Detection of integrins in human cataract lens epithelial cells and two mammalian lens epithelial cell lines

AIM: To compare the incidence of various integrin subunits in human cataract anterior lens epithelial cells (A-LEC) and in two mammalian LEC lines. METHODS: Circular sections of anterior capsules with attached LEC were obtained during cataract surgery. Integrin subunits were immunolocalised in these anterior LEC and in a human and rabbit LEC line, using four monoclonal antibodies specific for subunits alpha2, alpha3, and alpha5, and beta subunit 2. RESULTS: All of these subunits were found in at least a proportion A-LEC samples as follows: alpha2 71%, alpha3 92%, alpha5 62%, and beta2 24%. The human LEC line was immunoreactive for alpha2 and alpha3 only. The rabbit lens epithelial cell line was immunoreactive for alpha5 but there was no staining for alpha2, alpha3, or beta2. CONCLUSION: The A-LEC and mammalian LEC lines showed a similarity in their pattern of integrin expression. As these integrins are receptors for extracellular matrix (ECM) components, they are likely to be associated with the attachment and migration of LECs that precedes capsular opacification. Therefore these cell lines may be useful in the elucidation of mechanisms involved the pathogenesis of capsule opacification.     

体外培养人晶状体上皮细胞整合素的表达

目的:研究体外培养人晶状体上皮细胞整合素的表达。方法:对人晶状体上皮细胞株SRA01/04进行培养并传代,经间接免疫荧光标记法处理细胞后上流式细胞仪检测各整合素的阳性表达率。结果:整合素α2,α3,α5,β1,β2在SRA01/04的阳性表达率分别为85.3%,97.6%,74.0%,97.7%,3.65%。整合素α3与β1之间无统计学差异(P>0.05),其余两两间比较均有显著统计学差异(P<0.01)。结论:人晶状体上皮细胞株SRA01/04整合素呈阳性表达。     

骨桥蛋白在白内障晶状体上皮细胞中的表达

目的：探讨骨桥蛋白（osteopontin，OPN）在白内障晶状体上皮细胞中的表达水平及作用。方法：采用免疫组织化学法检测22例前囊下性白内障、17例核性白内障、14例皮质性白内障患者和11例正常晶状体上皮细胞中OPN的表达，并进行阳性细胞计数和各组间比较。结果：OPN在前囊下白内障患者晶状体上皮细胞中的表达明显增多，其阳性表达率与核性白内障、皮质性白内障及正常人晶状体比较，差异有显著意义（P〈0．01）。结论：OPN在前囊下性白内障晶状体上皮细胞中表达阳性，在前囊下白内障的形成过程中可能起重要作用。     

波形纤维蛋白在老年性白内障晶状体上皮细胞的表达

目的观察波形纤维蛋白在老年性白内障晶状体上皮细胞的变化.方法用卵白素-生物素过氧化物酶法对22例老年性白内障患者晶状体前囊膜上皮细胞进行波形纤维蛋白染色,采用包埋前免疫酶电镜技术处理6个晶状体前囊膜标本,并观察其超微结构;利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳及Westemnblot法分析4个晶状体表层组织(前囊膜、上皮细胞和表层皮质)中的波形纤维蛋白.结果老年性白内障晶状体上皮细胞的波形纤维蛋白表达减弱,与对照组比较差异有显著性(t=2.0948,P<     

人外伤性白内障晶状体上皮细胞的组织块培养

目的建立人外伤性白内障晶状体上皮细胞体外培养的简单有效方法，观察晶状体上皮细胞的体外生长规律和特点。方法应用改良组织块贴附培养法对儿童及成人外伤性白内障的晶状体上皮细胞进行体外培养，倒置显微镜下观察其生长规律。结果儿童和成人外伤性白内障晶状体上皮细胞都具有增生能力，晶状体上皮细胞均可传3代，晶状体上皮细胞多次传代后生长缓慢。结论儿童和成人外伤性白内障晶状体上皮细胞体外培养增生能力有限，儿童晶状体上皮细胞增生能力较强。改良组织块贴附培养法简单易行，重复性好，是晶状体上皮细胞体外培养的较好方法。     

白内障患者晶状体上皮细胞中Fas蛋白的表达

目的:了解不同类型白内障患者晶状体上皮细胞(lens epi-thelial cell,LECs)的凋亡情况及其与Fas蛋白(CD95)的关系。方法:取年龄相关性白内障、糖尿病性白内障及高度近视并发性白内障患者超声乳化手术中取下的前囊膜标本共73例。应用光镜、透射电镜及TUNEL标记法观察LECs的凋亡情况。同时应用免疫组化和流式细胞分别定性、定量检测Fas蛋白(CD95)在LECs中的表达。结果:光镜下3种白内障患者的LECs都呈透明样变性,细胞内有空泡。电镜下可见正常以及变性、坏死的LECs。部分LECs细胞内染色质凝集并边缘化,呈早期细胞凋亡改变。TUNEL检测发现3种白内障患者的LECs中均有TUNEL阳性细胞存在,LECs的凋亡率分别为(32.20±7.91)%、(31.00±9.43)%、(28.20±8.04)%,3种白内障间未见统计学差异。免疫组化结果表明在3种白内障患者的LECs中均有Fas蛋白(CD95)表达。流式细胞定量检测年龄相关性、糖尿病性及高度近视并发性白内障患者LECs中Fas蛋白(CD95)的表达量分别为(65.72±9.95)%、(63.46±13.30)%、(31.46±17.25)%,高度近视并发性白内障LECs中Fas蛋白(CD95)的表达与其他两种白内障间存在统计学差异(t检验,P<0.05)。结论:年龄相关性白内障、糖尿病性白内障及高度近视并发性白内障患者的LECs均出现凋亡,LECs的凋亡很可能是非先天性白内障发病的细胞病理学基础。Fas蛋白(CD95)介导的LECs的凋亡在年龄相关性白内障及糖尿病性白内障的形成中起重要的作用,而在高度近视并发性白内障LECs凋亡中可能存在其他通路。     

老年性和糖尿病性白内障晶状体上皮细胞的超微结构改变

目的 观察正常人,老年性白内障、糖尿病性白内障晶状体上皮细胞的超微结构,探讨细胞凋亡在白内障发生中的作用。方法 收集正常人、老年性白内障、糖尿病性白内障晶状体前囊膜送透射电镜扫描,取老年性白内障和糖尿病性白内障前囊膜各15例,用脱氧核苷酸末端转移酶缺口标记原位细胞检测法,检测晶状体上皮的凋亡细胞。结果 老年性和糖尿病性白内障晶状体上皮细胞的超微结构在形态上为扁平状,胞浆出现空泡变性,胞核出现固缩,染色质边聚,浓缩等改变。糖尿病性白内障晶状体上皮细胞的胞浆空泡变性更加严重。在光镜下老年性和糖尿病性白内障的晶状体上皮均有凋亡细胞。结论 老年性白内障和糖尿病性白内障的发生与晶状体上皮的细胞凋亡密切相关。     

大鼠半乳糖性白内障晶状体上皮细胞的病理变化

目的　探讨半乳糖性白内障大鼠晶状体上皮细胞的改变。方法　２４只ＳＤ雌性大鼠,分为正常对照组和白内障组,每组１２只。白内障组大鼠用半乳糖饲料喂养,正常对照组大鼠用普通颗粒饲料喂养。裂隙灯显微镜下观察大鼠晶状体混浊程度变化,观察至３０ｄ处死大鼠后取晶状体,在光镜和电镜下观察晶状体病理组织和超微结构改变。结果　观察至３０ｄ时,正常对照组大鼠晶状体保持透明,白内障组大鼠９眼（３７．５％）出现均一的皮质性混浊,１５眼（６２．５％）出现核混浊。白内障组光镜下可见晶状体皮质和核部大量纤维细胞水肿、崩解,前囊膜下及后囊膜下出现纤维细胞样的有核细胞堆积;透射电镜下可见晶状体上皮细胞变性、增生并突破晶状体上皮层向浅层皮质移行。结论　半乳糖性白内障不仅有晶状体纤维细胞水肿及结构破坏,还存在晶状体上皮细胞的异常增生、分化和移行。     

年龄相关性白内障晶状体上皮细胞的超微结构研究

目的:研究年龄相关性白内障晶状体上皮的超微结构变化。方法:用日本产HU-12A型电镜对8例年龄相关性白内障及3例正常透明晶状体上皮细胞的超微结构进行观察。结果:7例年龄相关性白内障晶状体前囊上皮细胞形状不规则,相邻细胞间以许多复杂的指状突超紧密镶嵌,部分上皮细胞内水肿,细胞间隙亦水肿呈较大的空泡状,细胞内线粒体等细胞器肿胀,个别区域上皮细胞坏死脱落,1例年龄相关性白内障晶状体前囊上皮细胞体积固缩,细胞核染色质分裂并出现凝聚的凋亡小体。结论:晶状体上皮细胞超微结构的病理形态学改变是年龄相关性白内障发生发展的重要原因之一。     

Human lens epithelial layer in cortical cataract

Normal and cataractous human eye lenses were studied by morphology and protein analysis. A marked decrease in protein sulfhydryl (PSH) and nonprotein sulfhydryl (NSPH) was observed in nuclear and cortical cataractous epithelia. Moreover, decrease in PSH contents and an increase in insoluble proteins were found to be correlated only in cortical cataractous epithelium which is also accompanied by various morphological abnormalities. In nuclear cataractous epithelium, however, there was very little insolubilisation of proteins. The epithelial morphology in nuclear cataracts was almost similar to normal lens epithelium. Hence, it is assumed that the protein insolubilisation and various morphological abnormalities are characteristics of cortical cataractous epithelium. This leads us to believe that opacification in cortical cataract might initiate in the epithelial layer.     

葛根素对糖尿病性白内障大鼠晶状体上皮细胞的保护作用

目的 通过葛根素全身与局部用药治疗糖尿病大鼠白内障疗效的比较,探讨葛根素的最佳用药途径.方法 实验研究.将192只健康无热源SD雄性大鼠按随机数字表法均分为对照组、链脲佐菌素(STZ)组、腹腔注射组及球旁注射组.采用STZ制作大鼠糖尿病模型.对照组与STZ组大鼠腹腔注射生理盐水,腹腔注射组和球旁注射组分别注射葛根素.分别于注射后20、40、60 d检查大鼠的全身情况和晶状体形态;光镜下观察晶状体上皮细胞(LEC)的形态;同时采用生物化学方法检测一氧化氮(NO)含量与一氧化氮合酶(NOS)活性;采用免疫印迹法及反转录聚合酶链反应法检测iNOS蛋白与iNOS mRNA在晶状体中的表达;应用透射电镜和扫描电镜观察LEC的亚细胞结构变化.数据采用两因素析因设计方差分析.结果 注射后20、40、60 d,对照组大鼠晶状体均透明,STZ组大部分晶状体核及核周皮质出现混浊,部分晶状体完全混浊;腹腔注射组和球旁注射组大鼠晶状体仅轻度混浊;光镜下LEC形态对照组无改变,STZ组细胞体积缩小,胞核浓缩,赤道部皮质出现大量特征性空泡性改变;而60 d腹腔注射和球旁注射组大鼠LEC形态为STZ组20 d时的改变;晶状体NO与NOS含量,iNOS蛋白与iNOS mRNA的表达对照组一直处于极低水平,未见表达及上调;随实验时间延长,STZ组NO2-/NO3-含量逐渐增多,NOS活性、iNOS蛋白与iNOS mRNA的表达与上调逐渐增强,在20、40及60 d均明显高于对照组(F=684.09,P＜0.01),腹腔注射及球旁注射组以上指标在各个时限点均明显低于STZ组,但仍高于对照组(F=1938.62,P＜0.05).透射电镜和扫描电镜观察,对照组大鼠LEC未见异常,STZ组LEC呈异常改变且病变逐渐加重.线粒体肿胀变性,粗面内质网结构不清,部分核固缩且大小不一,核周间隙加大(F值分别等于14 590.65和177.85,P＜0.001).晶状体纤维间窝球状结构逐渐消失,膜破裂脱落堆积形成球样小体;腹腔注射组和球旁注射组大鼠LEC细胞核与晶状体纤维正常,但可见少许线粒体肿胀、内质网扩张及细胞间隙增宽.结论 葛根素腹腔注射与球旁注射两种给药途径对糖尿病大鼠LEC均有保护作用并且疗效相当.

Abstract：

Objective The therapeutic effects of general and local applications of puerarin in the treatment of streptozotocin (STZ)-induced rat diabetic model were compared. Methods Experimental research. We equally divided normal Sprague-Dawley (SD) rats into a STZ group, a peritoneal injection group, a peribulbar injection group and a control group. STZ, peritoneal injection and peribulbar injection groups were first treated with STZ. Subsequendy, the STZ group was injected with normal saline intraperitoneally, while in the later two groups puerarin was injected through peritoneal and peribular routes,respectively. Control group only received peritoneal injection of saline. The morphology of lens epithelial cells (LEC) and their subcellular structure were examined by bright-field microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) 20, 40 and 60 days after the injection.Nitrogen oxide (NO) and nitric oxide synthase (NOS) were measured by biochemistry methods. Finally,inducible nitric oxide synthase (iNOS) protein and mRNA levels were monitored by Western blot and RTPCR, respectively. Data was processed with two factorial experiment analysis of variance. Results Twenty to sixty days after the injection, marked or complete lens opacities appeared in the STZ group, whereas only slight opacities appeared in the lens in peritoneal and peribulbar puerarin groups and the lens in the control group remained clear. At the 20th, 40th and 60th day after the injection, optical microscope detected pathological changes of LEC in the STZ group. The cell volume was decreased with a dense nucleus and many bubbles appeared around the equator ares. Under TEM, enlargement of cell gap, vacuoles in the cytoplasm, swelling of mitochondria and unclear structure of rough endoplasmic reticulum appeared in the LEC of the STZ group. Part of the nucleus was in karyopyknosis and peripheral nucleus gap was enlargement. Under SEM, normal fiber conjunction structure of the lens disappeared, fibers were swelling,part of fiber membranes were discontinuous, detached, and accumulated in certain areas. Mild lens opacities detected by bright-field microscope were developed in peritoneal and peribulbar puerarin injection groups.Nucleus and fibers in the lens cells of both groups appeared to be normal, with minor swelling of mitochondria, minor enlargement of endoplasmic reticulum and slight increase of intracellular space. NO,NOS and iNOS protein and mRNA of the lens were increased and up-regulated in STZ group. In the other two groups only minor changes were present and the changes were significantly less than that of the STZ group but greater than that in the control group. Conclusion Peritoneal and peribulbar injection of puerarin have similar therapeutic effects in the treatment of rat diabetic cataract.     

葛根素对糖尿病性白内障大鼠晶状体上皮细胞的保护作用

目的 通过葛根素全身与局部用药治疗糖尿病大鼠白内障疗效的比较,探讨葛根素的最佳用药途径.方法 实验研究.将192只健康无热源SD雄性大鼠按随机数字表法均分为对照组、链脲佐菌素(STZ)组、腹腔注射组及球旁注射组.采用STZ制作大鼠糖尿病模型.对照组与STZ组大鼠腹腔注射生理盐水,腹腔注射组和球旁注射组分别注射葛根素.分别于注射后20、40、60 d检查大鼠的全身情况和晶状体形态;光镜下观察晶状体上皮细胞(LEC)的形态;同时采用生物化学方法检测一氧化氮(NO)含量与一氧化氮合酶(NOS)活性;采用免疫印迹法及反转录聚合酶链反应法检测iNOS蛋白与iNOS mRNA在晶状体中的表达;应用透射电镜和扫描电镜观察LEC的亚细胞结构变化.数据采用两因素析因设计方差分析.结果 注射后20、40、60 d,对照组大鼠晶状体均透明,STZ组大部分晶状体核及核周皮质出现混浊,部分晶状体完全混浊;腹腔注射组和球旁注射组大鼠晶状体仅轻度混浊;光镜下LEC形态对照组无改变,STZ组细胞体积缩小,胞核浓缩,赤道部皮质出现大量特征性空泡性改变;而60 d腹腔注射和球旁注射组大鼠LEC形态为STZ组20 d时的改变;晶状体NO与NOS含量,iNOS蛋白与iNOS mRNA的表达对照组一直处于极低水平,未见表达及上调;随实验时间延长,STZ组NO2-/NO3-含量逐渐增多,NOS活性、iNOS蛋白与iNOS mRNA的表达与上调逐渐增强,在20、40及60 d均明显高于对照组(F=684.09,P＜0.01),腹腔注射及球旁注射组以上指标在各个时限点均明显低于STZ组,但仍高于对照组(F=1938.62,P＜0.05).透射电镜和扫描电镜观察,对照组大鼠LEC未见异常,STZ组LEC呈异常改变且病变逐渐加重.线粒体肿胀变性,粗面内质网结构不清,部分核固缩且大小不一,核周间隙加大(F值分别等于14 590.65和177.85,P＜0.001).晶状体纤维间窝球状结构逐渐消失,膜破裂脱落堆积形成球样小体;腹腔注射组和球旁注射组大鼠LEC细胞核与晶状体纤维正常,但可见少许线粒体肿胀、内质网扩张及细胞间隙增宽.结论 葛根素腹腔注射与球旁注射两种给药途径对糖尿病大鼠LEC均有保护作用并且疗效相当.     

白内障病人晶状体上皮细胞DNA损伤初探

目的欲研究白内障病人晶状体上皮细胞是否存在DNA损伤及损伤的程度.方法用单细胞电泳测定白内障手术中取下的白内障病人晶状体上皮细胞DNA损伤的情况.结果11例白内障病人晶状体上皮细胞中有6例确实存在DNA损伤,占54.54%.白内障病人晶状体上皮细胞中DNA损伤程度与年龄无关.每例白内障病人晶状体上皮细胞中DNA损伤程度的分布是轻＞中＞重.结论白内障的发生与晶状体上皮细胞DNA损伤有关,但只是众多诱因之一.     

葛根素对糖尿病性白内障大鼠晶状体上皮细胞的保护作用

目的 通过葛根素全身与局部用药治疗糖尿病大鼠白内障疗效的比较,探讨葛根素的最佳用药途径.方法 实验研究.将192只健康无热源SD雄性大鼠按随机数字表法均分为对照组、链脲佐菌素(STZ)组、腹腔注射组及球旁注射组.采用STZ制作大鼠糖尿病模型.对照组与STZ组大鼠腹腔注射生理盐水,腹腔注射组和球旁注射组分别注射葛根素.分别于注射后20、40、60 d检查大鼠的全身情况和晶状体形态;光镜下观察晶状体上皮细胞(LEC)的形态;同时采用生物化学方法检测一氧化氮(NO)含量与一氧化氮合酶(NOS)活性;采用免疫印迹法及反转录聚合酶链反应法检测iNOS蛋白与iNOS mRNA在晶状体中的表达;应用透射电镜和扫描电镜观察LEC的亚细胞结构变化.数据采用两因素析因设计方差分析.结果 注射后20、40、60 d,对照组大鼠晶状体均透明,STZ组大部分晶状体核及核周皮质出现混浊,部分晶状体完全混浊;腹腔注射组和球旁注射组大鼠晶状体仅轻度混浊;光镜下LEC形态对照组无改变,STZ组细胞体积缩小,胞核浓缩,赤道部皮质出现大量特征性空泡性改变;而60 d腹腔注射和球旁注射组大鼠LEC形态为STZ组20 d时的改变;晶状体NO与NOS含量,iNOS蛋白与iNOS mRNA的表达对照组一直处于极低水平,未见表达及上调;随实验时间延长,STZ组NO2-/NO3-含量逐渐增多,NOS活性、iNOS蛋白与iNOS mRNA的表达与上调逐渐增强,在20、40及60 d均明显高于对照组(F=684.09,P＜0.01),腹腔注射及球旁注射组以上指标在各个时限点均明显低于STZ组,但仍高于对照组(F=1938.62,P＜0.05).透射电镜和扫描电镜观察,对照组大鼠LEC未见异常,STZ组LEC呈异常改变且病变逐渐加重.线粒体肿胀变性,粗面内质网结构不清,部分核固缩且大小不一,核周间隙加大(F值分别等于14 590.65和177.85,P＜0.001).晶状体纤维间窝球状结构逐渐消失,膜破裂脱落堆积形成球样小体;腹腔注射组和球旁注射组大鼠LEC细胞核与晶状体纤维正常,但可见少许线粒体肿胀、内质网扩张及细胞间隙增宽.结论 葛根素腹腔注射与球旁注射两种给药途径对糖尿病大鼠LEC均有保护作用并且疗效相当.     

氧化性白内障中晶状体上皮细胞内Ca2+浓度的变化

目的 通过测定过氧化氢诱发的白内障中晶状体上皮细胞内Ca^2 浓度的变化，探讨Ca^2 浓度的改变在氧化性白内障发生机制中的作用。方法 用过氧化氢诱发实验组离体培养的大鼠晶状体产生白内障，用Fura-2／AM荧光标记法测定晶状体上皮细胞胞浆内Ca^2 的浓度，并与对照组正常晶状体相比。结果 经过氧化氢作用的实验组，6、12和24h时，晶状体上皮细胞胞浆内Ca^2 浓度与对照组相比明显升高，差异有显著性(P=0．001，0．000，0．000)。结论 本研究证实过氧化氢作用于大鼠晶状体后，其上皮细胞内Ca^2 浓度明显升高，从而为Ca^2 依赖的蛋白水解酶如钙蛋白酶等的激活创造必要条件。     

老年性白内障晶状体上皮细胞超微结构观察

目的 研究老年性白内障晶状体上皮细胞的超微结构,探讨老年性白内障与晶状体上皮细胞凋亡关系。方法 用透射电镜对20例老年白内障及10例正常透明晶状体上皮细胞的超微结构进行观察,拍照。结果 老年白内障组晶状体上皮细胞中发现了典型的凋亡小体,正常对照组未见凋亡细胞。结论 老年性白内障的发生与晶体上皮细胞的凋亡有关。     

老年性白内障晶狀體上皮細胞超微結構觀察

目的研究老年性白内障晶狀體上皮細胞的超微結構,探討老年性白内障舆晶狀體上皮細胞凋亡關系.方法用透射電鏡對20例老年白内障及10例正常透明晶狀體上皮細胞的超微結構進行觀察,拍照.結果老年白内障組晶狀體上皮細胞中發現了典型的凋亡小體,正常對照組未見凋亡細胞.結論老年性白内障的發生舆晶體上皮細胞的凋亡有關.