胚胎与成人视网膜神经细胞培养

目的 建立胚胎及成人视网膜神经细胞体外培养系统，为视网膜神经细胞的基础研究与药物开发提供实验模型。 方法 10～13周龄胎儿及20～40岁成人视网膜神经层用不同的酶进行消化，分散的细胞接种于预先经过包被的细胞培养盘内，加入或不加入表皮生长因子(epidermal growth factor, EGF)、成纤维细胞生长因子(fibroblast growth factor, FGF)、脑源性神经营养因子(brainderived neurotrophic factor, BDNF)、神经营养素 4(neurotrophic-4，NT-4)等培养。通过BrdU掺入、免疫组织化学及免疫荧光等方法检测培养细胞增殖并辨别细胞成分。 结果 胚胎及成人视网膜细胞可在体外连续培养100及180 d以上。加入EGF、FGF、BDNF或NT-4可明显促进胚胎与成人视网膜神经元存活，胎儿视网膜细胞增殖。与对照组相比，处理组神经元或神经节细胞的百分率较高。 结论 胚胎及成人视网膜神经细胞培养技术为视网膜神经细胞基础研究及药物开发提供了一种十分有价值的手段，加入外源性的生长因子可促进培养的视网膜神经细胞存活、增殖与分化。 (中华眼底病杂志, 2002, 18: 279-282)     

视网膜Muller细胞在视网膜病变中的作用和研究现状

视网膜Muller细胞是视网膜内最主要的神经胶质细胞，它贯穿整个视网膜，包绕与联系视网膜上的各类神经细胞，从而与视网膜神经细胞发生多种功能的交互作用。Muller细胞不仅起着支持和营养神经元的作用。还包括维持细胞外离子的稳定及参与谷氨酸盐循环，突触传递等作用。近年来。随着对视网膜Muller细胞的深入研究，发现Muller细胞还参与多种病理和生理过程，并且在视网膜的各种疾病中都发现伴有Muller细胞的神经胶质增生反应。对视网膜Muller细胞的形态和生理功能作了描述和分析，并对在各种病理状态下Muller细胞发生的改变和目前对Muller细胞的研究现状作一综述。     

视网膜细胞培养上清液对胚胎干细胞定向分化的神经样细胞钠通道动力学的影响

目的探讨不同视网膜细胞上清液对由胚胎干细胞诱导的神经细胞生理机能的影响。方法对拟胚体分别使用视黄酸（A组）、视黄酸+鼠视网膜胶质细胞和神经细胞培养上清液（B组）、视黄酸+人胎儿视网膜胶质细胞培养上清液（C组）进行次级诱导。在诱导后5~21 d，对各组中的细胞分别行细胞膜上的钠离子通道分析。结果诱导后5~21 d时，各组细胞钠电流变化均不明显。A组细胞钠通道呈爆发状的放电现象；B组呈现短促而频繁的状态；C组开放时程较长。三组细胞中，无电流细胞的比例分别为25%、11.4%和23.8%，但组间差异无统计学意义(P＞0.05)。A组Na+电流细胞数目表现为先升高后下降，B组表现为一直上升，C组表现为先下降后趋于平稳。A组细胞通道的开放时间最长，B组最短。三组的开放时间分布均可使用双指数拟和。结论在胚胎干细胞向神经细胞诱导中，视网膜细胞上清液对诱导细胞的生理功能会产生明显影响。(中华眼底病杂志，2007，23：91-93）     

人视网膜前体细胞的体外视网膜组织片下移植

目的研究人视网膜前体细胞移植到体外培养的人视网膜组织片下的细胞分化。方法取无眼部发育异常的4～5个月胚胎眼球，进行视网膜前体细胞分离培养。将传代的细胞移植到体外培养的视网膜神经上皮组织片下，通过光学显微镜和免疫组织化学观察细胞分化和组织整合情况。结果人视网膜前体细胞在体外培养时形成神经球样细胞团，传代后形成子代细胞团，表达神经干细胞标志Nestin。体外培养的视网膜组织片在5d、10d均能基本维持视网膜结构。移植到视网膜组织片下的视网膜前体细胞能够与其建立细胞连接。这些视网膜前体细胞分化后能够表达胶质纤维酸性蛋白、微管相关蛋白-2和视紫红质，分别为神经胶质细胞、神经元和光感受器细胞的特异蛋白。结论人视网膜前体细胞具有神经干细胞特征，在体外移植到培养的人视网膜组织片下，能够分化成相应的终末分化细胞。     

视网膜母细胞瘤的免疫组织化学特性及其分化潜势

应用６种单克隆和多克隆抗体对２７例视网膜母细胞瘤眼球石蜡切片用ＡＢＣ技术进行免疫组织化学研究。结果发现神经胶质细胞性标记物：神经胶质纤维酸性蛋白，白细胞抗原－７；可探测到视网膜固有成分及肿瘤内血管周围反应性神经胶质细胞成分。仅有４种肿瘤内随机分布的神经胶质细胞呈阳性反应。神经元性标记物：神经元特异性烯醇化酶，在多数病例（２１／２７）与不分化的肿瘤细胞呈免疫活性反应。用一种光感受细胞性标记物：视紫…     

体外无血清培养新生SD大鼠视网膜神经细胞

目的 采用无血清培养建立新生SD大鼠视网膜神经细胞的体外培养方法,为视网膜神经细胞的体外实验研究奠定基础.方法 取出生后3～5d的SD大鼠视网膜,用胰蛋白酶消化分离获取细胞悬液,使用无血清神经原培养基(Neurobasal TM-A Medium)和添加剂B-27 Supplement Minus AO进行培养.倒置相差显微镜观察细胞形态及轴突生长情况.培养至第5天,用抗微管相关蛋白2抗体、抗视紫红质蛋白抗体及抗胶质纤维酸性蛋白抗体,行免疫细胞化学鉴定并计算视网膜视杆细胞的纯度.结果 采用无血清法培养的视网膜神经细胞生长良好,细胞伸出突起,部分神经元的突起交织呈网状.生长至第5天经免疫细胞化学证实其中神经元细胞占95.6％,而38.6％为视网膜视杆细胞,同时神经胶质细胞的生长得到了很好地抑制.结论 无血清培养法可以获取纯度较高的视网膜神经细胞,为研究视网膜光感受器细胞提供了理想的体外实验模型.     

视网膜Müller细胞在视网膜病变中的作用和研究现状

视网膜Müller细胞是视网膜内最主要的神经胶质细胞,它贯穿整个视网膜,包绕与联系视网膜上的各类神经细胞,从而与视网膜神经细胞发生多种功能的交互作用.Müller细胞不仅起着支持和营养神经元的作用,还包括维持细胞外离子的稳定及参与谷氨酸盐循环、突触传递等作用.近年来,随着对视网膜Müller细胞的深入研究,发现Müller细胞还参与多种病理和生理过程,并且在视网膜的各种疾病中都发现伴有Müller细胞的神经胶质增生反应.对视网膜Müller细胞的形态和生理功能作了描述和分析,并对在各种病理状况下Müller细胞发生的改变和目前对Müller细胞的研究现状作一综述.     

培养鼠视网膜神经细胞的腺苷/腺苷酸受体研究

目的 检测体外培养鼠视网膜神经细胞上腺苷/腺苷酸受体的存在，方法 先进行兔视网膜Mueller细胞培养，而后依此细胞作基质培养鼠视网膜神经细胞，应用自由钙指示剂Fura-2AM及ARGUS-100系统，监测不同条件下细胞内自由钙的变化，从而确定腺苷/腺苷酸受体的存在。结果 1.0μmol/LATP可引起多量神经细胞内自由钙的变化，A2，P2x,P2y受体激动剂在不同浓度下也各自具有类似的作用。此作用不能被电压依赖性钙通道阻滞剂所抑制。结论 培养的视网膜神经细胞上具有A2，P2x,P2y的受体，表明ATP及去磷酸代谢物可能在视网膜功能活动中具有重要的神经介质功能。     

Müller细胞反应性胶质化在视网膜病变中作用

Müller细胞是脊椎动物视网膜内最主要的神经胶质细胞.它贯穿整个视网膜,与视网膜神经细胞及视网膜血管发生多种功能的交互作用,在视网膜神经递质调节、钾离子调节和pH值调节中都具有重要作用.Müller细胞受到各种急性或慢性病理因素刺激时被激活而发生不同程度活化的反应性胶质化对多种视网膜病变或病理状态均有影响.研究Müller细胞对于理解视网膜功能以及视网膜疾病的发生发展过程具有重要意义.     

APP17肽对糖尿病视网膜神经元表达NT-3和星形胶质细胞增殖的影响

目的　观察糖尿病早期视网膜神经元表达 NT- 3和星形胶质细胞增殖情况 ,并观察 APP17肽的作用。方法　用链脲佐菌素 (streptozotocin,STZ)复制 Wistar大鼠糖尿病模型 ,于模型建立 2周后 ,皮下注射 APP17肽 ,1月后处死大鼠取视网膜 ,进行神经免疫组化 ,观察 APP17肽对糖尿病早期视网膜神经元中神经营养因子 3(neurotrophin- 3,NT- 3)和星形胶质细胞中胶质原纤维酸性蛋白 (glial fibrillary acidi protein,GFAP)表达的影响。结果　同正常对照组相比模型组 (DM组 )视网膜神经元 NT- 3阳性细胞数明显减少 ,但APP17+DM组阳性细胞数多于 DM组 (P<0 .0 1) ,并且对照组与 APP17+DM组中星形胶质细胞 GFAP阳性数少于 DM组 (P<0 .0 1)。结论　在糖尿病视网膜病变早期存在视网膜神经细胞 NT- 3表达减少及星形胶质细胞增生 ,APP17肽可明显改善早期糖尿病大鼠视网膜神经元的表达 ,提示对神经视网膜具有保护作用     

A theoretical study on the regulation mechanism of Ca2+ concentration in lens related to the Ca2+ dependence of Ca2(+)-ATPase activity

A four-state model for the elementary process of the Ca2+ active transport system in lens was proposed, and based upon this model the Ca2+ dependence of Ca2(+)-ATPase activity was analyzed. The results indicated that the Ca2(+)-ATPase activity reaches a peak at approximately pCa 4-5, and decreases at higher and lower Ca2+ concentrations. In the range of pCa 5-6, ATPase activity increases with Ca2+ concentration. Therefore, the model accounts for the kinetic properties of Ca2(+)-ATPase is a qualitative way. Moreover, it is proved that the Ca2+ dependence of Ca2(+)-ATPase activity is theoretically based upon the changes in Ca2+ affinity of Ca2+ binding sites and the Ca2+ dependence of Ca2(+)-ATPase activity is possibly involved in the regulation of intracellular Ca2+ concentration in vivo as a switching mechanism.     

糖尿病大鼠视网膜组织中Ca2+-Mg2+-ATPase活性变化的实验研究

目的:观察糖尿病大鼠视网膜组织中钙镁离子三磷酸腺苷酶(Ca2 -Mg2 -ATPase)活性的变化,探讨糖尿病视网膜病变发生发展的有关因素。方法:用孔雀绿比色分析法对糖尿病大鼠发病后不同时期视网膜组织中Ca2 -Mg2 -ATPase活性进行了检测,并与空腹血糖水平、糖基化血红蛋白含量、血脂、视网膜组织中Ca2 和Mg2 含量及糖尿病病程的关系进行了分析。结果:糖尿病大鼠视网膜组织中Ca2 -Mg2 -ATPase活性明显下降,Mg2 含量下降,Ca2 含量增高。Ca2 -Mg2 -ATPase活性与空腹血糖水平、糖基化血红蛋白含量、视网膜组织中Ca2 和Mg2 含量及糖尿病病程密切相关。结论:糖尿病大鼠视网膜组织中Ca2 -Mg2 -ATPase活性下降可能是其糖尿病视网膜病变的病理基础之一。     

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.     

Activator of Ca2+-transport in the lens

It has been hypothesized that an activator of Ca-ATPase co-exists with Ca-ATPase in the mouse lens. Though the Ca-ATPase activity could be measured from mouse lens homogenate, its activity could not be determined in individual soluble and insoluble fractions. The Ca-ATPase activity, however, could be detected when an activator of this enzyme such as calmodulin was added to the lens insoluble fraction. This enzyme in the lens insoluble fraction was activated also by addition of soluble fraction. The Ca-ATPase in homogenate was inhibited by chlorpromazine and N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which are calmodulin antagonists. When the mouse lens was incubated with W-7, the degree of the lens opacification increased in relation to W-7 concentration.     

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.     

Ca2+-ATPase activity in the human lens

A membrane-rich preparation of paired human lenses was prepared in such a manner as to preserve ATPase activity. The lipid:protein ratio of these preparations was increased 12-fold with an 85% recovery of total phospholipid. The pattern of stimulation of ATPase activity by a range of calcium concentrations was found to be similar in membrane preparations of epithelium and cortex. The concentration of calcium necessary for half-maximal simulations of ATPase activity was approximately 10(-6) M. Ca2+-ATPase activity is undetectable in the lens nuclear region. A shift in the sensitivity of lens epithelial Ca2+-ATPase activity was observed with increasing age concomitant with a general increase in Ca2+-ATPase activity suggesting age related modifications of the membrane.     

蓝光所致人视网膜色素上皮细胞损伤与细胞内钙离子含量变化的关系

背景 对蓝光照射所致视网膜色素上皮(RPE)细胞损伤与细胞内钙离子含量变化关系的深入研究可能对探讨视网膜变性类疾病的发生机制及防治具有重要意义. 目的 建立人RPE细胞蓝光损伤模型,探讨RPE细胞损伤与细胞内钙离子含量变化的关系.方法 将人RPE细胞系培养传代,锥虫蓝染色评估细胞活力.第4代人RPE细胞分别用(2000±500) lx蓝光照射3、6、9、12h,终止细胞培养24 h后,采用TUNEL法检测不同时间点细胞的凋亡情况,确定最适宜的光照度.然后将RPE细胞分为无光照组、光照组、硝苯地平组、光照+硝苯地平组、(-)BayK8644组、光照+(-)BayK8644组.除无光照组外,其他各组细胞均用蓝光照射6h,终止细胞培养24h,采用激光扫描共焦显微镜检测细胞内游离钙离子含量,激发光波长为488 nm,发射光波长为505 nm,每组随机选取30个细胞,扫描细胞图像,采用分析软件分析各组细胞内游离钙离子的荧光强度.结果 锥虫蓝染色证实RPE细胞的活力均在90％以上,TUNEL染色和免疫组织化学染色证实光照3h组未见细胞凋亡,而光照后6、9、12h发现不同数量的凋亡细胞.硝苯地平组RPE细胞内游离钙离子荧光强度比无光照组和光照+硝苯地平组低,差异均有统计学意义(均P=0.000);光照组、(-)BayK8644组、光照+(-)BayK8644组RPE细胞内钙离子荧光强度高于无光照组,差异均有统计学意义(均P=0.000);光照+硝苯地平组与无光照组相比较,(-)BayK8644组与光照+(-)BayK8644组比较,差异均无统计学意义(P=0.339、0.410). 结论 光照度(2000±500)1x、光照6h、终止培养24 h为建立蓝光照射致人RPE细胞损伤模型的最适合条件.蓝光照射所致的人RPE细胞损伤与细胞内游离钙离子含量增加有关.