糖尿病视网膜病变患者红细胞膜Ca~(2 )-Mg~(2 )-ATPase活性变化及相关研究

目的探讨糖尿病视网膜病变发生发展的有关因素。对40例糖尿病机网膜病变患者红细胞膜Ca~(2 )－Mg~(2 )－ATPasc活性进行了检测，并与红细胞内Ca~(2 )－Ms~(2 )－ATPasc含量、空腹血糖、糖基化血红蛋白及红细胞变形指数等进行了分析。结果糖尿病机网膜病变患者红细胞膜Ca~(2 )－Ms~(2 )－ATPasc活性显著下降（P＜0．01），细胞内Ca~(2 )含量增加（P＜0．01）而Mg~(2 )含量则显著下降（P＜0．01）。糖尿病视网膜病变者红细胞膜Ca~(2 )－Ms~(2 )－ATPasc活性下降与空腹血糖、糖基化血红蛋白、红细胞变形指数及红细胞内Ca~(2 )－Ms~(2 )－ATPasc含量等变化密切相关。结论糖尿病视网膜病变患者红细胞膜Ca~(2 )－Ms~(2 )－ATPasc活性异常下降可能是导致或加速视网膜病变发生发展的原因之一。     

糖尿病视网膜病变患者红细胞膜Ca^2＋—Mg^2＋—ATPase活性变化及…

目的 探讨糖尿病视网膜病变发生发展的有关因素，对４０例糖尿病视网膜病变患者红细胞膜Ｃａ＾２＋－Ｍｇ＾２＋－ＡＴＰａｓｅ活性进行了检测，并与红细胞内Ｃａ＾２＋，Ｍｇ＾２＋含量，空腹血糖，糖基化血红蛋白及红细胞变形指数等进行了分析，结果 糖尿病视网膜病变患者红细胞膜Ｃａ＾２＋－Ｍｇ＾２＋－ＡＴＰａｓｅ活性显著下降（Ｐ〈０．０１），细胞内Ｃａ＾２＋含量增加（Ｐ〈０．０１），而Ｍｇ＾２＋含量则显著下降（Ｐ     

基于社区的2型糖尿病患者糖尿病视网膜病变相关危险因素

目的:了解本地区社区2型糖尿病患者糖尿病视网膜病变(diabetic retinopathy,DR)的相关危险因素,为社区2型糖尿病并发症的预防和治疗提供理论依据。方法:对本地区常住居民中2型糖尿病的住院患者,进行糖尿病视网膜病变的分期并对相关因素如病程、血压、血糖水平、血脂等进行统计学分析。结果:糖尿病患者176例中,DR者53例,患病率为30.1%,糖尿病病程、糖化血红蛋白是DR发生的危险因素(均为P<0.05)。年龄、收缩压、舒张压、空腹血糖、餐后2h血糖、胆固醇、甘油三脂、高密度脂蛋白、低密度脂蛋白、谷丙转氨酶、谷草转氨酶、尿肌酐、尿素氮未成为DR发生的危险因素(均为P>0.05)。结论:糖尿病的病程、糖化血红蛋白是糖尿病视网膜病变发生的主要危险因素。     

蒲黄提取物对糖尿病视网膜病变大鼠的保护作用

目的:探讨蒲黄提取物对糖尿病视网膜病变(DR)大鼠的保护作用。方法:将50只SPF级大鼠随机分为对照组:正常饲养;DR组:给予等量的生理盐水灌胃;实验A组:蒲黄提取物50mg/(kg·d);实验B组:蒲黄提取物100mg/(kg·d);实验C组:蒲黄提取物200mg/(kg·d)。测定各组大鼠空腹血糖;HE染色观察各组大鼠视网膜病理学变化;ELISA检测各组大鼠血清中IL-6、TNF-α含量;Western blot检测视网膜组织VEGF、VEGFR2和Ang-1蛋白表达水平;q RT-PCR法检测视网膜组织VEGF、VEGFR2和Ang-1 mRNA表达水平。结果:与对照组相比,DR组和各实验组大鼠空腹血糖、血清中IL-6、TNF-α含量及视网膜组织VEGF、VEGFR2和Ang-1的蛋白及mRNA表达水平均显著升高(P<0.05);与DR组相比,各实验组大鼠空腹血糖均有下降,且实验B组和实验C组大鼠空腹血糖显著降低(P<0.05);此外,实验B组和实验C组大鼠血清中IL-6、TNF-α含量、视网膜组织VEGF、VEGFR2和Ang-1的蛋白及mRNA表达水平与DR组相比均降低(P<0.05)。HE结果发现,DR组大鼠视网膜光感受器细胞层结构被明显破坏,细胞出现水肿、间隙增宽;实验B组和实验C组大鼠视网膜组织病理学均有不同程度改善。结论:蒲黄提取物可下调DR大鼠炎症水平及VEGF、VEGFR2和Ang-1的表达,从而改善视网膜组织病变。     

社区2型糖尿病视网膜病变的相关危险因素分析

目的:分析2型糖尿病患者糖尿病视网膜病变(diabetic retinopthy,DR)相关危险因素。方法:2型糖尿病患者300例,根据有无DR及病变程度分为3组:正常视网膜(NDR)组、非增殖型视网膜病变(NPDR)组和增殖型视网膜病变(PDR)组进行临床分析。结果:和NDR组比较,NPDR、PDR组病程、收缩压(SBP)、舒张压(DBP)、空腹血糖(FPG)、2h血糖(2hPG)、糖化血红蛋白(HbA1c)、24h尿蛋白排泄量显著升高,差异有统计学意义(P<0.05)。Logistic回归分析表明:病程和SBP是DR发生的独立危险因素。结论:病程、收缩压(SBP)、舒张压(DBP)、空腹血糖(FPG)、2h血糖(2hPG)、糖化血红蛋白(HbA1c)、24h尿蛋白排泄量等因素与DR的发生有关,其中糖尿病病程及SBP是DR的独立危险因素。     

2型糖尿病发生增生性糖尿病视网膜病变的 危险因素 

目的探讨2型糖尿病发生增生性糖尿病视网膜病变( proliferative diabetic retinopathy，PDR) 的患病率和危险因素。方法对1994~2001年首次在我院糖尿病中心就诊的2型糖尿病患者 2 739例，进行眼并发症筛查中散瞳检查眼底和荧光素眼底血管造影 (fundus fluorescein angiography, FFA) 确定为糖尿病性视网膜病变（diabetic retinopathy, DR）患者的临床资料进行回顾性分析。同期检测患者的血压、空腹和餐后血糖，糖化血红蛋白（HbA1c）、血脂、肌酐和尿白蛋白含量。结果2型糖尿病DR总患病率为27.8%（761/2 739），PDR的患病率为4.2%（114/2 739），占所有DR患者的15%。PDR病变组的病程、空腹血糖、糖化血红蛋白、血压水平和尿白蛋白含量均高于对照组（P＜0.01，糖化血红蛋白低于0.05）。Logistic多元回归分析显示，影响PDR发病的危险因素是病程（r=0.15, P＜0.01）和尿白蛋白含量（r=0.08, P＜0.05）。病程为5年和5年以上的患者，并发PDR的危险因素是尿白蛋白含量和空腹血糖水平（r=0.13, P＜0.05）。进一步分析表明病程为5年以内、5~10年和大于10年的患者，PDR的患病率分别为2.3%、5.9%、12.4%。无蛋白尿、微量蛋白尿和显性蛋白尿组PDR的患病率分别为2.1%、5.3%、18.8%。结论2型糖尿病并发PDR与糖尿病病程、尿白蛋白含量、空腹血糖、糖化血红蛋白和血压水平增高有关。其中病程、尿白蛋白含量和空腹血糖水平是影响PDR发病的危险因素。(中华眼底病杂志,2003,19:338-340)     

基于社区健康管理档案视角下糖尿病视网膜病变危险因素分析

目的：：调查本地社区2型糖尿病患者糖尿病视网膜病变情况,分析2型糖尿病视网膜病变的危险因素,为预防和治疗糖尿病视网膜病变提供参考。方法：随机选取2015-01/03社区健康档案2型糖尿病患者268例资料,通过远程阅片系统进行阅片,回顾性分析糖尿病视网膜病变患者人口学资料与实验室指标。结果：在268例2型糖尿病患者中,检出糖尿病视网膜病变85例(31.7%);糖尿病视网膜病变患者与非糖尿病视网膜病变患者性别、病程、收缩压、空腹血糖、糖化血红蛋白、尿肌酐等比较,差异有统计学意义(P<0.05);多因素Logistic回归分析表明,病程、收缩压、空腹血糖、糖化血红蛋白是糖尿病视网膜病变的独立危险因素(P<0.05)。结论：糖尿病患者长病程、高血压、高血糖是发生糖尿病视网膜病变的主要危险因素,应加强对糖尿病患者血压、血糖指标的监测,预防糖尿病视网膜病变的发生。     

糖尿病大鼠视网膜组织中Na+-K+-ATPase活性变化的研究

近年来,组织细胞膜中ATPase活性异常在糖尿病的慢性并发症中的作用已引起了人们高度重视.为探索影响糖尿病视网膜病变发生发展的有关因素,我们动态地观察了糖尿病大鼠发病后不同时期视网膜组织中Na+ -K+ -ATPase活性的变化. 1 材料与方法 1.1 实验动物成年雄性Wistar大鼠48只,体重210～240 g,适应性饲养7天,禁食12 h后按体重随机分成实验组与对照组,每组24只.实验组腹腔注射链脲佐菌素(65 mg/kg,用0.1mol/L pH 4.5柠檬酸盐缓冲液配制)诱发糖尿病,对照组空腹注射等量柠檬酸盐缓冲液.单笼喂养.于注射链脲佐菌素后的第3天起测空腹血糖,血糖≥16.5 mmol/L且尿糖持续阳性时定为实验组.在糖尿病发病后4,6,12和16周4个时点,每组各取6只动物,禁食12 h后麻醉下尾静脉采血作空腹血糖及糖化血红蛋白检测,并摘取双侧眼球,冰台固定眼球壁取出视网膜组织,精密天平称重,以0.05 mol/L冰冷磷酸缓冲液(pH7.8,含0.1 mmol/L EDTA),冰浴下制成10%组织匀浆.     

糖尿病视网膜病变发生危险因素的病例对照分析

目的探讨糖尿病视网膜病变(DR)发生的相关危险因素。方法收集107例糖尿病视网膜病变(DR)患者和不伴有视网膜病变的糖尿病(NDR)患者102例,分别就其可能诱发DR的危险因素如病程、空腹血糖、糖化血红蛋白、血压、血脂、纤维蛋白原水平和尿白蛋白等因素进行病例对照研究。结果通过单因素方差分析发现,糖尿病患者的病程长、空腹血糖升高、血中糖化血红蛋白升高、高血压病、尿白蛋白升高和血浆纤维蛋白原升高与DR的发病呈显著正相关,血清甘油三酯、胆固醇与DR发病无显显相关关系。结论糖尿病视网膜病变的发生和发展与患者的病程、血糖水平、糖化血红蛋白、合并高血压病、尿蛋白以及血浆纤维蛋白原水平升高有关,预防上述危险因素,可减少DR的发生发展。     

妊娠期糖尿病视网膜病变患者血中糖基化血红蛋白和果糖胺的水平及其临床意义

目的　探讨妊娠期糖尿病视网膜病变的发病与进展因素。方法　随机检测 32例DR孕妇、 32例NDR孕妇和 32例正常孕妇的血糖、糖基化血红蛋白和果糖胺。结果　 (1)妊娠期糖尿病视网膜病变的发病率为 1 4 4 % ;(2 )妊娠期糖尿病视网膜病变的进展率为 31 2 5 % ;(3)妊娠中期 ,DR孕妇组血中糖基化血红蛋白和果糖胺水平明显高于正常孕妇组 (P <0 0 5 ) ;(4)妊娠晚期 ,DR孕妇和NDR孕妇组血中糖基化血红蛋白和果糖胺水平明显高于正常孕妇组 (P <0 0 1)。结论　妊娠会促进糖尿病孕妇的视网膜病变加重 ,可能与血中糖基化血红蛋白和果糖胺水平增高有关。     

Localization of Na+, K+-ATPase and Ca2+-activated Mg2+-dependent ATPase in retinal rods.

Distribution of Na⁺, K⁺-ATPase, K⁺-nitrophenylphosphatase. Ca²⁺-activated, Mg²⁺dependent ATPase (Ca²⁺, Mg²⁺-ATPase) and Mg²⁺-ATPase over subfractions of rod outer segments (ROS) of cattle retina has been studied. These enzymes appear to concentrate in the heavy subfractions which, as indicated by their high succinate dehydrogenase (SDGase) activity, consist of, mainly, rod inner segment (RIS) membraneous fragments. On the contrary, rhodopsin—marker of ROS disc membranes and ROS envelopes—peaks in the light subfractions showing simultaneously extremely low level both ATPase and SDGase activities.The conclusion is made that ROS disc membranes and ROS envelopes lack Na²⁺- and typical Ca²⁺-transporting systems and that both the pumps are located in the RIS zone of a rod photoreceptor.     

Identification of low-Ca2+- and high-Ca2+-requiring neutral proteases in rat retina

Two low-Ca2+-requiring proteases (calpain I) and one high-Ca2+-requiring protease (calpain II) have been separated from the cytosol of rat retina by DEAE-cellulose column chromatography. Calpain I was half-maximally activated at 3 microM free Ca2+ and fully activated at 10 microM free Ca2+. Half-maximal activation of calpain II was at 0.4 mM free Ca2+ while full activation was observed at 0.8 mM free Ca2+. Calpain activity has also been demonstrated in sealed rod outer segments. The soluble fraction of the rod outer segments contained 89% of the enzyme activity. The possible role of calpain in retina is discussed.     

H2O2-modification of Na,K-ATPase. Alterations in external Na+ and K+ stimulation of K+ influx

Studies, at steady state, of the Na,K-ATPase dependent influx of K+ into bovine lenses in organ culture are used to characterize further the H2O2-modification of the Na+ pump. Control lenses display constants for interaction with external Na+ and K+ similar to those obtained for the erythrocyte. H2O2 treatment of the bovine lens leads to total loss of external Na+ stimulation and alteration of external K+ stimulation.     

Analysis of rat lens 45Ca2+ fluxes: evidence for Na(+)-Ca2+ exchange

The influx and efflux kinetics of 45Ca2+ were studied in the rat lens in vitro. Both data sets could be fitted by a multi-compartment mathematical model and were interpreted in terms of extracellular, cytosolic and slowly-exchanging (bound) components. At the end of a 16-hr influx period, when uptake into the extracellular and cytosolic compartments is complete, the 45Ca2+ exchanged fraction is less than 20% of the total calcium determined by atomic absorption. The bound compartment is therefore by far the largest in the lens. The efflux rate constant determined from the model for the cytosolic compartment was approximately 8 x 10(-3) min-1 and its origin was confirmed by its sensitivity to temperature, absence of external sodium and presence of the amiloride-analogue, dichlorobenzamil. A 55% reduction in efflux was obtained in sodium-free solution, indicating that Na(+)-Ca2+ exchange is responsible for a large proportion of calcium movement from the lens against its electrochemical gradient. This was confirmed in influx studies where, reduction of the lens sodium gradient by either exposure to sodium-free medium or 0.1 mM ouabain significantly elevated the 45Ca2+ content of the lens relative to the control level. Exposure to sodium-free conditions also rendered the lens opaque, which did not occur in the absence of external calcium. These experiments suggest a critical role for Na(+)-Ca2+ exchange in maintaining a low internal Ca2+ and hence transparency.     

Partial characterization of a high affinity [Ca2+ + Mg2+]-dependent adenosinetriphosphatase from bovine retina

Examination of retinal tissue homogenates indicated the presence of a [Ca2+ + Mg2+]-dependent adenosinetriphosphatase activity that exhibited high affinity for Ca2+ (K0.5 = 0.17 microM) and moderately high affinity for Mg2+ and ATP (K0.5 = 12.5 microM and Km = 22.8 microM, respectively). Maximum ATP hydrolysis occurred at pH 7.4. Under conditions of optimal substrate, cation and hydrogen ion concentrations, specific activity ranged from 15 to 18 nmol phosphate released min-1 mg-1 protein. Although the retinal [Ca2+ + Mg2+] adenosinetriphosphatase hydrolyzes both ATP and dATP, other nucleotides (CTP, GTP, ITP and UTP) were not hydrolyzed to any great extent. The monovalent cations, Li+, K+ and Na+, had no effect upon hydrolysis of ATP; whereas Cs+ and NH4+ ions were moderately (approximately 30%) inhibitory. All divalent cations tested were stimulatory. With the exception of rotenone which inhibited ATP hydrolysis approximately 25%; retinal adenosinetriphosphatase activity was insensitive to mitochondrial inhibitors (NaN3, KCN, ruthenium red and oligomycin). Adenosinetriphosphatase activity was observed to be very sensitive to low concentrations (I50 approximately 2 microM) of vanadate; whereas, lanthanum administration resulted in no inhibition. Removal of calmodulin (80%) resulted in reducing adenosinetriphosphatase activity 60% but addition of exogenous calmodulin back to calmodulin deficient membranes did not restore activity to starting levels. Calmodulin antagonists trifluoperazine and calmidazolium reduced significantly Ca2+ stimulated, Mg2+ dependent ATP hydrolysis. We conclude that the [Ca2+ + Mg2+]-dependent adenosinetriphosphatase of bovine retina is a non-mitochondrial protein exhibiting very high affinity for Ca2+ and appears to require calmodulin for maximum activity. Because of its high affinity for Ca2+, this protein may play an important role in reducing intracellular Ca2+ to nanomolar levels.     

培养的胚胎视网膜神经细胞Ca2+分布与Ca2+通道特征

目的 检测分析培养的胚胎早期视网膜神经细胞游离钙（Ca^2 )分布与钙（Ca^2 ）通道特征。方法 体外培养11－15周胎儿视网膜神经细胞，在含Ca^2 与不含Ca^2 的Hepes缓冲液中与钙荧光指示剂Fluo3孵育染色，同时加入或不加入异博定、佩尔地平或地塞米松，共聚焦显微镜观察记录游离Ca^2 分布，以及受不同浓度K^ 刺激后Ca^2 通道开放与Ca^2 转移情况。结果 培养的11－15周胎儿视网膜神经元及神经胶质细胞受K^ 刺激后均出现明显的Ca^2 转移与再分布，在缺乏细胞外Ca^2 的情况下，细胞浆内钙库仍可迅速释放Ca^2 并转移至细胞核内。异博定、佩尔地平及地塞米松能够抑制细胞外Ca^2 进入视网膜神经细胞内。结论 胚胎早期视网膜神经元及神经胶质细胞存在L型Ca^2 通道，并已基本发育成熟。     

Ca2+/Na+ exchanger and Na+,K+ 2Cl- cotransporter in lens fiber plasma membrane vesicles.

When the Ca(2+)-sensitive fluorescent probe, Fura-2, or the Na(+)-sensitive probe, SBFI, in their cell permeable forms or the Cl(-)-specific probe, SPQ, were incubated with plasma membrane vesicles prepared from dogfish and bovine lenses fibers, there was a selective accumulation of the ion-specific probes within the vesicles. The SBFI and Fura-2 fluorescent excitation ratios of 340 nm to 380 nm (em: 505 nm) in the presence of an outwardly-directed Na+ gradient across the vesicles membrane, indicate that the influx of Ca2+ is increased by 152.5% and 147.4% for dogfish and bovine vesicles, respectively. The Na+ influx into the vesicles is also enhanced by 154.1% for dogfish and 149.1% for bovine lens when an outwardly-directed Ca2+ was present. This stimulation is not affected when either 50 microM valinomycin, or 50 mM K+ is present. The activity of this bidirectional Ca2+/Na+ exchanger could be inhibited by 100 microM bepridil or 200 microM La3+. The entrance behaviour of Cl- as monitored by the SPQ fluorescent signal indicates that, the Cl- influx is Na(+)-dependent. The Cl- influx is stimulated 152.8% and 187.6% for dogfish and bovine lens, respectively, when an inwardly-directed Na+ gradient is present, and is further enhanced when a K+ gradient is also present. The stoichiometry of Na+ to Cl- entering the vesicles was 1:2. This Na+,K+ 2Cl- cotransporter is not affected by 20 microM valinomycin or 50 mM K+. However, the transporter is completely inhibited by 50 microM furosemide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Ca2+ Signalling in Postnatal Mouse Retinal Ganglion Cells: Involvement of OPA1 in Ca2+ Clearance

Purpose: To identify the genetic defect associated with keratoconus (KC) in an Ashkenazi Jewish family and to evaluate its nature and its phenotypic expression within carriers. Methods: A three generation Ashkenazi Jewish family with KC was ascertained. Diagnosis was based on clinical examination and corneal topography. Segregation analysis was performed using micro-satellite polymorphic markers in close proximity to 7 previously associated KC loci and genes. Mutation analysis of the VSX1 gene was performed by direct sequencing of PCR-amplified exons, and a BseR1 restriction assay. In selected cases, where the genotype was consistent with KC, additional effort to detect subtle corneal changes was made by computerized Orbscan measurements. Results: We found co-segregation between the KC phenotype and a polymorphic marker close to the VSX1. Sequencing revealed a previously described missense mutation (D144E). All of the mutation carriers manifested pathologic corneal findings; some had overt KC while others had subtle corneal alterations identifiable only by Orbscan. Conclusions: These findings support the pathogenic role of VSX1 gene in KC. The variable expression among the carriers, suggests the involvement of other factors in determining the final phenotype.     

Excitation mapping with the organic cation AGB2+

Excitation mapping is a method of visualizing the signaling history of neurons with permeant organic cations. It is compatible with high-resolution imaging, allowing concurrent visualization of all neuronal classes and their glutamate-gated excitation histories. Excitation mapping documents the stability and precision of neuronal signaling within a given neuronal class, arguing that single unit electrophysiological sampling accurately reflects neuronal diversity. We here review the theory of excitation mapping, provide methods and protocol links; outline imaging concepts; provide parametric data on the temporal range and physiological sensitivity of excitation mapping; and show that immunocytochemical methods for macromolecules are compatible with excitation mapping.     

Identification and spatiotemporal characterization of spontaneous Ca2+ sparks and global Ca2+ oscillations in retinal arteriolar smooth muscle cells

PURPOSE: To identify spontaneous Ca(2+) sparks and global Ca(2+) oscillations in microvascular smooth muscle (MVSM) cells within intact retinal arterioles and to characterize their spatiotemporal properties and physiological functions. METHODS: Retinal arterioles were mechanically dispersed from freshly isolated rat retinas and loaded with Fluo-4, a Ca(2+)-sensitive dye. Changes in [Ca(2+)](i) were imaged in MVSM cells in situ by confocal scanning laser microscopy in x-y mode or line-scan mode. RESULTS: The x-y scans revealed discretely localized, spontaneous Ca(2+) events resembling Ca(2+) sparks and more global and prolonged Ca(2+) transients, which sometimes led to cell contraction. In line scans, Ca(2+) sparks were similar to those previously described in other types of smooth muscle, with an amplitude (DeltaF/F(0)) of 0.81 +/- 0.04 (mean +/- SE), full duration at half maximum (FDHM) of 23.62 +/- 1.15 ms, full width at half maximum (FWHM) of 1.25 +/- 0.05 mum, and frequency of 0.56 +/- 0.06 seconds(-1). Approximately 35% of sparks had a prolonged tail (>80 ms), similar to the Ca(2+)"embers" described in skeletal muscle. Sparks often summated to generate global and prolonged Ca(2+) elevations on which Ca(2+) sparks were superimposed. These sparks occurred more frequently (2.86 +/- 025 seconds(-1)) and spread farther across the cell (FWHM = 1.67 +/- 0.08 microm), but were smaller (DeltaF/F(0) = 0.69 +/- 0.04). CONCLUSIONS: Retinal arterioles generate Ca(2+) sparks with characteristics that vary during different phases of the spontaneous Ca(2+)-signaling cycle. Sparks summate to produce sustained Ca(2+) transients associated with contraction and thus may play an important excitatory role in initiating vessel constriction. This deserves further study, not least because Ca(2+) sparks appear to inhibit contraction in many other smooth muscle cells.