半乳糖性大鼠白内障晶体内前列腺素生物合成的研究

对半乳糖性白内障晶体内微粒体生物合成前列腺素能力变化分析发现：用５０％半乳糖喂养２４小时后晶体内ＰＧＦ２α水平降至对照组的３１．１１％；４８小时后为对照组的５８．０４％，第５天时为对照组的５２．１９％，在第九天时为对照组的４８．０２％，第２１天时为对照组的３２．１５％，研究发现半乳糖性白内障晶体内前列腺素合成能力受到明显影响，而这可能与白内障形成有关。     

半乳糖对离体晶状体上皮细胞游离钙影响的实验研究

目的 观察高浓度半乳糖诱导正常离体晶状体上皮细胞中游离钙的变化以及钙离子阻滞剂维拉帕米对它的阻滞作用。方法 钙离子指示剂fluo-3与离体的正常品状体上皮细胞中游离钙螯合后，用激光扫描共聚焦显微镜观察35mmol／L半乳糖在不同条件下致细胞内荧光强度的变化。结果 35mmol半乳糖作用于离体品状体上皮细胞后可见细胞内与钙离子的荧光强度呈持续上升趋势，各个细胞上升的幅度及反应的速度有细微差别，但100s左右均出现上升趋势。先加2mg／mL的维拉帕米，2min后加半乳糖溶液至终浓度为35mmol，观察5min只见基本正常的钙波动，未见明显的钙上升或下降改变。结论 半乳糖溶液能致离体晶状体上皮细胞中游离钙升高，维拉帕米能有效地抑制此过程。     

后发性白内障形成中晶体上皮细胞PCNA的表达

探讨后发性白内障形成过程中晶体上皮细胞的增殖性变化。方法 :采用免疫组化SABC法对兔后发性白内障形成过程中晶体上皮细胞PCNA的表达进行检测。结果 :①术后各组增殖指数 (PI值 )均大于对照组 ,除术后 5个月时赤道部P =0 0 1外 ,其余均P <0 0 1。②术后第 1天晶体上皮细胞PCNA表达明显增强 ,赤道部PI值为 0 6 1± 0 0 6 ,前囊下部为 0 5 5± 0 0 5 ,与对照组相比P <0 0 1;术后 1天 ,后囊出现PCNA阳性细胞 ;术后 2个月后囊纤维化。③赤道部PCNA阳性表达强于前囊下部 ,术后 1天、 3天、 1个月与前囊下相比P <0 0 1,术后 1周、 2～ 5个月P >0 0 5。结论 :晶体上皮细胞的增殖、移行发生在术后早期 ,术后 1天增殖能力最强 ;赤道部细胞增殖能力强于前囊下部 ;术后晶体上皮细胞移行、增殖、分化导致后发性白内障的形成。     

实验性半乳糖性白内障的拉曼光谱研究

为了探讨半乳糖性白内障形成过程中晶体核部的水合现象，我们对喂养半乳糖３～１７天的实验组及相应对照组Ｗｉｓｔａｒ大白鼠的晶体核进行激光拉曼光谱研究。结果表明：（１）实验织晶体核部发生明显的水合现象。（２）水合程度与晶体核部的混浊成正比。认为半乳糖性白内障晶体核部的水合现象主要由Ｎａ￣＋／Ｋ￣＋失衡所致。     

肝素抑制后发性白内障形成的实验研究

肝素抑制后发性白内障形成的实验研究上海第二医科大学新华医院眼科夏小平，陆道炎，王丽天，陈才根，吴念祖目前有关减少后发性白内障形成的方法涉及二个方面。手术方面包括：尽可能多地清除晶体皮质和前囊膜上皮细胞、术中冷冻后囊、现在中山医科大学第三临床学院眼科尽...     

人晶体上皮细胞密度及形态学观察

本文对正常及白内障晶体上皮细胞的密度及形态学进行了观察，对比分析了晶体上皮细胞形态学特点与白内障类型的关系，结果证明核性白内障晶体上皮细胞密度较高，且形态规则，是其晶体混浊发展缓慢的形态学基础，后囊下型白内障发生与晶体上皮存在的增生活跃的重叠细胞有关。     

国产谷胱甘肽滴眼剂防治动物萘及半乳糖诱发的白内障疗效观察

观察国产谷胱苷肽滴眼剂(GSHc)对萘及半乳糖诱发的家兔及大鼠白内障模型的疗效,并探讨体内增高GSH浓度对白内障防治的影响。在裂隙灯追踪下显示预防给GSH可防止或延迟白内障发生,效果显著。对于轻度及中度晶体混浊,无论是萘或半乳糖模型均有显著性不同于对照组。大鼠滴眼加腹腔注射GSH可提高血中GSH浓度,疗效高于单纯局部滴眼用药组。对于成熟期白内障,本药无治疗效果。     

晶状体上皮细胞凋亡与硒性白内障关系的实验研究

目的 探讨晶状体上皮细胞凋亡与亚硒酸钠诱发的硒性白内障形成的关系。方法 给ＳＤ大鼠注射亚硒酸钠后１，３，７天剥取日状体吓膜。用流式细胞仪（ＦＣＭ）计数晶状体上皮细胞凋亡百分率，ＤＮＡ片断凝胶电泳检测晶状体上皮细胞核提取物，并应用透射电镜观察晶状体上皮细胞凋亡的开矿学变化。结果 给药后第１天晶状体上皮细胞凋亡百分率即显著增高，第３天达高峰，以后逐渐下降。而晶状体混浊程度持续加重，给药后第７天形成典型     

大鼠半乳糖性白内障晶状体中TGF-β1基因的表达

目的 研究转化生长因子-β1(TGF-β1)基因在大鼠白内障发生过程中的表达。方法 给3周龄大鼠单眼球后注射20％半乳糖生理盐水诱导白内障的发生，运用RT-PCR方法测定在不同时相大鼠晶状体中TGF-β1基因的表达。结果 半乳糖注射后晶状体上皮细胞TGF-β1 mRNA于8h开始上升，24h达高峰，是正常对照的4倍，2d后迅速下降，4d完全恢复到正常水平。结论在白内障形成的早期，晶状体细胞中TGF-β1呈高表达，提示TGF-β1基因可能在半乳糖性白内障的发生过程中起重要作用。     

豚鼠d—半乳糖性白内障的实验模型研究

用ｄ－半乳糖１２５ｍｇ／ｋｇ／天给豚鼠球后注射，连续３２天，造成糖性白内障模型，用裂隙灯检分级记分，扫描电镜微观形态分析，晶体ＭＤＡ含量及不溶性和高分子量蛋白病理生化测定，对多项指征进行了综合评价和分析，证实该模型成功率在９０％以上，对考察药物对白内障的防治作用具有一定意义。     

The unfolded protein response in lens epithelial cells from galactosemic rat lenses

PURPOSE: Diabetic complications are associated with hypoglycemia and hyperglycemia. The purpose of this study was to investigate the effect of both glucose deprivation and hyperglycemia on the induction of endoplasmic reticulum (ER) stress and the subsequent activation of the unfolded protein response (UPR) that results in apoptosis in in vitro cultured lens epithelial cells (LECs) and in vivo cataract formation in galactose-fed rats. METHODS: Lenses from rats fed a standard diet containing 50% galactose with or without an aldose reductase inhibitor (ARI) were investigated. Transformed human LECs were cultured in standard 10% FCS-DMEM containing various concentrations of sugar. UPR-specific proteins from both the rat lenses and lens cultures were quantified by protein blot analysis. Cell death was evaluated with TUNEL staining and ethidium homodimer-1 (EthD) dyes. Reactive oxygen species (ROS) were quantified with H2-DCF, and free glutathione (GSH) levels were measured with a commercial GSH quantification kit. RESULTS: Increased apoptosis of the LECs was observed in the lenses of rats fed the galactose diet for 5 to 9 days, and nuclear cataracts subsequently developed in these lenses after 13 to 15 days. Protein blot analysis of the LECs from these galactose-fed rats showed higher levels of the UPR-specific proteins Bip/GRP78, ATF4, and CHOP. These LECs also demonstrated activation of the UPR-specific procaspase-12 and the increased presence of ROS, whereas GSH was reduced. Because these results indicate that the UPR is activated in LECs along with the production of ROS and apoptosis during cataract formation in the galactose-fed rats, subsequent studies were conducted to determine the role of nonenzymatic glycation, osmotic stress, and oxidative stress on these biochemical processes. In vitro cultures of human LECs showed that the UPR was induced by osmotic and oxidative stress, but not by glycation. In addition, the UPR and apoptosis in LECs was induced by glucose deprivation. The ARI blocked the induction of the UPR, cell death, and cataract formation. CONCLUSIONS: The UPR that is induced by abnormally high or low concentrations of sugar is linked to the production of ROS, increased apoptosis in LECs, and cataract formation. The inhibition of the UPR induction by ARI suggests that osmotic stress may be the primary inducer of the UPR. Modulation of the UPR pathways may offer novel methods for the development of therapeutic tools to delay cataracts.     

c—myc反义寡核苷酸抑制半乳糖性白内障晶状体上皮细胞凋亡的机制

目的研究c-myc反义寡核苷酸（ASODN）对半乳糖性白内障晶状体上皮细胞（LECs）凋亡的影响。方法将Wistar大鼠按随机数字表法分为生理盐水组、半乳糖组和半乳糖＋c-myc ASODN组,每组36只。半乳糖组及半乳糖＋c—mycASODN组每日球后注射O．2mL20％半乳糖,制作大鼠半乳糖性白内障模型,半乳糖＋c—mycASODN组球后注射半乳糖4h后加注Lipo—mycASODN复合液0．2mL,隔日1次。分别于给药后7、14、24d处死动物,取出晶状体,检测LECs的凋亡情况,采用TUNEL法检测c—mycASODN对半乳糖诱导LECs凋亡的影响,透射电镜下观察LECs超微结构的改变。结果各组在7、14、24d的LECs凋亡率比较,差异均有统计学意义（F7 days=3418．495,P〈0．01;F14 days=1137．555,P〈0．01;F14 days=2198．871,P〈0．01）;24d时半乳糖组LECs的凋亡率为56．57±3．20,生理盐水组为0．37±0．11,差异有统计学意义（P〈0．01）;半乳糖＋c．mycASODN组细胞凋亡率为29．35±1．63,较半乳糖组明显降低（P〈0．05）。透射电镜下观察发现,半乳糖组可见LECs呈凋亡细胞的早期改变;随着高糖诱导时间的延长,凋亡细胞逐渐增多;生理盐水组几乎未见到凋亡细胞;半乳糖＋c—mycASODN组凋亡细胞数量较同期半乳糖组少。结论在白内障形成过程中半乳糖能诱导LECs凋亡,c．mycASODN能够抑制半乳糖诱导的LECs凋亡。     

Effects of calcium on lens epithelial cells in rabbits

PURPOSE: The action of lens epithelial cells (LECs) is important for cataract and posterior subcapsular cataract after cataract surgery. In this study, we analyzed the effects of calcium on the characteristics of LECs. METHODS: The LECs were collected using albino rabbits and incubated in minimum essential medium [MEM, Introgen Corp. (12% fetal bovine serum: FBS)] (37 degrees C, 5 % CO2) for a week to induce their proliferation. Cell culture dishes (35 mm) were prepared and 7 mm cylindrical pipes were placed in them. After that, around 10,000 cultured LECs were placed in the pipes and incubated. After 2 hours incubation, the pipes were removed and various doses of MEM (0, 2, 10 and 20 mM) replaced the calcium. Proliferation and shapes of LECs were observed using a confocal microscope and immunohistological analysis [alpha-smooth muscle actin (alpha-SMA) and bromodeoxyuridine (BrdU)]. The LECs were incubated with collagen gel and different calcium doses (0, 2, 10 and 20 mM) of MEM to calculate the contraction rate. RESULTS: It was observed that the LECs changed to fibroblast-like cells at high doses of calcium using a confocal microscope. Histological studies showed that the BrdU positive cells were increased by using 10 and 20 mM calcium MEM, but the positive cells were decreased by using 0 and 2 mM calcium MEM. Increase of alpha-SMA stained cells was recognized when using 0, 10 and 20 mM calcium MEM. The contraction rate of collagen gel was increased by using the 10 and 20 mM calcium MEM. CONCLUSION: The changes of calcium concentration might be an important factor for the development of cataract, posterior subcapsular opacification, and contraction of the lens capsule after cataract surgery.     

Presence of alpha smooth muscle actin in lens epithelial cells of aphakic rabbit eyes.

AIMS: To determine whether alpha smooth muscle actin (alpha-SMA), a marker for myofibroblastic cells, is present in lens epithelial cells (LECs) in rabbit aphakic eyes. METHODS: Phacoemulsification was performed in rabbit eyes, which were enucleated after surgery. Immunohistochemical methods were used to detect alpha-SMA in LECs. RESULTS: Five days after surgery, the presence of alpha-SMA positive LECs was observed mainly around the adhesive portion of the anterior capsule margin and the posterior capsule. The posterior capsule was wrinkled at the adhesive portion. The alpha-SMA positive LECs were flattened with spindle-shaped cross sections. Seven days after surgery, the alpha-SMA positive LECs covered most of the central posterior capsule. They disappeared 10 days after surgery. On the other hand, the cuboidal LECs in the capsular bag were negative for alpha-SMA. CONCLUSION: The flattened LECs with spindle-shaped cross sections observed 5 days after cataract surgery contained alpha-SMA. Such LECs were distinguished biochemically from the cuboidal LECs, which lacked alpha-SMA.     

The Relationship between Osmotic Stress and Calcium Elevation: in vitro and in vivo Rat Lens Models

Bothin vivoandin vitromodels were employed in the present study to assess the relative contribution of osmotic stress and increasing calcium levels to the development of sugar cataracts. In galactose cataract obtained from galactosemic weanling rats, the concentration of total calcium increased by nearly 10% at the first sign of visible opacification observed on the fourth day post-galactose feeding. After 7 days of galactose feeding, calcium levels continued to rise, to 0.8 mm. During the first 10 days, loss of lens transparency and calcium elevation was gradual and steady, with precipitous changes occurring on days 11 and 12. In groups of rats where galactose feeding was stopped after 7 days, cataract reversal was followed during the next 5 weeks. During the initial first week of recovery, calcium influx and elevation in the lens continued but began to decline steadily thereafter. After 3 weeks of recovery, lens transparency had returned to almost normal. Calcium levels continued to decline and reached normal levels between day 34 and 42, nearly 4 weeks after removal of the galactose diet.The relationship between osmotic stress and calcium elevation was investigated more directly by culturing normal rat lenses in hypoosmotic medium (280 mOsm) to create osmotic gradients similar to that in galactosemic lenses. The results showed that during the first day of culture (12 hr), osmotically stressed lenses gained 3 mg of water, became opaque and gained excess calcium (7 mmcompared to 0.7 mm). Microscopic vacuoles appeared to accompany the process of opacification and contributed to increased light scattering and the loss of lens transparency.Additional experiments were designed to further distinguish between the effects of osmotic stress and calcium elevation on the opacification process. Thus, lenses were incubated in control and high-calcium medium (20 mm) at 300 mOsm. Within 12 hr of incubation, calcium elevation progressed to 1.37 mm, nearly doubling the normal value. Although opacification was observed in these lenses, no sign of vacuoles was evident. Collectively, the findings from this study support the premise that an early influx of calcium is brought about by osmotic stress and is responsible for the observed loss in transparency in osmotic (sugar) cataract.     

Ultrastructural changes during the development and reversal of galactose cataracts

Ultrastructural alterations in rat lens that accompany the induction of galactose cataract and the reinstatement of tissue transparency that occurs subsequent to removal of the animals from galactose diet were documented. Lenses examined at different times after the initiation of galactose diet exhibited edematous, liquefied fibers and cellular cysts. Electron dense aggregates were initially found in sections obtained from the pre-equatorial region and subsequently in the tissue from the anterior polar region. The location of the aggregates in the galactose-fed animals followed a spatio-temporal pattern which paralleled cataract maturation at the macroscopic level. The aggregates were not present in either the epithelium of the cataractous lenses or in the epithelium or fibers of lenses from animals that were fed glucose or laboratory chow alone. Mature cataracts were observed in rats fed galactose for 20 days. However, a reinstatement of fiber morphology and lens transparency was realized if these animals were subsequently placed on a galactose free diet. Normalization of fiber morphology and alignment was noted after the animals had been on a galactose free diet for 28 days. However, a nuclear opacity persisted throughout the 3 month observation period. The extent to which the repair of existing fibers and/or the formation of newly formed fibers contribute to the reversal of lens opacity remains to be ascertained.     

Biochemical changes associated with the development and reversal of galactose cataracts.

The rate of synthesis of glutathione (GSH) in lens extracts during cataract formation has been determined and found to be the same as that of extracts from normal lenses. It is concluded that unlike X-ray-induced cataracts the potential mechanism for GSH synthesis in galactose cataracts is unaffected. In addition, the levels of GSH, free amino acids, dulcitol, cations and the degree of hydration of the lens during the development and reversal of galactose cataract have been determined.Feeding of a normal diet even after the development of mature cataracts (20 days) leads to the disappearance of cortical opacities almost completely leaving an essentially clear lens with only a very fine pinhead nuclear opacity.The levels of GSH, taurine and other free amino acids, which fall rapidly dudring cataract formation, return to near normal values during the reversal phase even though the lenses are hydrated. In cnntrast to the near complete recovery of these constituents, sodium ion concentration remains four times higher than in the control lenses almost 4 weeks after diet reversal. The recovery of potassium ion during the same period is about 70% of controls. The continued hydration of the lenses during the reversal phase of cataract, despite the disappearance of dulcitol, is apparently related to the elevated sodium ion concentration.A possible explanation for the recovery of GSH and free amino acids in hydrated lenses has been suggested.     

Effect of the cytokines on the prostaglandin E2 synthesis by lens epithelial cells of human cataracts.

BACKGROUND--Lens epithelial cells (LECs) derived from human cataracts have been reported to produce various cytokines and prostaglandin E2 (PGE2) in culture. The effects of IL-1, TGF-beta, and b-FGF on the PGE2 synthesis by LECs have been studied. METHODS--A circular piece of the anterior capsule with attached LECs was obtained by capsulotomy during cataract surgery and cultured. The primary, almost confluent, cultures were used for the study. The PGE2 concentration of the culture media for 24 h was measured after the addition of recombinant human IL-1 alpha, TGF-beta 2, or b-FGF at various concentrations. The PGE2 concentration was also measured in the media to which each cytokine and rabbit polyclonal anti-human antibodies against the corresponding cytokine had been added. RESULTS--The PGE2 concentration of the culture media after addition of IL-1 alpha at the concentration of 100 or 500 pg/ml (1765 (768) and 3071 (1121) pg/10(4) cells) or TGF-beta 2 at the concentration of 10 or 100 ng/ml (689 (264) and 750 (189) pg/10(4) cells) was significantly increased compared with that in the controls (67 (20) pg/10(4) cells). These effects were suppressed by the corresponding anticytokine antibodies. Basic FGF and anti-human b-FGF showed no significant effect on the PGE2 concentration. IL-1 and TGF-beta increased but b-FGF did not affect the PGE2 synthesis by LECs in culture. CONCLUSION--IL-1 and TGF-beta may participate in postoperative inflammation after cataract surgery by increasing PGE2 synthesis by residual LECs.