晶状体上皮细胞凋亡与白内障发生的分子生物学机制

目的:观察白介素转化酶3(Caspase-3)在白内障晶状体上皮细胞中的表达及与凋亡的关系。方法:经手术收集25只白内障晶状体前囊膜以及胚胎晶状体(6只)和成人透明晶状体(4只)的前囊膜,采用免疫组织化学染色的方法观察Caspase-3的表达及染色强度。并对Caspase-3在年龄相关性白内障与并发性白内障中的表达进行比较。结果:Caspase-3在白内障组有很好的表达,在年龄相关性白内障组16只中14只呈阳性表达,在并发性白内障组9只中8只呈阳性表达,在透明晶状体没有表达;在年龄相关性白内障与并发性白内障之间没有显著性差异(P>0.05)。结论:凋亡在白内障的发生中存在。     

水通道蛋白0和水通道蛋白1在老年性白内障及透明晶状体前囊膜上皮细胞的表达

【目的】明确水通道蛋白0（aquaporin 0,AQP0）和水通道蛋白1（aquaporin 1,AQPl）在透明晶状体及老年性白内障晶状体上皮细胞及前囊膜的表达及分布,并初步探讨AQPO和AQPl在老年性白内障发病中的重要作用。【方法】收集45例老年性白内障（白内障组）及10例正常透明晶状体（对照组）前囊膜,通过免疫组化方法检测AQP0和AQPl的表达。【结果】白内障及对照组均可见AQPO和AQPl的表达,对照组AQPO的阳性细胞表达率（100％）显著高于白内障组（47％）（P〈0．05）,对照组中AQPl的阳性细胞表达率（100％）亦显著高于白内障组（55％）（P〈0．05）。【结论】正常晶状体上皮细胞有AQPO和AQPl的活性表达,对保证晶状体的生理动态平衡和晶状体的透明性起到了重要的作用,AQPO和AQPl在老年性白内障晶状体前囊膜及上皮细胞中的异常表达与老年性白内障的发生密切相关。     

Telomere‐dependent senescent phenotype of lens epithelial cells as a biological marker of aging and cataractogenesis: the role of oxidative stress intensity and specific mechanism of phospholipid hydroperoxide toxicity in lens and aqueous

Cataract formation represents a serious problem in the elderly and has a large impact on healthcare budget. Aging and cataract formation are relatively complex phenomena, both in vivo and in vitro. Telomeres are special structures at the end of chromosomes. They shorten during each round of replication, and this has been characterized as a mitotic counting mechanism. Our review analysis in this work shows that the rate of telomere shortening in human lens epithelial cells during aging and cataract formation is modulated by oxidative stress as well as by differences in antioxidative defense capacity of the normal and cataractous crystalline lenses. Presented in this review studies suggest that telomere shortening in human lens cells and increased oxidative stress are the result of the peroxidative damage to the lens cell membranes and biomolecules induced in the lack of reductive detoxification of phospholipid hydroperoxides as the triggering mechanism of cataractogenesis. Lipid peroxidation (LPO) is a causative factor of cataract. The increased concentrations of primary molecular LPO products (diene conjugates, lipid hydroperoxides) and end fluorescent LPO products were detected in the lipid moieties of the aqueous humor samples obtained from patients with senile and complicated cataracts when compared to normal donors. The progressive accumulation of oxidative damage may act as an important mechanism for organism aging and cataractogenesis. The oxidative stress form and intensity might determine the lens senescence rate and cataract type, making efforts in the cataract prevention challenge more complex. The analyzed challenge in this work is that the reduction in telomere shortening rate and damages in telomeric DNA make an important contribution to the anticataract and life‐extension effect of carnosine administered systemically in the formulations stabilizing a dipeptide from the enzymatic hydrolysis with carnosinase, or topically administered to the eye with carnosine ophthalmic prodrug N‐acetylcarnosine and lubricant formulations thereof including corneal absorption promoters. Telomere length in the human crystalline lens cells is a reflection of aging, cataractogenesis, and lifespan in biogerontological studies.     

先天性白内障BALB／c小鼠的晶体纤维扫描电镜观察

用扫描电镜对先天性白内障ＢＡＬＢ／ｃ小鼠的晶体纤维进行了观察。结果显示，白内障晶体纤维的病变特点主要为纤维肿胀和基质的絮状网络样改变，并对上述病变以及先天性白内障与其他类型白内障的病理变化结果之间的关系进行了探讨。     

Lens superoxide dismutase and catalase activities in diabetic cataract

OBJECTIVE: Biochemical evidence suggests that the oxidative damage of the lens proteins is involved in the genesis of senile cataract and the development of diabetes-related pathologic changes such as the formation of cataracts. In particular, lens proteins are subject to extensive oxidative modification. Oxidative damage either decreases the antioxidant capacity or decreased antioxidant capacity results in oxidative damage. The purpose of this study was to analyze the activities of the antioxidant enzymes such as Cu,Zn Superoxide Dismutase (Cu,Zn-SOD) and catalase in the cataractous lenses of the type 2 diabetic group and cataractous lenses of the senile group. METHOD: Eighteen diabetic cataractous lenses and twenty six senile cataractous lenses were studied. Cu,Zn-SOD activity was measured in lenses by enzymatic method and catalase activity was measured by colorimetric method. RESULTS: Cu,Zn-SOD levels were significantly lower in the diabetic cataractous lenses than senile cataractous lenses (respectively 8.052 +/- 0.818, 18.216 +/- 4.217 microg/g prot. p < 0.05). Similarly, catalase levels were significantly lower in the diabetic cataractous lenses than senile cataractous lenses (respectively 0.326 +/- 0.134, 0.665 +/- 0.322 kU/g prot. p < 0.001). CONCLUSION: The results of the present study indicate that the antioxidant capacity in the diabetic cataractous lenses were decreased and this result suggests a role of antioxidant enzymes in the genesis of diabetic cataracts.     

Inhibition of rabbit keratocyte and human fetal lens epithelial cell proliferation by retrovirus-mediated transfer of antisense cyclin G1 and antisense MAT1 constructs

The aim of this study is to evaluate the potential of gene transfer of cell cycle control genes as treatment of corneal haze or secondary cataract formation. The guiding hypothesis is that strategic modulation of the cyclin G1 or MAT1 gene by retrovirus-mediated gene transfer will inhibit proliferation of rabbit keratocytes (RabK) and fetal human lens epithelial (FHLEpi) cells in vitro. RabK and FHLEpi cell cultures were transduced in triplicate with retroviral vectors bearing either a nuclear-targeted beta-galactosidase, an antisense cyclin G1 (aG1), an antisense MAT1 (aMAT1) construct, or the neo(r) gene. The presence of beta-galactosidase activity in the transduced cultures was detected by immunohistochemical X-Gal staining, while cyclin G1 and MAT1 protein expression levels were evaluated by Western analysis. Proliferation of RabKs and FHLEpi cells was analyzed by counting the number of cells in the aG1 and aMAT1 vector-transduced cultures over 5 days. The mean transduction efficiency was 34.4% (SD 1.41) for RabKs and 19.7% (SD 1.83) for FHLEpi cells. Downregulation of cyclin G1 and MAT1 protein expression was noted 24 hr after transduction of RabK cultures with the respective vectors. Cytostatic effects of the aG1 and aMAT1 vectors in both RabKs and FHLEpi cells were most pronounced on the fifth day (RabKs, p < 0.0007; FHEpi cells, p < 0.001). An increased incidence of apoptosis was identified in both aG1 and MAT1-transduced FHLEpi cells. Taken together, these data suggest the potential utility of developing aG1 and aMAT1 retroviral vectors in gene therapy protocols for corneal haze and secondary cataract formation.     

糖尿病性白内障晶状体上皮细胞的Fas、P53基因表达

目的探讨糖尿病性白内障及老年性白内障晶状体上皮细胞(LEC)的凋亡与Fas、P53基因的关系。方法在白内障超声乳化手术中取糖尿病性及老年性白内障患者的晶状体前囊膜标本45例,应用光镜,电镜观察LEC的凋亡情况,应用RT-PCR定量检测Fas蛋白,P53蛋白在白内障LEC中的表达。结果两种白内障LEC中均有Fas,P53蛋白表达。Fas蛋白在糖尿病性白内障LEC中的表达平均是老年性白内障的13.34倍,P53蛋白在糖尿病性白内障LEC中的表达平均是老年性白内障的7.3倍,两者差异有统计学意义(P0.05)。结论两种白内障LEC均出现凋亡,Fas、P53基因在糖尿病性白内障LEC凋亡中起重要作用。     

Nonenzymatic addition of glucocorticoids to lens proteins in steroid-induced cataracts

A frequent manifestation of long-term glucocorticoid administration is the occurrence of posterior subcapsular cataracts. The molecular basis for this effect has not yet been elucidated. The addition of prednisolone to the rat lens in culture results in a time- and concentration-dependent lens opacification that correlates with the formation of covalent prednisolone-lens protein adducts. Prednisolone adduct formation was analyzed by [3H]prednisolone incorporation and by immunoprecipitation with antiserum specific for proteins modified by the nonenzymatic addition of prednisolone. In the rat lens, these adducts were localized in both the water-soluble and urea-soluble lens protein fractions. Gel electrophoresis and fluorography revealed that the most extensively modified proteins were two crystallins subunits. Lens proteins from 33 normal and cataractous human lenses were fractionated and analyzed for the presence of prednisolone-protein adducts by competitive radioimmunoassay. Adducts were detected only in those samples derived from glucocorticoid-induced cataractous lenses. We conclude that elevated glucocorticoid levels lead to the formation of glucocorticoid-lens protein adducts both in vitro and in vivo. Lens protein modification by glucocorticoids may lead to sufficient biochemical or structural alterations so as to result in cataract formation. The ability of glucocorticoids to form adducts with proteins in vivo also may play a role in some of the other toxic manifestations of long-term glucocorticoid therapy.     

Identification of four chromosomal loci determining obesity in a multifactorial mouse model.

We previously described a new mouse model for multigenic obesity, designated BSB. We now report the use of a complete linkage map approach to identify loci contributing to body fat and other traits associated with obesity in this model. Four loci exhibiting linkage with body fat, or with the weights of four different fat depots, residing on mouse chromosomes 6, 7, 12, and 15, were identified and confirmed by analysis of additional BSB mice. Each of the four loci differed with respect to their effects on the percent of body fat, specific fat depots and plasma lipoproteins. The loci exhibited allele-specific, non-additive interactions. A locus for hepatic lipase activity was co-incident with the body fat and total cholesterol loci on chromosome 7, providing a possible mechanism linking plasma lipoproteins and obesity. The chromosome 7 locus affecting body fat, total cholesterol and hepatic lipase activity was isolated in congenic strains whose donor strain regions overlap with the chromosome 7 BSB locus. These results provide candidate genes and candidate loci for the analysis of human obesity.     

ERK1/2信号传导通路介导碱性成纤维细胞生长因子诱导人晶状体上皮细胞中环氧合酶2的表达

背景:碱性成纤维细胞生长因子在各种白内障形成中都起着重要的作用,可促进晶状体上皮细胞的增殖并化生为纤维细胞,但其信号通路尚不清楚。目的:探讨ERK1/2信号转导通路在碱性成纤维细胞生长因子诱导的人晶状体上皮细胞环氧合酶2表达中的作用。方法:使用10μg/L的碱性成纤维细胞生长因子干预培养的人晶状体上皮细胞0,1,3,6,12,24h,RT-PCR检测刺激不同时间人晶状体上皮细胞环氧合酶2mRNA的表达,Western blot检测细胞中环氧合酶2及磷酸化ERK1/2的表达。在阻断实验中应用特异性ERK1/2的阻断剂PD98059阻断ERK信号转导通路1h,再用10μg/L碱性成纤维细胞生长因子刺激细胞6h,Western blot检测细胞中环氧合酶2的表达。结果与结论:碱性成纤维细胞生长因子刺激后的人晶状体上皮细胞中环氧合酶2mRNA及其蛋白的表达显著增加(P<0.01),同时碱性成纤维细胞生长因子诱导人晶状体上皮细胞磷酸化ERK1/2活性增强,表达水平随作用时间而增加,30min时达到最高峰(P<0.01),6h后恢复至基线水平;PD98059可抑制人晶状体上皮细胞环氧合酶2的表达(P<0.01)。说明ERK1/2信号转导通路参与了碱性成纤维细胞生长因子诱导的人晶状体上皮细胞中环氧合酶2的表达,在后发性白内障的形成过程中起着重要作用。     

ERK1/2信号传导通路介导碱性成纤维细胞生长因子诱导人晶状体上皮细胞中环氧合酶2的表达

背景：碱性成纤维细胞生长因子在各种白内障形成中都起着重要的作用,可促进晶状体上皮细胞的增殖并化生为纤维细胞,但其信号通路尚不清楚。目的：探讨ERK1/2信号转导通路在碱性成纤维细胞生长因子诱导的人晶状体上皮细胞环氧合酶2表达中的作用。方法：使用10μg/L的碱性成纤维细胞生长因子干预培养的人晶状体上皮细胞0,1,3,6,12,24h,RT-PCR检测刺激不同时间人晶状体上皮细胞环氧合酶2mRNA的表达,Western blot检测细胞中环氧合酶2及磷酸化ERK1/2的表达。在阻断实验中应用特异性ERK1/2的阻断剂PD98059阻断ERK信号转导通路1h,再用10μg/L碱性成纤维细胞生长因子刺激细胞6h,Western blot检测细胞中环氧合酶2的表达。结果与结论：碱性成纤维细胞生长因子刺激后的人晶状体上皮细胞中环氧合酶2mRNA及其蛋白的表达显著增加（P〈0.01）,同时碱性成纤维细胞生长因子诱导人晶状体上皮细胞磷酸化ERK1/2活性增强,表达水平随作用时间而增加,30min时达到最高峰（P〈0.01）,6h后恢复至基线水平;PD98059可抑制人晶状体上皮细胞环氧合酶2的表达（P〈0.01）。说明ERK1/2信号转导通路参与了碱性成纤维细胞生长因子诱导的人晶状体上皮细胞中环氧合酶2的表达,在后发性白内障的形成过程中起着重要作用。     

高功率微波对兔眼晶状体上皮细胞P21WAF1及Cyclin E表达的影响

目的　观察高功率微波 (highpowermicrowave ,HPM)对实验动物兔眼晶状体上皮细胞P2 1WAF1 、CyclinE表达水平的影响 ,以探讨防护HPM眼损伤的机理及方法。方法　采用功率密度为 80 0mW cm2 的微波持续照射青紫兰兔眼组织 2 0min ,并于微波照射后 3 0d ,90d ,180d及 3 60d时应用免疫组化、原位杂交及图像分析技术对兔眼晶状体上皮细胞P2 1WAF1 、CyclinE蛋白及其mRNA表达水平进行检测。结果　HPM照射可影响晶状体上皮细胞的正常增殖过程 ;当HPM照射后 3 0d时 ,P2 1WAF1 表达明显升高 ,对细胞增殖周期抑制作用增强 ;CyclinE表达则明显降低 ,其正性调节细胞生长作用减弱 ;当HPM照射后 90 ,180及 3 60d时 ,微波照射组上述各指标与对照组比较 ,差异均无显著性意义 ,而且各项指标的mRNA表达均与其蛋白表达同步且一致。结论　HPM照射可干扰细胞正常的生长增殖周期进程 ,而晶状体上皮细胞增殖异常可能是导致白内障的重要原因之一。     

Drugs designed to maintain the transparence of the ocular lens

Summary— Research into the biological basis of lens transparency has demonstrated the implication of lens sugar stress in the diabetic cataract whereas senile cataract is the result of natural degeneration which is enhanced by various external factors such as cosmic and ionizing rays, or oxidative processes. Drugs have been developed which are aimed at being effective on lens pathological physiology and metabolism, concurrently. Such molecules: aldose reductase inhibitors (ARIs: sorbinil, AD-5467, CT-112 and imirestat), acetyl salicylic acid (ASA), salicylate (SA) and sodium monomethyl trisilanol orthohydroxybenzoate (SMB, a prodrug for salicylate) have undergone pharmacodynamic, pharmacokinetic and/or clinical studies which are presented here. ARIs have shown efficacy in slowing down and preventing the progression of experimental sugar cataracts; sorbinil can partially reverse the very early morphological signs of sugar cataract. Sorbinil and imirestat have also demonstrated anti-oxidant properties. ARIs administration (per os or by topical instillation) generally results in lens levels compatible with concentrations that are efficient on biochemical mechanisms of cataract formation. However, at the present time, clinical evaluations are in progress and as yet, there is no confirmation of their efficacy in man. ASA and SA can prevent various mechanisms of lens protein denaturation; they inhibit AR and prevent, in vitro, the formation of some pigments found in the aged cataractous lens. Extrapolation of the ASA ocular pharmacokinetics results in animal to man, suggest that ASA administration per os could result in efficacious levels in the lens. This is also sustained by the observation of a reduced frequency of cataracts in ASA treated diabetic rhumatoid arthritis patients. SMB pharmacokinetic studies have shown small but persistent levels of the active principle in the lens. They suggest that the capsule slows down SA diffusion into the lens and that, on the contrary, lens epithelium facilitates its penetration. Preliminary results of pharmacodynamic studies are given.     

Coincidence of genetic loci for plasma cholesterol levels and obesity in a multifactorial mouse model.

We have examined backcross progeny derived from a cross of Mus spretus with C57BL/6J, that range from 1 to 50% carcass lipid (n = 215), and from 22 to 130 mg/dl plasma total cholesterol (n = 238). Statistical analysis revealed that distal mouse chromosome 7 exhibits significant linkage both to plasma total cholesterol (likelihood of the odds [LOD] 5.8) and to carcass lipid (LOD 3.8). A locus on chromosome 6 also shows significant linkage to plasma total cholesterol (LOD 5.6), but no linkage to carcass lipid. Neither chromosomal region contains any previously mapped genes likely to influence lipoprotein metabolism, indicating that novel genetic factors contributing to plasma lipoprotein levels have been identified.     

转化生长因子β2对人晶状体上皮细胞E-钙黏素和α-平滑肌肌动蛋白表达的影响

背景：有研究表明,人晶状体上皮细胞的间质转分化是后发性白内障的主要病理改变,转化生长因子β2可以诱导间质转分化的发生。目的：体外培养人晶状体上皮细胞,观察转化生长因子β2对人晶状体上皮细胞转分化相关蛋白E-钙黏蛋白和α-平滑肌肌动蛋白表达的影响。方法：利用组织块法体外培养人晶状体上皮细胞并传代,选择传5代的细胞进行实验,采用100ng/L转化生长因子β2诱导48h,反转录-聚合酶链反应方法检测E-钙黏蛋白、α-平滑肌肌动蛋白mRNA的表达,应用蛋白质印迹法Westernblot检测其蛋白表达。结果与结论：转化生长因子β2处理人晶状体上皮细胞48h后,细胞E-钙黏蛋白表达明显减弱,α-平滑肌肌动蛋白的表达明显增强。提示转化生长因子β2可以成功诱导晶状体上皮细胞间质转分化,转化生长因子β2处理的晶状体上皮细胞可以作为间质转分化的细胞模型。     

Trib1 is a lipid- and myocardial infarction-associated gene that regulates hepatic lipogenesis and VLDL production in mice

Recent genome-wide association studies have identified a genetic locus at human chromosome 8q24 as having minor alleles associated with lower levels of plasma triglyceride (TG) and LDL cholesterol (LDL-C), higher levels of HDL-C, as well as decreased risk for myocardial infarction. This locus contains only one annotated gene, tribbles homolog 1 (TRIB1), which has not previously been implicated in lipoprotein metabolism. Here we demonstrate a role for Trib1 as a regulator of lipoprotein metabolism in mice. Hepatic-specific overexpression of Trib1 reduced levels of plasma TG and cholesterol by reducing VLDL production; conversely, Trib1-knockout mice showed elevated levels of plasma TG and cholesterol due to increased VLDL production. Hepatic Trib1 expression was inversely associated with the expression of key lipogenic genes and measures of lipogenesis. Thus, we provide functional evidence for what we believe to be a novel gene regulating hepatic lipogenesis and VLDL production in mice that influences plasma lipids and risk for myocardial infarction in humans.     

Farnesyl analogues inhibit vasoconstriction in animal and human arteries.

Recent studies have suggested that nonsterol, mevalonate-derived metabolites are implicated in the control of vascular tone and blood pressure. Because of the metabolic importance of farnesyl pyrophosphate, a 15-carbon (C15) intermediate of the cholesterol pathway, the vasoactive properties of the farnesyl motif were investigated. Two farnesyl analogues were used: farnesol, the natural dephosphorylated form of farnesyl pyrophosphate, and N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC), a synthetic mimic of the carboxyl terminus of farnesylated proteins. Both compounds inhibited NE-induced vasoconstriction in rat aortic rings at micromolar concentration. Their action was rapid, dose dependent, and reversible. Shorter (C10) and longer (C20) isoprenols as well as N-acetyl-S-geranyl-L-cysteine (C10) did not inhibit the response to NE. In contrast, N-acetyl-S-geranylgeranyl-L-cysteine (C20), exhibited vasoactive properties similar to AFC. It was further demonstrated that AFC and farnesol inhibited KCl and NaF-induced contractions, suggesting a complex action on Ca2+ channels and G protein-dependent pathways. Finally, the effect of farnesol and AFC on the NE response was reproduced in human resistance arteries. In conclusion, mevalonate-derived farnesyl analogues are potent inhibitors of vasoconstriction. The study suggests that farnesyl cellular availability is an important determinant of vascular tone in animals and humans, and provides a basis for exploring farnesyl metabolism in humans with compromised vascular function as well as for using farnesyl analogues as regulators of arterial tone in vivo.     

Adenovirus-mediated suicide gene transduction: feasibility in lens epithelium and in prevention of posterior capsule opacification in rabbits.

The most common complication of cataract surgery is the development of posterior capsule opacification (PCO). Hyperplasia of the lens epithelium is one of the main cellular events following phacoemulsification, and has been found to be an important feature contributing to opacification of the posterior capsule. Adenoviral vector-mediated transfer is a suitable method for transducing the herpes simplex virus thymidine kinase gene (HSV-tk) into proliferating cells, allowing for the selective killing of these cells by ganciclovir (GCV) treatment. To determine the potential of gene transduction for lens epithelial cells, we studied the transduction of rabbit lens epithelial cells with adenoviral vectors containing either the Escherichia coli beta-galactosidase (lacZ) gene or the HSV-tk gene in vitro and in vivo in an experimental model of PCO. The efficiency of lacZ gene transfer in rabbit lens epithelial cells was at least 95% both in vitro and in vivo. In vivo transduction with HSV-tk adenoviral vector followed by GCV treatment significantly inhibited the development of PCO (p<0.001). These results suggest that adenoviral vector-mediated transfer of HSV-tk into the proliferating lens epithelial cells is feasible and may provide a novel therapeutic strategy for PCO.