nuclei were large (average 9 microns diameter). Variation in morphological changes like vacuolization of cytoplasm and nuclei, pyknotic nuclei, and superimposed cells was more evident in the mixed type of cataracts.     

Lens epithelial apoptosis and cell proliferation in human age-related cortical cataract

PURPOSE: To probe the presence of apoptosis in the epithelium of human lenses with age-related cortical cataract as well as to assess cell proliferation, a predicted consequence of apoptotic cell death, in this specific cell population. METHODS: DNA fragmentation was assessed using terminal digoxigenin-labeled dUTP nick end labeling (TUNEL) in capsulotomy specimens obtained from patients who underwent either extracapsular cataract extraction for the removal of adult-onset cortical cataract (n=27) or clear lens extraction for the correction of high myopia (n=25). Cell proliferation was assayed in 23 epithelia of cataractous lenses, and 20 epithelia of non-cataractous lenses with the proliferation marker MIB1, a monoclonal antibody against the nuclear antigen Ki-67 that is detected throughout the cell cycle but is absent in the resting (G0) cell. RESULTS: TUNEL staining was observed in 25 (92.6%) specimens of cataractous lenses, whereas cells undergoing apoptosis were identified in 2 (8%) of the epithelia from non-cataractous lenses. Only two MIB1-positive samples were detected, one of which was a capsule obtained during intracapsular cataract extraction. CONCLUSIONS: The epithelium of human lenses with cortical cataract undergoes low rate apoptotic death. This limited epithelial apoptosis is unlikely to result in any significant cell density decrease since epithelial gaps are likely to be replaced by cell proliferation at the germinative zone of the anterior lens capsule. Nevertheless, the accumulation of small-scale epithelial losses during lifetime may induce alterations in lens fiber formation and homeostasis and result in loss of lens transparency.     

Calcium-induced high molecular weight proteins in the intact rabbit lens

We have investigated the ability of Ca2+ to induce the formation of high molecular weight (HMW) proteins in the intact lens. Ca2+ cataracts were produced in rabbit lenses by culturing the lenses for either four days in medium containing 20 mM Ca2+ or for three days in medium containing 100 mM Ca2+. Lenses cultured in 20 and 100 mM Ca2+ medium became opaque after 20 hr and contained 30 and 200 times higher levels of Ca2+, respectively, than transparent lenses cultured in medium containing 1 mM Ca2+. Lenses exposed to 100 mM Mg2+ did not lose transparency. The opacification of the lenses extended to a depth of 1 mm into the cortical layer and did not involve the nucleus. No significant differences were found in the concentrations of either soluble or insoluble proteins present in freshly excised lenses and Ca2+ cataracts. Soluble HMW proteins, greater than 1.5 X 10(6) daltons, were in two- and five-fold greater amounts in the 20 and 100 mM Ca2+ cataracts, respectively, compared to controls. HMW protein present in the 100 mM Ca2+ cataract amounted to approximately 3% of the total soluble protein in the lens. The amount of Ca2+ present in the HMW fraction was 1 Ca2+ per 5 X 10(5) daltons, no higher than that present in the unaggregated crystallins. No evidence was found for covalent bonding in the aggregate. Results of polyacrylamide gel electrophoresis and double immunodiffusion indicated the presence of alpha- and beta- but not gamma-crystallin in the HMW protein.(ABSTRACT TRUNCATED AT 250 WORDS)