老年性白内障晶状体上皮细胞超微结构的研究

目的观察老年性白内障晶状体上皮细胞超微结构的改变，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24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结构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，部分细胞溶解坏死或发生皱缩，未见凋亡细胞。结论老年性白内障的发生与晶状体上皮细胞变形坏死密切相关，与晶状体上皮细胞凋亡无关。     

晶状体上皮细胞凋亡与白内障形成的实验研究

目的：探讨氧化损伤致晶状体上皮细胞凋亡与白内障形成的关系。方法：离体家兔晶状体于M199培养液中培养,实验组另加过氧化氢使其最后浓度为0．9mM,对照组不加过氧化氢。实验组与对照组均不同时限分为3、6、12、18、24、36、48、60、84、108小时10个组。每组晶状体6－9只。利用白色背景下黑色方格作为判断晶状体混浊度的客观指标,其方法是透过晶状体观察方格线的清晰度并拍照。利用TUNEL技术检测晶状体上皮细胞凋亡率,在电子显微镜下观察细胞超微结构的改变并对凋亡细胞予以确认。结果：在过氧化氢损伤下随着培养时间的延长,晶状体混浊度加重,6小时开始轻微混浊,108小时几乎完全混浊;过氧化氢损伤可导致晶状体上皮细胞凋亡,且随时间延长,凋亡率增高,3 亡率为4．45％,84小时凋亡率达93．89％,108小时几乎全部凋亡。结论：过氧化氢可诱发体外培养的晶状体上皮细胞凋亡;细胞凋亡是实验性白内障形成的细胞学基础;进一步探讨保护晶状体免受氧化损伤及其他可致晶状体上皮细胞凋亡的各种损伤因素的措施,就有可能防止白内障的发生。     

老年性白内障的不同类型与晶状体上皮细胞密度的改变

【目的】探讨晶状体上皮细胞密度的改变与不同类型老年性白内障的关系。【方法】选取超声乳化白内障摘除手术病人的晶状体前囊膜标本 ,其中白内障的类型被分为皮质型 (成熟期 )、核硬化型 (≥NⅢ 级 )、后囊下型和混合型。健康成年人透明晶状体的前囊膜标本来源于眼库的供体眼球。所有前囊膜标本经铺片、固定染色后 ,细胞计数测定前囊膜中央区晶状体上皮细胞密度 (LECD)。【结果】健康成年男性透明晶状体LECD为 (4 15 2± 75 0 )个 /mm2 ,女性为 (4 874± 5 5 0 )个 /mm2 (P<0 0 1) ;核硬化型白内障 (≥NⅢ 级 )的LECD为 (5 12 0± 45 0 )个 /mm2 ,核硬化型白内障 (≥NⅢ 级 )晶状体上皮细胞密度高于其他各组及正常对照 (P <0 0 1) ,其中男性与女性的LECD分别为 (5 15 0± 35 0 )个 /mm2 和 (5 0 0 5± 480 )个 /mm2 (P >0 0 1)。【结论】健康成年女性的透明晶状体上皮细胞密度高于男性 ;核硬化型白内障 (≥NⅢ 级 )中男女晶状体上皮细胞密度无显著差异 ;以上结果结合当前流行病学的研究资料提示晶状体上皮细胞密度高可能是核硬化型白内障发生的易感因素 ,为研究老年性白内障的病理机制提供了新的线索     

晶状体上皮细胞凋亡的研究进展

细胞凋亡（apoptosis）又称程序性细胞死亡（programmed cell death，PCD），它是由Kerr等学者于1972年首次发现并命名。细胞凋亡是由基因控制的细胞自主性死亡．其发生对机体维持稳态和组织器官正常生理功能至关重要。晶状体上皮细胞（lens epithelial cell，LEC）是眼晶状体前囊膜的单层上皮细胞，是晶状体内代谢最活跃的部位，担负着晶状体的生长、分化及损伤修复，     

老年性白内障晶状体上皮细胞的形态学变化

目的 观察老年性白内障晶状体上皮细胞的形态学改变,探讨其与白内障形成的关系。方法 采集老年性白内障晶状体囊外除术中取出的前囊膜,采用铺片及ＨＥ染色、平板包埋及制备超薄切片等技术,分别在光镜和电镜下观察晶状体上皮细胞的密度、细胞形态和超微结构变化。结果 白内障 晶状体上皮细胞密度较正常晶状体明显减小（Ｐ〈０．０１）,６０岁以上组细胞低于６０岁以下组（Ｐ〈０．０１）。光镜下,形态异常的细胞散在分布,     

年龄相关性白内障患者晶状体上皮细胞的死亡方式及超微结构改变

目的:探讨年龄相关性白内障晶状体上皮细胞(lens epithelial cell,LEC)的死亡方式和细胞超微结构改变.方法:取年龄相关性白内障患者晶状体前囊共12例,其中6例皮质性白内障,6例核性白内障.分别行透射电镜观察.结果:比C形状扁平,正常细胞和变性坏死细胞间隔存在,线粒体广泛空泡化.所有标本均发现胀亡细胞,3例核性白内障及4例皮质性白内障标本发现典型paraptosis样细胞,仅有1例核性白内障标本发现早期凋亡细胞,细胞内偶见髓样体存在.结论:线粒体空泡化可能在年龄相关性白内障LEC的发生发展中起重要作用,胀亡和paraptosis可能是LEC的主要死亡方式.     

人类晶体上皮细胞的体外培养与观察

目的：建立人类晶体上皮细胞培养的简便方法并观察原代和传代细胞的生物学特征和组织学变化。方法：将人工晶体植入术中取下的前囊膜分割成小碎片，吸管转移至培养瓶底部进行原代培养，7—10d后常规方法进行传代培养，采用相差显微镜观察活体细胞的增殖活动和形态学变化。结果：人类晶体上皮细胞在体外培养时可以存活并传代，但是其生存能力与供体年龄有关。传代后期细胞发生纤维化改变。结论：本方法简便，有效。可用于实验研究。     

The Apoptosis of Bovine Lens Epithelial Cells Induced by Proteasome Inhibitor MG132

To investigate the effect of proteasome inhibitor MG132 on the apoptosis of bovine lens epithelial cells （BLECs）, the cells were treated with MG132 at different concentrations for12, 24 and 36 h. The cell viability was analyzed by MTT assay and the effect of MG132 on the apoptosis of BLECs was assessed by flow cytometry （FCM）. The results showed that after treatment for the same period, the inhibitory effect of MG132 on BLECs proliferation was enhanced with the increment of the concentration of MG132 （0, 2, 5, 10, μmol/L） （P〈0.05）. The 50% inhibiting concentration （IC50） was 2.03 μmol/L when the BLECs were treated with MG132 for 36 h. MG132 also induced the apop- tosis of BLECs obviously. FCM showed that the apoptosis index of the cells treated by MG132 at 2 μmol/L for 12 h was （20.24±1.51）%, and that of the control was （0.98±0.20）% respectively （P〈0.01, n=3）. It was concluded that MG132 could lead to apoptosis of BLECs. The decrease of proteasome activity may play an important role in the formation and development of cataract.     

紫外线对人晶状体上皮细胞凋亡的诱导及Bcl-2,Bax基因的影响

目的:研究紫外线照射体外培养人晶状体上皮细胞(human lens epithelial cell,HLEC)对凋亡的诱导、凋亡调控基因(Bax,Bcl-2)表达的变化,探讨紫外线诱导HLEC凋亡的机制。方法:以实验室培养的HLEC细胞株为研究模型,采用同一紫外线光源对HLEC进行照射。按紫外线照射时间将HLEC分为0,5,10,15及30 min组。采用Annexin V+PI双染流式细胞计数对HLEC凋亡进行检测,用原位杂交的方法检测各组Bax,Bcl-2 mRNA的表达。结果:随紫外线照射时间的延长HLEC凋亡率增加,Bcl-2阳性细胞率逐渐降低;而Bax阳性细胞率逐渐增加。HLEC凋亡率与Bcl-2和Bax的比率呈负相关(r=-0.874,P<0.05)。结论:紫外线照射可诱导HLEC凋亡,Bax和Bcl-2可能参与了紫外线诱导的HLEC凋亡的基因调控过程。     

Expression of P53 during lens epithelial cell apoptosis induced by ultraviolet

Cataract is a disease induced by multi- factorsand its pathologic mechanism isstill unknown.Ithasbeen accepted that ultraviolet irradiation ( UVR) isone of the most important pathogenesis.Li etal hasreported that UVR could induce the apoptosis of lensepithelial cells( LECs) and resulted in lens opacities.In order to study the pathologic mechanism of UVR-induced cataract at molecular and cellular levels,weinvestigated the apoptosisof LECs and the expressionof P5 3.1　 MATERIALSAND M…     

Mitochondrial proteomic analysis of isopsoralen protection against oxidative damage in human lens epithelial cells

Objective:To investigate the protective effects of the natural medicinal monomer isopsoralen（ISR） with estrogenic activity against oxidative damage in human lens epithelial cells B3（HLE-B3） caused by hydrogen peroxide（H₂O₂） and to pursue the possible mitochondrial proteomic regularity of the protective effects.Methods: HLE-B3 cells were treated with H₂O₂（300μmol/L）,β-estradiol(E₂:10^-8 mol/L) and H₂O₂,ISR(10^-5 mol/L) and H₂O₂,or left untreated.Altered expressions of all mitochondrial proteins were analyzed by protein array and surfaceenhanced laser desorption ionization time of flight mass spectrometry（SELDI-TOF-MS）.The mass/charge（m/z） ratios of each peak were tested by the Kruskal-Wallis rank sum test,and the protein peak value of the m/z ratio for each treatment by pair comparison was analyzed with the Nemenyi test.Results:H₂O₂ up-regulated the expressions of two protein spots（with m/z of 6532 and 6809）.E₂ mitigated the oxidative damage,and the expression of one protein spot（m/z 6532） was down-regulated.In contrast,ISR down-regulated both of protein spots（m/z 6532 and 6809）.Conclusions:ISR could effectively inhibit H₂O₂-induced oxidative damage in HLE-B3 cells.The protein spot at m/z of 6532 might be the target spot of ISR against oxidative damage induced by H₂O₂.     

Effects of rapamycin on expression of Bcl-2 and Bax in human lens epithelial cells and cell cycle in rats

The effects of rapamycin on the expression of Bcl-2 and Bax protein in in vitro cultured human lens epithelial cells(LECs) and cell cycle were investigated in order to provide the theoretical basis for the development of new inhibitory drugs for clinical prevention and treatment of after-cataract.The cultured LECs of second and third passages were collected and treated with rapamycin.The LECs were transferred into 96-well culture plates and divided into 6 groups,and each group was set to have 8 duplicate wells.In the negative control group,the LECs were given culture medium only,and in the blank control group,only culture medium was given.In the four rapamycin-treated groups,different concentrations(20,40,60 and 80 ng/mL) of rapamycin were given.After treatment for 24,48 and 72 h,the absorbance(A) values in each well were determined by MTT assay.The cell cycles of all groups were detected by using flow cytometry.Real-time fluorescent quantitative polymerase chain reaction(RFQ-PCR) and Western blot were used to detect the mRNA and protein expression of Bcl-2 and Bax respectively.MTT assay showed that rapamycin could inhibit proliferation of LECs in a time-and dose-dependent manner.Flow cytometry revealed that rapamycin could block the conversion of LECs from G1 phase to S phase,resulting in the increase of cells in G1 phase and the decrease of the cells in S phase.RFQ-PCR indicated that rapamycin could down-regulate the expression of Bcl-2 mRNA,but up-regulate the expression of Bax mRNA,suggesting it could induce apoptosis of LECs.Western blot demonstrated that rapamycin could suppress the expression of Bcl-2 protein,but promote the expression of Bax protein.It is concluded that rapamycin could inhibit proliferation of LECs probably not only by blocking the progression of cell cycle,but also by promoting the induction of apoptosis.     

核因子E2相关因子2在晶状体上皮细胞抗氧化损伤中的作用*

目的 探讨核因子E2相关因子2（Nrf2）在晶状体上皮细胞抗过氧化氢（H2O2）氧化损伤和凋亡中的作用。方法 通过MTT测定不同浓度H2O2对人晶状体上皮细胞（HLECs）活力的影响;流式细胞术检测正常对照组（不加H2O2）,不同浓度H2O2处理组（100、200及300?μmol/L）和Nrf2抑制组（GSK-3β+H2O2 300?μmol/L）活性氧（ROS）及超氧化物酶歧化酶（SOD）的含量;Western blotting检测凋亡相关蛋白剪切型Caspase-3（Cleaved-Caspase-3）、Bax、Bcl-2、Nrf2总蛋白、Nrf2核蛋白及Nrf2浆蛋白的表达;免疫荧光法检查Nrf2在晶状体上皮细胞的表达和定位。结果 当H2O2浓度为400?μmol/L时,晶状体上皮细胞不可逆死亡。细胞经H2O2处理48?h后,各组ROS的表达量比较,差异有统计学意义（P?<0.05）,与正常对照组比较,各处理组ROS含量升高（P?<0.05）,Nrf2抑制组与H2O2高浓度组比较,差异无统计学意义（P?>0.05）。处理48 h后,各组SOD的含量比较,差异有统计学意义（P?<0.05）;与正常对照组比较,各处理组的SOD含量降低（P?<0.05）,与H2O2高浓度组比较,Nrf2抑制组SOD含量下降（P?<0.05）。处理48?h后,各组Cleaved-Caspase-3、Bax、Bcl-2表达的比较,差异有统计学意义（P?<0.05）,与正常对照组比较,H2O2各个浓度组Caspase-3、Bax降低（P?<0.05）,Bcl-2升高（P?<0.05）,与H2O2高浓度组比较,Nrf2抑制组的Cleaved-Caspase-3、Bax升高（P?<0.05）,而Bcl-2降低（P?<0.05）;H2O2各浓度组中Nrf2相对表达相应增多且有核内转移的趋势,Nrf2抑制组无Nrf2表达。结论 Nrf2在人晶状体上皮细胞中的表达及核易位能增加抗氧化酶和抗凋亡蛋白的含量,降低促凋亡蛋白含量,从而起到自身抗氧化损伤和抗凋亡的作用。     

晶状体上皮细胞DNA损伤的SCGE测定法

目的 研究晶状体上皮细胞DNA的损伤.方法 应用单细胞凝胶电泳(SCGE)法测定氧化、辐射及细胞外高糖、高钙环境对晶状体上皮细胞DNA的损伤.结果 对照组中晶状体上皮细胞呈圆形无拖尾,表明其DNA未受损伤.各处理组细胞呈不同程度的典型彗星图像,头尾分明,与对照组相比差异具有显著性(P＜0.001).结论 以SCGE法测定出多种因素均可导致体外培养的晶状体上皮细胞DNA损伤,考虑其可能与白内障的形成有关.