白藜芦醇对过氧化氢诱导人晶状体上皮细胞氧化损伤的保护作用

目的 探讨白藜芦醇(resveratrol,RES)对过氧化氢(H2O2)诱导人晶状体上皮细胞(human lens epithelial cells,HLEC)氧化损伤的保护作用.方法 HLEC传代培养24h后,分别加入不同浓度(5μmol·L-1、10 μmol·L-1、20 μmol·L-1、40 μmol·L-1) RES预处理12 h后,加入100 μmol·L-1 H2O2继续孵育24 h,倒置相差显微镜观察细胞形态改变,MTT比色法检测RES对H2O2诱导的HLEC活力的影响,流式细胞仪检测HLEC细胞凋亡率,比色法检测凋亡相关因子caspses-3及caspase-9的表达.结果 氧化损伤可以诱导HLEC形态改变,RES处理后,细胞形态逐渐得到改善.MTT结果显示RES对HLEC活性无抑制作用,RES(5μmol·L-1、10 μmol· L-1、20 μmol· L-1、40 μmol·L-1)孵育24 h后细胞存活率分别为(101.30±4.49)％、(100.31±3.53)％、(101.71±3.33)％、(99.30±3.00)％,与对照组(99.67±2.67)％比较,差异均无统计学意义(均为P＞0.05);模型组HLEC经氧化损伤处理后,细胞存活率(34.33±3.71)％明显下降,用20 μmol·L-1及40 μmol·L-1 RES处理后,HLEC存活率分别提高到(57.33±5.61)％和(72.67±6.98)％,与模型组比较差异均有统计学意义(均为P＜0.05).流式细胞计数结果显示:对照组HLEC凋亡率为(1.99±0.17)％,经H2O2处理后,模型组HLEC凋亡率为(51.73±4.97)％,20μmol·L-1、40 μmol·L-1 RES处理后,HLEC凋亡率分别为(34.43±3.67)％、(26.55±2.07)％,与模型组比较,差异均有统计学意义(均为P＜0.05).此外,RES还可以减少H2O2所致HLEC内caspses-3及caspase-9的表达.结论 RES可以明显抑制HLEC凋亡,其抑制凋亡的作用可能是其防止和延缓白内障发生发展的细胞学基础,从而为寻求有效的防治白内障药物提供可靠的实验依据.     

丹参抗晶状体上皮细胞DNA氧化损伤的研究

目的研究丹参对晶状体上皮细胞（LECs）DNA氧化损伤的保护作用。方法应用单细胞凝胶电泳法分别测定0．1、0．2、0．4、0．8mg／mL丹参液对体外培养的LECs氧化损伤的保护作用，并与牛磺酸的保护作用相比较。结果各质量浓度组丹参液培养后的LECs对H2O2的损伤均有抵抗作用，尤其0．8mg／mL质量浓度组的保护作用优于40mmol／L牛磺酸组，除0．1mg／mL丹参组外各处理组细胞的尾长、彗星细胞百分率与H2O2损伤组相比差异有统计学意义（P〈0．01）。结论丹参中的有效成分对体外培养的LECs氧化损伤有明显的保护作用，且呈现质量浓度依赖的量效关系。     

晶状体上皮细胞培养及Ⅳ型胶原表达

目的体外培养小牛晶状体上皮细胞，免疫组织化学SABC法检测1V型胶原表达。方法小牛晶状体前囊组织块培养，晶状体上皮细胞原代和传代培养。传代2次后免疫组织化学法检测细胞内1V型胶原表达。结果原代培养2-3周后细胞成单层后传代培养，传代细胞易贴壁，9～10d后可再传代，免疫组织化学检测发现细胞质内有大量Ⅳ型胶原表达，形成细胞的骨架成分。结论晶状体上皮细胞在增生时有大量Ⅳ型胶原表达，其与晶状体后囊混浊有关。     

Na,K-ATPase of cultured bovine lens epithelial cells: H2O2 effects.

Na,K-ATPase function was studied in cultured bovine lens epithelial cells under confluent and non-confluent conditions. The affinity of the Na,K-ATPase for the cardiac glycoside, ouabain, differs between the confluent and non-confluent cultures. The confluent cells have a higher affinity for ouabain than do the non-confluent cells. The ouabain affinity of the confluent cells is similar to that for the Na,K-ATPase isolated from the bovine axolemma and the bovine lens cortex. The ouabain affinity of the non-confluent cells is similar to that for the Na,K-ATPase of the renal medulla and bovine lens epithelium. Similar results are not found with confluent and non-confluent MDCK cells. H2O2 treatment of confluent and non-confluent lens epithelial cell cultures has differing effects on the Na,K-ATPase function. In the confluent cell preparations, H2O2 affects K(+)-dependent dephosphorylation of the intermediate phosphoenzyme. In the non-confluent preparations. H2O2 appears to inhibit K(+)-occlusion.     

金钗石斛提取物对晶状体上皮细胞氧化损伤防护作用

目的:探讨中药金钗石斛提取物中4种不同极性的生物碱(脂溶性、水溶性、低极性、弱极性)对人晶状体上皮细胞(HLEC)氧化损伤防护作用的影响。方法:将H2O2与传代培养的HLEC共同孵育复制氧化损伤模型,同时加入不同极性的金钗石斛提取物作用24h后,用四甲基偶氮唑蓝法(MTT)检测HLEC的增殖情况,流式细胞术检测其凋亡情况,并探讨不同极性及同一极性不同浓度的金钗石斛提取物对氧化损伤后细胞增殖及凋亡的影响。结果:H2O2组LEC增殖下降,金钗石斛脂溶性生物碱和水溶性生物碱高剂量组可促进氧化损伤的LEC的增殖,其中脂溶性生物碱显著增强氧化损伤的LEC的增殖(P<0.01)。脂溶性生物碱可以明显抑制H2O2诱导的LEC凋亡(P<0.01)。结论:金钗石斛脂溶性生物碱低剂量组通过抗氧化损伤而促进LEC的增殖,抑制LEC的调亡。     

半胱氨酸天冬氨酸酶在过氧化氢诱导大鼠晶状体上皮细胞凋亡中的表达

目的 探讨半胱氨酸天冬氨酸酶（caspase3)在氧化损伤致大鼠晶状体上皮细胞凋亡过程中的表达及意义。方法 离体大鼠晶状体于MEM培养液中培养，实验组加入终浓度为2mmol/L的过氧化氢（H2O2），对照组不加H2O2，观察培养过程中两组晶状体混浊程度；透射电镜下观察晶状体上皮细胞超微结构改变；免疫组织化学方法测定细胞中caspase3表达。结果 在H2O2作用下随培养时间延长，晶状体混浊度加重，伴随晶状体上皮细胞凋亡增加；caspase3表达量随细胞凋亡及晶状体混浊度的增加而增加。结论 晶状体上皮细胞凋亡是氧化损伤诱发白内障模型的细胞学基础；caspase3可能参与晶状体上皮细胞凋亡和白内障的形成，在白内障的发病机制中占有重要地位。     

枸杞多糖对H2O2诱导人晶状体上皮细胞氧化应激损伤的保护作用

目的 探讨枸杞多糖(lycium barbarum polysaccharides,LBP)对H2O2诱导人晶状体上皮细胞(SRA01/04)氧化应激损伤的保护作用.方法 将SRA01/04细胞传代培养24h后,加入不同浓度(50 mg·L-1、100 mg·L-1、200 mg·L-1、400 mg·L-1、800 mg·L-1和1600 mg·L-1)LBP预处理24 h,之后加入200 μmol·L-1H2O2继续培养24 h,用CCK-8检测LBP对H2O2诱导的SRA01/04细胞活力的影响并筛选出LBP最佳保护浓度用于后续实验.采用流式细胞仪检测SRA01/04细胞凋亡率和线粒体膜电位变化情况,Western blot检测各组凋亡相关蛋白Bcl-2及Bax的表达情况.结果 CCK-8检测结果显示:LBP浓度为200 mg·L-1和400 mg·L-1时,能够促进SRA01/04细胞增殖,细胞存活率分别为(121.10±5.56)％和(128.20 ±3.79)％,与对照组(99.98±4.73)％比较,差异均有统计学意义(均为P＜0.05).H2O2模型组SRA01/04细胞经氧化应激损伤后,细胞存活率(51.67±4.91)％明显降低,与对照组(99.67±2.52)％相比,差异有统计学意义(P ＜0.001).用200 mg·L-1和400mg·L-1 LBP预处理后,SRA01/04细胞存活率明显提高至(74.01±3.21)％及(84.67±4.33)％,与H2O2模型组比较差异均有统计学意义(均为P＜0.01).400 mg·L-1 LBP对RA01/04细胞氧化应激损伤保护作用最大.流式细胞仪检测结果显示:对照组细胞凋亡率为(5.1±1.2)％;H2O2模型组细胞凋亡率为(25.9±1.5)％;400 mg·L-1 LBP预处理后,细胞凋亡率降至(13.8±1.2)％,与H2O2模型组比较,差异有统计学意义(P＜0.01).线粒体膜电位检测结果显示:对照组阳性细胞数最多,线粒体膜电位高;H2O2模型组阳性细胞数最少,线粒体膜电位低;400 mg·L-1 LBP预处理后,阳性细胞数增多及线粒体膜电位升高,与H2O2模型组相比,差异有统计学意义(P＜0.001).Western blot检测显示:与对照组相比,H2O2模型组Bcl-2表达下降,Bax表达上升(P＜0.01);经400 mg·L-1 LBP预处理后,Bcl-2表达上升,Bax表达下降,与H2O2模型组比较,差异有统计学意义(P＜0.01).结论 LBP对H2O2诱导人晶状体上皮细胞的氧化应激损伤具有保护作用,可抑制细胞凋亡.     

miR-34a/SIRT1在H2O2诱导人晶状体上皮细胞氧化应激中的表达

目的 观察微小RNA-34a(microRNA-34a,miR-34a)和沉默信息调节因子1(silent information regulator 1,SIRT1)在不同浓度H2O2诱导的人晶状体上皮细胞(SRA01/04细胞)氧化应激中的表达变化.方法 用不同浓度的H2O2(0μmol·L-1、100 μmol·L-1、200 μmol·L-1、300 μmol·L-1、400 μmol·L-1)处理细胞24 h后,用CCK-8检测细胞存活率,流式细胞仪检测细胞凋亡率和RT-PCR检测miR-34a/SIRT1的表达.结果 CCK-8检测结果显示:100～400 μmol·L-1 H2O2对SRA01/04细胞有增殖抑制作用,且呈剂量依赖关系,与0μmol·L-1 H202组相比差异均有统计学意义(均为P＜0.01);流式细胞仪凋亡检测结果显示:0μmol·L-1 H2O2组细胞凋亡率为(6.1±1.2)％;100 μmol·L-1、200μmol·L-1、300 μmol·L-1 H2O2组细胞凋亡率分别为(26.3±1.8)％、(32.5±2.2)％、(64.7±5.3)％,各组与0μmol·L-1H2O2组比较,差异均有统计学意义(均为P＜0.01).RT-PCR检测结果显示:不同浓度H2 O2处理SRA01/04细胞后,细胞中的miR-34a表达水平呈剂量依赖性升高,而SIRT1表达水平呈相应下降,与0μmol·L-1 H2O2组相比差异均有统计学意义(均为P ＜0.001).结论 在一定浓度H2O2诱导的人晶状体上皮细胞氧化应激中,miR-34a表达水平显著增加,而SIRT1表达水平显著下降.下调miR-34a表达可增加氧化应激人晶状体上皮细胞存活率.     

Repair of H2O2-induced DNA damage in bovine lens epithelial cell cultures

H2O2 concentrations only slightly higher than normal physiological levels found in the lens and aqueous fluid produce a significant number of DNA single-strand breaks in lens epithelial cell cultures. In this investigation, the repair of DNA damaged by short-term, H2O2-induced oxidation was examined in bovine lens epithelial cell cultures. Repair was rapidly initiated and was almost completed in 30 min. A drop in NAD concentration was associated with the DNA damage. 3-Aminobenzamide inhibition of poly(ADP-ribose) polymerase, an enzyme believed to be stimulated by DNA oxidation and involved in DNA repair, prevented the loss of NAD. In contrast, a similar drop in ATP concentration was only slightly lessened by the presence of this inhibitor. Inhibition of the polymerase by 3-aminobenzamide primarily affected only the early recovery period. Overall, recovery occurred almost as effectively in the presence of the inhibitor as in its absence. Preincubation of lens cultures with o-phenanthroline, an iron chelator, prevented the drop in NAD levels associated with DNA damage. Since a hydroxyl radical is produced from H2O2 by a Fenton type reaction, this result supports the concept that the H2O2-induced oxidation of DNA is caused by hydroxyl radical. In contrast, peroxide-induced loss of activity of a cytosolic enzyme, glyceraldehyde-3-phosphate dehydrogenase, was unaffected by the presence of o-phenanthroline, suggesting direct H2O2 oxidation of this enzyme. The results of these experiments suggest that lens epithelium contains enzymes that rapidly repair single-strand DNA breaks induced by H2O2 insult.     

Polyol accumulation in cultured human lens epithelial cells

Human lens epithelial (HLE) cells in tissue culture accumulated significant levels of galactitol when they were cultured for 72 hr in medium containing 30 mM D-galactose. Polyol accumulation was accompanied by the appearance of vacuoles as seen by transmission electron microscopy. The number and size of intracellular vacuoles increased when the culture period was extended to 7 days. In addition, polyol accumulation was accompanied by loss of myoinositol. None of these changes occurred in cells exposed to 30 M L-galactose which is not a substrate for aldose reductase. The accumulation of galactitol, intracellular vacuole formation and loss of myoinositol observed in D-galactose-exposed cells were prevented by the inclusion of the aldose reductase inhibitor, sorbinil, in the culture medium. Comparison of the relative efficacies of two aldose reductase inhibitors indicate that AL 1576 is nearly 20 times more potent than sorbinil in inhibiting the human lens enzyme. It is concluded that vacuole formation in HLE cells is due to the osmotic effect of polyol formation brought about by the action of aldose reductase and that the etiology of human diabetic cataract may also involve the polyol pathway.     

Characteristics of iris and retinal pigment epithelial cells cultured on collagen type I membranes

PURPOSE: Transplantation of pigment epithelial cells is a promising treatment modality to repair retinal damage in age-related macular degeneration. For this purpose, it is necessary to establish cell culture techniques that allow acquisition of proper functional and morphological characteristics by the cells to be transplanted. METHODS: Primary retinal pigment epithelial (RPE) and iris pigment epithelial (IPE) cells grown to confluence on collagen membranes were examined for morphology, adhesion, proliferation, apoptosis, as well as viability after ex vivo transplantation. RESULTS: Pigment epithelial cells adhere, proliferate, form monolayers, acquire differentiated properties, and remain viable during transplantation to the subretinal space. CONCLUSIONS: Pigment epithelial cells cultured on collagen membranes acquire differentiated characteristics and are amenable to be transplanted as cell monolayers.     

过氧化氢对体外培养的兔晶体上皮细胞凋亡的诱导

目的研究H2O2诱导兔晶体上皮细胞凋亡.方法采用终浓度250μmol/LH2O2处理兔晶体上皮细胞,分别用TUNEL法、流式细胞仪和透射电镜检测H2O2处理后的兔晶体上皮细胞.结果用H2O2处理后的兔晶体上皮细胞,TUNEL法可见胞核呈棕褐色着染,苏木素不能复染;流式细胞仪检测到约9.6×105细胞出现亚二倍体峰,二倍体峰几近消失;透射电镜下细胞膜完整,但表面微绒毛消失,细胞质浓缩,胞浆内细胞器结构不清晰,并见大量大小不等的空泡,核质比例增大,胞核呈圆形,核膜双层结构不清,呈光滑状,核内染色质聚集成块,部分染色质边集.结论终浓度250μmol/LH2O2可以诱导兔晶体上皮细胞发生凋亡.     

Effect of H(2)O(2)on human lens epithelial cells and the possible mechanism for oxidative damage repair by thioltransferase

Human lens epithelial (HLE) B3 cells were used to study the oxidative damage and cellular repair with respect to the redox homeostasis, the oxidative defense enzymes and the glucose metabolic pathway. The effect of oxidative stress on cell growth was initially analyzed by culturing the cells with a bolus amount (0.02--0.1m M) of hydrogen peroxide (H(2)O(2)) in minimal essential medium (MEM) containing 20% fetal bovine serum (FBS) for 1 week. Concentration of H(2)O(2)greater than 0.03m M showed progressive inhibition of cell growth. However, the cells were also shown to tolerate H(2)O(2)concentrations up to 0.5m M by detoxifying the exogenous oxidant within 3hr without any detectable DNA damage. Therefore, this short-term H(2)O(2)exposure model was chosen to study the effect of oxidative stress on the cellular redox homeostasis. HLE B3 cells were first grown to confluence in MEM with 20% FBS. Approximately 1.6 million cells were gradually weaned off serum by subculturing in 2% FBS overnight, followed by serum-free medium for 30 min before subjecting to a bolus of 0.1m M H(2)O(2)for up to 180 min. These cells were used for biochemical analysis, which included H(2)O(2)detoxification (H(2)O(2)in the medium), glutathione (GSH) level and lactate production. Activity measurements were conducted on the oxidation defense enzymes: glutathione-S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx); the dethiolating enzyme, thioltransferase (TTase); and a key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (G-3PD). While the B3 cells were shown to tolerate and detoxify 0.1m M H(2)O(2)within 60 min, the GSH pool was transiently depleted in the first 60 min before fully recovered. GPx suffered more than 80% loss in activity and was unable to recover fully. GST showed slight inactivation but neither GR nor TTase was affected. G-3PD was inactivated to < 50% within 15 min of oxidative stress and was reactivated gradually to 80% of normal at the end of 180 min, concurrent with the transient loss of lactate production in the same cells. The reactivation of G-3PD was both temperature- and GSH-dependent, occurring only at physiological temperature and failing to reactivate when the intracellular GSH pool was depleted by BCNU (GR inhibitor) pretreatment. The inactivated cellular G-3PD in the cell extract could be partially reactivated by DTT (6m M) or by recombinant human lens thioltransferase (RHLT) but not by GSH (1m M), GR or GST. HLE cells cultured in the presence of L-(35)S-cystine and cycloheximide displayed an extra radiolabelled protein band on the autoradiograph in the H(2)O(2)treated cells. The labelled band was positively reacted with G-3PD antibody and could be removed by RHLT, indicating that S-thiolation of G-3PD occurred. The H(2)O(2)pre-exposed cells also transiently accumulated proteins modified by thiolation, including protein-S-S-glutathione (PSSG) and protein-S-S-cysteine (PSSC). It can be concluded that HLE could endure up to 0.1m M of H(2)O(2)oxidative stress since the cell could be protected by its effective repair systems, including dethiolating the inactivated key SH-sensitive enzymes. TTase may play a role in this. One of the mechanisms may be through preserving glucose metabolism and supplying ATP needed for maintaining cell viability.     

Propyl gallate is a superoxide dismutase mimic and protects cultured lens epithelial cells from H2O2 insult

n-Propyl gallate (nPG) is a food preservative that is generally regarded as safe by the US FDA. It suppresses oxidation in biological systems. The mechanism by which nPG acts in biological systems is uncertain. We investigated whether nPG protected cultured lens epithelial cells from H(2)O(2)-induced damage. Cells were treated with H(2)O(2) or with nPG and then H(2)O(2). H(2)O(2) inhibited growth, caused membrane blebbing, decreased lactate production, increased the level of GSSG, decreased the levels of GSH, ATP and NAD(+), and G3PDH activity, stimulated the hexose monophosphate shunt and induced single-strand breaks in DNA. nPG prevented the H(2)O(2)-induced growth inhibition, membrane blebbing, drop in NAD(+) and single-strand breaks in DNA.The mechanism by which nPG acts at the chemical level was investigated using electron paramagnetic resonance (EPR), direct spectrophotometric kinetic measurements, and cyclic voltammetry. When nPG at low concentrations (nM to microM) was mixed with a large excess of O(2)(-)*, the superoxide signal was destroyed as indicated by UV visible spectroscopy and EPR. Kinetic analysis indicated that nPG dismutated O(2)(-)* in repetitive additions of superoxide with little loss of activity. The rate constant for the overall reaction of nPG with O(2)(-)* was ca. 10(6)M(-1)s(-1). nPG had a very low specific binding constant for Fe(2+) as determined by cyclic voltammetry. The evidence indicates that nPG dismutates the superoxide ion in a catalytic manner.