特殊表型先天性白内障小鼠晶状体中细胞缝隙连接蛋白的表达

目的观察γD-晶状体蛋白点突变导致的小鼠先天性白内障表型,检测该特殊表型先天性白内障小鼠晶状体中细胞缝隙连接蛋白（Cx）的表达。方法观察突变小鼠出生后不同时间晶状体的形态学变化;应用免疫荧光染色法分析晶状体内Cx46和Cx50的表达和分布。结果突变小鼠模型呈稳定一致的显性遗传,出生后7 d即表现为明显的核性白内障,出生后21 d纯合子小鼠晶状体混浊严重,后囊膜自然破裂;免疫荧光染色分析发现突变小鼠晶状体内Cx46和Cx50的表达均出现下降,越靠近晶状体中心部下降越明显。结论γD-晶状体蛋白点突变可导致晶状体后囊膜破裂这种特殊表型的先天性白内障,白内障的形成和后囊膜破裂的产生与晶状体内Cx46和Cx50的表达下降有关。     

水通道蛋白-1在高糖培养人晶状体上皮细胞中的表达

目的研究水通道蛋白-1（AQP-1）在糖尿病性白内障发病机制中的作用。方法从健康的供体眼中分离出人晶状体囊膜,用组织块培养法将人晶状体囊膜分别置于1g/L葡萄糖＋体积分数15%胎牛血清的DMEM培养液中（对照组）或4.5g/L葡萄糖＋15%胎牛血清的DMEM培养液中进行培养（高糖组）。培养3d、28d后观察2组人晶状体上皮细胞（LECs）的生长状态。采用免疫荧光技术和流式细胞术检测AQP-1在人LECs中表达的平均荧光强度,应用流式细胞术检测2组人LECs的凋亡和坏死情况,并进行比较。结果培养第3天2组LECs的生长形态近似,但培养28d高糖组坏死细胞增加。培养3d后高糖组人LECs的AQP-1免疫荧光表达较对照组明显增强,2组间平均荧光强度的差异有统计学意义（t=3.934,P=0.004）,但2组间LECs的凋亡率和坏死率差异均无统计学意义（t=1.681,P=0.131;t=1.064,P=0.318）。培养28d后高糖组人LECs的AQP-1免疫荧光表达较对照组弱,差异有统计学意义（t=3.069,P=0.015）,LECs凋亡率、坏死率较对照组升高,差异均有统计学意义（t=11.213,P〈0.01;t=15.778,P〈0.01）。结论 AQP-1的异常表达在糖尿病性白内障的发生发展中起重要作用。     

Egr-1在硒性白内障中的表达及意义

目的 探讨早期反应因子(Egr-1)在硒性白内障发生各个时期的晶状体上皮细胞(LECs)中的表达及意义.方法 45只SD大鼠随机分为实验组、对照组和正常组,每组15只.实验组隔日颈部皮下注射亚硒酸钠(3.46 mg/kg),共3次.对照组在相应部位注射等量生理盐水.裂隙灯下观察大鼠晶状体混浊情况,并在注药后第1、3、7 d用免疫组织化学法测定各时间点大鼠LECs中Egr-1蛋白的表达.结果 注药后实验组大鼠晶状体混浊情况持续加重,第7 d形成典型严重的核性白内障,对照组及正常组晶状体始终未见混浊.LECs中Egr-1在给药后第1 d呈弱阳性表达,平均光密度值为0.29±0.03,给药后第3 d呈阳性及强阳性表达,平均光密度值为0.46±0.05,给药后第7 d大多数为弱阳性表达,平均光密度值为0.33±0.04.结论 Egr-1在硒性白内障发生的早期有表达,可能是硒性白内障发生的早期影响因子之一.     

大鼠激素性白内障中Na+-K+ATP酶活性的变化

目的：观察激素性白内障中Na^＋-K^＋ATP酶活性变化,探讨激素性白内障发病机制.方法：大鼠的96只透明晶状体随机分为对照组（DMEM）、地塞米松诱导的白内障组（DMEM＋地塞米松10 u mol/L）,体外培养7d,动态观察晶状体混浊情况,1,3,5,7d分别从各组取12只晶状体,测定晶状体中的Na＋-K＋ATP酶活性.结果：7d对照组晶状体呈雾状混浊,白内障组晶状体出现重度核混浊.白内障组培养3,5,7d,Na^＋-K^＋ATP酶活性分别下降约23.5%（P=0.002）,49.6%（P＜0.001）,75.8%（P＜0.001）,而对照组Na^＋-K^＋ATP酶活性下降无显著性意义.结论：地塞米松可诱导离体大鼠晶状体产生核性白内障,离子转运障碍可能参与激素性白内障的形成.     

HSP70在STZ-糖尿病性白内障发病机制中的作用

目的：探讨HSP70在链脲佐菌素－糖尿病性白内障发生、发展中的作用。方法：将60只SD大鼠随机分为两组，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2w末、4w末、8w末，分别摘取眼球，检测热休克蛋白－70（HSP70）在晶状体上皮细胞（LECs）中的表达情况。结果：对照组晶状体一直保持透明，白内障组晶状在2w末出现空泡，8w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增加，结论：HSP70可能通过调节LECs的生产在糖性白内障的发生，发展中起重要作用。     

人8-羟基鸟嘌呤糖苷酶1与年龄相关性白内障关系的临床研究

目的探讨与年龄相关性白内障（ARC）患者晶状体上皮细胞（LECs）中参与DNA损伤后碱基清除修复途径的氧化损伤修复基因—人8-羟基鸟嘌呤糖苷酶1（HOGG1）水平与ARC的关系。方法收集三种ARC（皮质性、核性、后囊下性）LECs样本,以透明晶状体LECs为对照组,用免疫组化、RT-PCR方法测定HOGG1在LECs的表达情况。结果对照组LECs中可见HOGG1的表达,三种ARC患者LECs中可见HOGG1表达较对照组增高（F=107.62,P〈0.01）,但三种ARC之间没有统计学差异。对照组与ARC组HOGG1均位于细胞质和细胞核。说明ARC LECs的细胞核和细胞质中HOGG1的表达量上调。结论 HOGG1表达上调参与ARC的发生发展。     

核因子抑制剂对眼表碱烧伤后白内障形成的影响

目的探讨大鼠眼表碱烧伤后应用核因子抑制剂对晶状体上皮细胞（LECs）核转录因子-κB（NF-κB）mRNA表达的影响。方法制造大鼠眼表碱烧伤模型。实验组每天给予PDTC球结膜下注射,对照组每天给予生理盐水球结膜下注射,分别于烧伤后1、3、5、7d处死大鼠,取晶状体行免疫组织化学染色及RT-PCR检测。结果碱烧伤后1d,对照组大鼠晶状体上皮脱落,但实验组大鼠LECs完整。碱烧伤后5～7d,2个组的晶状体上皮和皮质变得致密浓缩。碱烧伤后1～3d,对照组LECs中NF-κB表达的灰度值明显低于核因子抑制剂组（t=2.836,P=0.036;t=4.932,P=0.004）,但对照组NF-κBmRNA的表达（NF-κB/β-actin）明显高于核因子抑制剂组（t=31.563,P=0.000;t=17.837,P=0.000）。碱烧伤5d后,2组间NF-κB及其mRNA表达量的差异均无统计学意义（P〈0.05）。结论眼表碱烧伤可以导致白内障的发生,在碱烧伤后早期使用核因子抑制剂对白内障的形成有一定的抑制作用。     

维甲酸对人RPE细胞胞间通讯功能和Cx43表达的影响

目的观察培养的人视网膜色素上皮（RPE）细胞中连接蛋白Cx43的表达特点，以及维甲酸（RA）对人RPE细胞生长和细胞间缝隙连接功能、细胞间通道蛋白Cx43表达的影响及其相互关系。方法免疫组织化学方法观察缝隙连接蛋白（Cx43）在培养的人RPE细胞中的表达特点；不同浓度的RA作用于培养的第4代人的色素上皮细胞，用MTT方法观察RA对体外培养人RPE细胞生长的抑制；用LY染料传输方法测定细胞间隙连接功能，观察这三组不同浓度的RA对人RPE细胞缝隙连接功能的影响；免疫组织化学方法鉴别不同浓度RA影响后的缝隙连接蛋白Cx43的表达，用计算机灰度测量的方法评估表达的强度并作统计学分析。结果免疫组织化学染色显示体外培养的人RPE细胞表达缝隙连接蛋白Cx43，表达的部位主要在细胞浆和细胞膜上；RA对人RPE细胞的生长有明显的抑制作用，其抑制作用呈剂量依赖性；LY染料传输试验表明在RA的作用下RPE细胞间通讯功能明显增强；经计算机图像分析表明RA使连接蛋白Cx43表达增强，增强的程度与RA的浓度呈正相关。结论体外培养的人RPE细胞可以表达Cx43蛋白，RA抑制培养的色素上皮细胞的生长，提高细胞间隙连接通讯功能，上调Cx43蛋白在人RPE细胞中的表达。     

P38在体外培养鸡胚胎晶状体半乳糖性白内障形成中的作用

目的:探讨P38参与的晶状体上皮细胞凋亡的调节在体外培养的鸡胚胎晶状体半乳糖性白内障中的作用。方法:用30mmol/L半乳糖诱导体外培养的鸡胚胎晶状体建立半乳糖性白内障模型,在培养10d里,通过每日观察、照相、计算晶状体的混浊面积,观察MAPK-P38抑制剂SB203580对半乳糖引起的晶状体混浊程度的影响,并用TUNEL原位凋亡细胞染色方法,观察晶状体冰冻组织切片上细胞凋亡情况。结果:半乳糖30mmol/L能诱导体外培养的鸡胚胎晶状体产生周边皮质性混浊,随着半乳糖暴露时间的延长,晶状体的混浊面积增加,P38抑制剂SB203580能有效减轻半乳糖性晶状体的混浊。TUNEL检测显示,在培养2,5,10d的半乳糖性白内障晶状体切片上,凋亡细胞的百分数分别为1.7%,4.5%和12%,而SB203580处理的晶状体,凋亡细胞数明显减少,在相应点分别是0.7%,1.5%和1.4%。结论:P38可能通过参与晶状体上皮细胞凋亡调控在半乳糖性白内障的形成中起作用。抑制P38激活能阻止晶状体细胞凋亡,减轻白内障形成。     

EGF对人视网膜色素上皮细胞胞间通讯功能和Cx43表达的影响

目的探讨培养的人视网膜色素上皮(retinalpigmentepithelium,RPE)细胞中连接蛋白Cx43的表达,以及表皮生长因子(epidermalgrowthfactor,EGF)对人视网膜色素上皮细胞间缝隙连接功能、细胞间通道蛋白Cx43表达的影响。方法免疫组化的方法观察Cx43在培养的人RPE细胞中的表达特点,用10mg·L-1、20mg·L-1、30mg·L-1不同浓度的EGF作用于培养的第4代人RPE细胞后,观察缝隙连接蛋白Cx43的表达强弱变化,用计算机灰度测量的方法评估表达的强度并作统计学分析。用LY染料传输方法测定细胞间隙连接功能,观察这3组不同浓度的EGF对人RPE细胞缝隙连接功能的影响。结果免疫组化染色显示体外培养的人RPE细胞表达缝隙连接蛋白Cx43,经计算机图像分析表明EGF使连接蛋白Cx43表达减弱,下调的程度与EGF的浓度呈正相关。LY染料传输试验显示在EGF作用下视网膜色素上皮细胞间通讯功能明显降低。结论体外培养的人RPE细胞表达Cx43,EGF可以减弱细胞间隙连接通讯功能、下调Cx43在人RPE细胞中的表达。     

The role of Src family kinases in cortical cataract formation

PURPOSE: The goal of this study was to determine the role of Src family kinases (SFKs) in the development of lens cataract. This question was particularly significant, because these tyrosine kinases mediate the stress pathways known to lead to cataract formation. The experiments were focused on whether the inhibition of SFK activity suppresses the formation of lens opacities. METHODS: A whole-lens culture system was developed, in which cortical opacities formed within 5 days, in embryonic day (E)10 lenses grown in medium containing 10% fetal bovine serum. SFK activity was blocked in the cultured lenses by growth in the presence of the SFK-specific inhibitor PP1. Control cultures were grown in medium without inhibitor or in the presence of PP3, the inactive analogue of PP1. Lenses were cultured for 10 days, observed, and photographed daily. Opacification was quantified with image-analysis software. Tissue architecture was determined after hematoxylin and eosin staining and cellular organization by fluorescent localization of filamentous actin with fluorescein-conjugated phalloidin. RESULTS: Almost all lenses in the control cultures developed cortical opacities covering approximately 50% of the lens area by day 10. Similar to control cultures, PP1-treated lenses showed mild posterior opacities during the first 5 days in culture, but then became strikingly transparent. Only 7% of the PP1-treated lenses showed development of cortical cataract, and the average area of opacity was just 0.5% by culture day 10. In all cultured lenses, even in the presence of the PP1 inhibitor, the bow region of the lens extended to the posterior pole, and distribution of nuclei from the posterior pole toward the anterior aspects of the lens suggested that newly added fiber cells were misdirected. However, neither this feature, nor the presence of vacuoles appeared to correlate with the development of opacity in the cultured lenses. Instead, the lens opacities appeared to result from gross abnormalities in the shape and organization of cells in the equatorial and cortical fiber zones, as observed by F-actin staining. Culturing the lenses in the presence of the SFK inhibitor prevented these lens cell aberrations as well as the development of lens opacity. CONCLUSIONS: The formation of cataract can involve activation of SFK-mediated pathway(s) leading to disorganization of developing lens fiber cells, and inhibiting these tyrosine kinases blocks cataract progression.     

Prenatal lens development in connexin43 and connexin50 double knockout mice.

PURPOSE: To determine the roles of intercellular communication in embryonic eye growth and development, mice with a targeted deletion of the Cx43 gene were examined, and mice without both Cx43 and Cx50 were generated and analyzed. METHODS: Embryonic eyes and lenses from wild-type mice, or mice deficient in Cx43, Cx50, or both Cx43 and Cx50 were collected and analyzed structurally by light and electron microscopy, immunohistochemically using connexin-specific antibodies, biochemically by Western blot analysis, and physiologically by measuring patterns of junctional communication revealed by iontophoretic injection of junction-permeable reporter molecules. RESULTS: Cx50 expression was limited to the ocular lens and was not detected in either the cornea or the retina. Cx43(-/-) embryos showed development of structurally normal lenses and eyes when examined by light and electron microscopy through embryonic day (E)18.5. In addition, Cx43(-/-) lenses synthesized four different markers of lens differentiation: MIP26, alphaA-crystallin, alphaB-crystallin, and gamma-crystallin. Double-knockout lenses were also histologically normal through E18.5 and synthesized the four lens differentiation markers. When assayed by intracellular injection with Lucifer yellow (Molecular Probes, Eugene, OR) and neurobiotin at E15.5, Cx43(-/-)/Cx50(-/-) lenses retained gap junction-mediated dye transfer between fiber cells. In contrast, dye transfer in double-knockout lenses was dramatically reduced between epithelial cells and was eliminated between epithelial cells and fibers. CONCLUSIONS: These data indicate that the unique functional properties of both Cx43 and Cx50 are not required for prenatal lens development and that connexin diversity is required for regulation of postnatal growth and homeostasis.     

Na^＋-K^＋-ATP酶活性及其α1催化亚基在鸡晶状体中的分布

目的对Na^＋-K^＋-ATP酶催化亚基蛋白在鸡晶状体的上皮层、周边部和中央部纤维的分布和酶活性进行检测和比较。方法将部分胚胎第10天（E10）的鸡晶状体显微解剖为晶状体上皮部、周边部纤维和中央部纤维3个部分，剩余部分制备冰冻切片。利用免疫荧光染色和Western blot方法检测Na^＋-K^＋-ATP酶催化亚基（α）在晶状体各部分的表达，通过检测哇巴因敏感的ATP水解率测定Na^＋-K^＋-ATP酶的活性。结果在晶状体切片上，α1催化亚基蛋白在晶状体上皮细胞（LECs）膜上有很强的表达，而在晶状体纤维细胞膜的表达明显减弱，在周边部上皮细胞的表达有明显的极性，而在中央部上皮细胞中未见表达。Western blotting检测也再次证实α1催化亚基蛋白在LECs中的表达明显高于在晶状体纤维中的表达。上皮层Na^＋-K^＋-ATP酶的活性是周边部纤维酶活性的2倍，是中央部纤维酶活性的11倍。结论Na^＋-K^＋-ATP酶α1催化亚基蛋白在鸡晶状体的上皮细胞和纤维上表达；在鸡晶状体上Na^＋-K^＋-ATP酶及其活性的分布是不均一的。     

Src-家族酪氨酸激酶特异性抑制剂对H2O2介导的晶状体上皮细胞Ca2＋内流的影响

目的观察Src-家族酪氨酸激酶（SFK）抑制剂PP1对H2O2诱导的晶状体上皮细胞（LECs）内游离钙离子（Ca2＋）浓度变化的影响。方法用Ca2＋荧光探针Fluo-3/AM负载人晶状体HLE-B3,用激光共焦显微镜观察在不同浓度H2O2刺激后细胞内Ca2＋的荧光强度变化;进一步用0.1nmol/LPP1、0.3μmol/（L·min）过氧化氢酶和DMSO分别预处理细胞,观察在常规Ca2＋、低Ca2＋和高Ca2＋培养基条件下H2O2刺激后细胞内Ca2＋荧光强度的变化,评价PP1对H2O2诱导的LECs内Ca2＋的作用。结果不同浓度H2O2刺激后细胞内游离Ca2＋浓度升高,呈剂量依赖效应。用0.1mmol/LH2O2刺激细胞,在常规Ca2＋培养基中,PP1组和过氧化氢酶组细胞内Ca2＋荧光强度增长幅度与DMSO组比较分别降低（28.5±4.2）%、（33.8±3.7）%,差异均有统计学意义（q=3.73,P〈0.05;q=4.21,P〈0.05）。在低Ca2＋培养基中,3个组细胞内Ca2＋荧光强度增强均不明显;在高Ca2＋培养基中,PP1组、过氧化氢酶组的荧光强度增加幅度较DMSO组分别降低（13.5±1.8）%和（21.3±2.4）%（q=5.58,P〈0.01;q=7.11P〈0.01）。常规Ca2＋和高Ca2＋培养基中,PP1组和过氧化氢酶组细胞内Ca2＋荧光强度增长幅度的差异均无统计学意义（q=3.04,P〉0.05;q=2.76,P〉0.05）。结论 SFK特异性抑制剂PP1能有效抑制H2O2诱导的LECs的Ca2＋内流,从而阻断依赖Ca2＋激活的信号转导途径,阻止皮质性白内障的发生。     

Protein kinase C-gamma activation in the early streptozotocin diabetic rat lens

PURPOSE: The purpose of this study is to demonstrate the early activation of the protein kinase C-gamma (PKC-gamma) pathway in the streptozotocin (STZ)-induced diabetic rat lens. METHODS: Twelve-week-old male and female Sprague-Dawley rats were injected with 80 mg/kg (body weight) of STZ (N-[methylnitrosocarbamoyl]-D-glucosamine) intraperitoneally. Very high glucose (VHG) diabetes was defined as a nonfasting blood glucose level of at least 450 mg/dl, confirmed by daily monitoring with Accu-Check Advantage test strips, and occurred about 2 weeks after STZ administration. All assayed lenses were from VHG or age-matched control rats, harvested within 24 hr of VHG detection. PKC-gamma activation was measured by enzyme activity assay and by Western blotting to show autophosphorylation on Thr514. Cellular insulin-like growth factor-1 (IGF-1), PKC-gamma phosphorylation of Cx43 on Ser368, and activation of phospholipase C-gamma 1 (PLC-gamma 1), extracellular signal-regulated kinase (ERK1/2), and caspase-3 were determined by Western blotting. Endogenous diacylglycerol (DAG) levels were measured with a DAG assay kit. Lens gap junction activity was determined by the microinjection/Lucifer yellow dye transfer assay. Electron microscopy was applied to affirm fiber cell damage in the VHG diabetic lenses. RESULTS: In the lenses of VHG diabetic rats, PKC-gamma enzyme was activated. PKC-gamma could be further activated by 400 nM phorbol-12-myristate-13-acetate (PMA), but the PKC-gamma protein levels remained constant. No elevation of IGF-1 level was observed. Western blots showed that activation of PKC-gamma may be due to activation of PLC-gamma 1, which synthesized endogenous DAG, a native PKC activator. The level of PKC-gamma -catalyzed phosphorylation of Cx43 on Ser368 and resulting inhibition of lens gap junction dye transfer activity was increased in the VHG diabetic lenses. At this early time period, the diabetic lens showed no activation of either caspase-3 or ERK1/2. Only a single fiber cell layer deep within the cortex (approximately 90 cell layers from capsule surface) showed vacuoles and damaged cell connections. CONCLUSIONS: Early activation of PLC-gamma 1 and elevated DAG were observed within VHG diabetic lenses. These were correlated with activation of PKC-gamma, phosphorylation of Cx43 on Ser368, and inhibition of dye transfer. Abnormal signaling from PKC-gamma to Cx43 in the epithelial cells/early fiber cells, observed within VHG diabetic lenses, may be responsible for fiber cell damage deeper in the lens cortex.     

Coordinate signaling by Src and p38 kinases in the induction of cortical cataracts

PURPOSE: The goals of this study were to determine whether MAP kinase signaling pathways play a role in the formation of lens cataracts and to examine the potential signaling relationship between Src and MAP kinases in signaling the induction of lens opacities. METHODS: Embryonic day (E)10 chick lenses were cultured in Medium 199 containing 10% fetal bovine serum. The activation state of Src kinases and the MAP kinases extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK), and p38 in the lens epithelium was determined over a time course from 10 minutes to 10 days in culture by immunoblot analysis. Src kinase activation was suppressed by exposure to the Src family kinase-specific inhibitor PP1. To examine the role of specific MAP kinases in the development of lens opacities, lenses were grown for 10 days in the presence or absence of inhibitors of ERK (U0126), JNK (SP600125), and p38 (SB203580). Lenses were observed and photographed daily, and the degree of opacification was quantified by using image-analysis software. RESULTS: Within a short time after placing embryonic lenses in culture conditions that induce the formation of cataracts, there occurred a great increase in the activation state of the MAP kinase ERK. Activation of ERK was both rapid and transient. No activation of the MAP kinase JNK was observed in the cataract cultures beyond that which occurred in normal lens epithelium, even though JNK activation is often linked to the cellular response to stress. In contrast, although p38 activation was barely detected in the normal embryonic lens, this stress-activated protein kinase exhibited a robust activation in cataract cultures that was sustained throughout the culture period. Studies conducted to map the cataract signaling pathways indicate that the p38 MAP kinase functions upstream of the Src kinase. To analyze the potential role of ERK, JNK, and p38 in cataract induction, lenses were cultured in the presence of specific MAP kinase inhibitors. Although the inhibitors of ERK and JNK did not interfere with the formation of cataract, p38 inhibitors blocked the development of lens opacities with an efficacy similar to that of the Src kinase inhibitor PP1. CONCLUSIONS: Activation of both Src and p38 kinases lead to the induction of cataract.     

Regulation of lens cell-to-cell communication by activation of PKCgamma and disassembly of Cx50 channels

PURPOSE: Lens fiber gap junctions comprise approximately equal molar amounts of connexin46 (Cx46) and connexin50 (Cx50), both of which contribute significantly to coupling in the lens cortex and nucleus. The current study was conducted to test the hypothesis that regulation of lens coupling by activation of protein kinase Cgamma (PKCgamma) affects the number of channels composed of Cx46, Cx50, or both connexins. METHODS: Whole rat lenses were treated with phorbol-12-myristate-13-acetate (TPA) to activate PKCgamma or the inactive analogue 4alpha-phorbol,12,13-didecaneote (PDD) as a control. The superficial cortical fibers were studied morphologically by quantitative freeze-fracture immunolabeling (FRIL); functionally by Lucifer yellow dye transfer assay; and chemically by measuring PKCgamma activity, connexin phosphorylation and coimmunoprecipitation. RESULTS: Treatment with TPA activated PKCgamma and uncoupled the lens cortex by approximately 60%. PDD had no effect. Activation of PKCgamma decreased the density of Cx50 channels assembled in gap junctions, increased the density of Cx50 hemichannels in the plasma membrane and induced circular voids measuring 22 to 300 nm in diameter within the remaining plaques. Coimmunoprecipitation studies indicated that the soluble PKCgamma was translocated into membrane fractions that contained Cx46, Cx50, and the lipid raft marker caveolin (Cav)-1. In the membrane environment, PKCgamma phosphorylated Cx50 at serines and threonines and Cx46 only at threonines. CONCLUSIONS: The studies provide experimental support for the hypothesis that gap junctions comprising mixtures of Cx46 and Cx50 channels provide malleable communicating pathways between the lens nucleus and the metabolically active fibers in the surface. The findings also suggest that Cx50 channel disassembly occurs in distinct lipid microdomains.     

茶多酚对高糖条件下大鼠晶状体上皮细胞死亡率及线粒体膜电位的影响

目的探讨茶多酚对高糖条件下培养的大鼠晶状体上皮细胞死亡率（CDR）及线粒体膜电位（MMP）的影响,阐明糖尿病性白内障的发生机制及茶多酚干预的疗效及机制。设计实验研究。研究对象体外培养的大鼠晶状体上皮细胞。方法体外培养大鼠晶状体上皮细胞,培养基中葡萄糖浓度分别为5、30mmol／L（L1组和L2组）,另在30mmol／L葡萄糖浓度培养基中加入0．1μM和0．3μM的茶多酚进行干预（L3组和L4组）。流式细胞仪检测各条件下大鼠品状体上皮细胞MMP和CDR的变化。主要指标大鼠晶状体上皮细胞CDR及MMP。结果LI、L2、L3、L4组大鼠晶状体上皮细胞CDR分别为（0．68±0．32）％、（13．50±4．48）％、（0．64±0．48）％、（0．50±0．30）％,L1与L2组比较、L2与L3组比较差异均有统计学意义（P．0．04,P=0．04）,L3与L4组比较差异无统计学意义（P=0．99）。L1、L2、L3、L4组大鼠晶状体上皮细胞MMP分别为95．53±4．61、37．38±3．56、110．02±8．04、102．63±9．48,L1与L2组比较、L2与L3组比较差异均有统计学意义（P=0．01,P=0．01）,L3与L4组比较差异无统计学意义（P=1．00）。结论高糖条件下培养的大鼠晶状体上皮细胞线粒体膜电位降低、细胞死亡率明显升高。茶多酚具有稳定线粒体膜电位的作用,可延缓细胞凋亡,降低细胞死亡牢：（眼科,2009,18：104—107）     

人眼Tenon囊成纤维细胞缝隙连接细胞间通讯的检测

目的:观察体外培养的人眼Tenon囊成纤维细胞(human Tenon’s capsule fibroblasts,HTFs)缝隙连接细胞间通讯(gap junctional intercellular communication,GJIC)功能及其构成蛋白connexin43(Cx43)的表达及分布。方法:锐刀划痕罗氏黄(lucifer yellow)荧光染料传输法观察GJIC功能;RT-PCR及Western blot分别检测Cx43 mRNA及蛋白表达;免疫细胞化学方法检测Cx43的分布。结果:荧光染料自划痕线向邻近未损伤细胞传输达6级细胞以上,同时检测到Cx43 mRNA与蛋白的表达;免疫细胞化学法观察到Cx43分布于细胞膜。结论:培养的HTFs具有较强的GJIC功能,通过Cx43介导GJIC赋予细胞能够相互之间传递信息的能力,提示在创伤愈合过程中GJIC对成纤维细胞功能同步化方面发挥重要作用。