摘要
白内障的发病机制较为复杂,与多种因素相关。活性氧簇(reactive oxygen species,ROS),包括过氧化氢(hydrogen peroxide,H_2O_2)、超氧阴离子等都可参与这一致病过程。ROS可以引起很多生物学变化,使晶状体的水不溶性蛋白增加而形成白内障。
晶状体纤维细胞的质膜含有丰富的胆固醇。高胆固醇有利于晶状体膜结构维持于生理状态。胆固醇的消耗可以破坏细胞内的质膜微区—脂筏(lipid raft)。甲基-β-环糊精(methyl-β-cyclodextrin,MβCD)可以与胆固醇结合,从而消耗细胞质膜上的胆固醇。
小窝(caveolae)是一种由非笼型蛋白包被的细胞质膜微区,含有丰富的胆固醇、鞘糖脂和蛋白质。Caveolae存在于多种细胞中,可在细胞质膜上向内凹陷,形成50~100nm大小的囊泡。小窝蛋白(caveolin)是caveolae的标记蛋白。Caveolae及caveolin具有重要的功能,可以参与细胞的物质转运和信号转导过程。哺乳动物细胞的caveolin家族成员包括三种亚型:caveolin-1,caveolin-2和caveolin-3。
一些研究观察到人和动物的晶状体有caveolae及cavcolin的存在;并且通过离心发现在人的白内障晶状体中,缺乏含有膜成分的非沉淀条带。但是目前对于人晶状体上皮细胞(human lens epithelial cells,HLECs)系的caveolae及caveolin的研究甚少,国内尚未见有报道。我们知道H_2O_2所造成的氧化损伤是一种比较成熟的研究白内障发病机制的模型,因此我们选用这一模型来研究氧化损伤和caveolae/caveolin的关系,以进一步探讨白内障的发病机制。
目的:通过H_2O_2刺激HLECs,观察H_2O_2对HLECs增殖活性的影响、caveolin分布与表达的变化,以及caveolin-1的磷酸化;同时用MβCD消耗HLECs的胆固醇后,观察caveolin表达的变化;从而评估caveolin及caveolin-1的磷酸化对于HLECs内的细胞信号转导及胆固醇运输的作用,并探讨caveolin与白内障发生的可能关系。
方法:用不同浓度的H_2O_2及10mM MβCD刺激SRA01/04 HLECs,刺激时间分别为0、5、10、15、30和60分钟。用MTT法观察H_2O_2对HLECs增殖活性的影响。用荧光显微镜和激光共聚焦显微镜观察H_2O_2和MβCD刺激细胞以后,caveolin的分布改变和磷酸化caveolin-1的分布。通过western blot免疫印迹实验观察caveolin表达的变化,并观察磷酸化caveolin-1的表达。用透射电子显微镜观察正常HLECs内caveolae的分布。
结果:0.1mM H_2O_2刺激HLECs时间达10分钟以上时,或1.0mM H_2O_2刺激时间达5分钟以上时,MTT法即发现对细胞的增殖活性有影响。与对照组相比,存在显著性差异(p<0.05,F=11.63;或p<0.05,F=185.984)。1.0mM H_2O_2刺激HLECs 5分钟以后,在激光共聚焦显微镜下,可以观察到细胞浆内caveolin的分布增多。当用10mM MβCD刺激细胞5分钟以后,在荧光显微镜下,观察到细胞形态变小,发生皱缩。通过western blot免疫印迹实验,可以观察到0.1、0.2、0.5和1.0mM H_2O_2刺激HLECs 30分钟以后,与对照组相比,细胞膜蛋白和总蛋白的caveolin表达都下调(p<0.05,F=6.149;或p<0.05,F=14.489)。此外,0.1mM H_2O_2(p<0.05,F=6.843;或p<0.05,F=7.944)和1.0mM H_2O_2(p<0.05,F=6.242;或p<0.05,F=5.457)刺激达到不同时间以后,细胞膜蛋白和总蛋白的caveolin表达也下调。当用10mM MβCD刺激细胞达到不同时间后,细胞膜蛋白和总蛋白的caveolin表达也表现为下调,但是膜蛋白在刺激时间达15分钟以后,与对照组相比,存在统计学显著性意义(p<0.05,F=9.890),而总蛋白没有统计学显著性意义(p>0.05,F=1.480)。用1.0mM H_2O_2刺激HLECs达60分钟时,western blot免疫印迹实验显示,caveolin-1的酪氨酸14位点发生了磷酸化。通过透射电子显微镜,观察到呈Ω样或瓶颈样外观的caveolae的分布,但数量并不丰富。
结论:HLECs对H_2O_2的刺激反应包括细胞增殖活性的降低、细胞内caveolin的重新分布、caveolin表达的下调,以及caveolin-1的磷酸化。当用MβCD消耗细胞的胆固醇后,细胞形态变化,caveolin的表达也下调。以上结果提示我们:HLECs内有caveolae的存在,并有caveolin/caveolin-1的分布;高水平的胆固醇对于维持caveolae的结构和功能是必须的。在H_2O_2的刺激下,caveolin可能参与细胞信号的转导过程,而caveolin-1的磷酸化是其参与H_2O_2诱导的信号转导过程的一个关键因素;氧化损伤可能通过下调caveolin的表达而参与白内障的发生。
The mechanisms of cataract formation are very complicated. Oxidative stress is believed to be an important contributing factor in maturity onset of cataract formation. Reactive oxygen species (ROS), such as hydrogen peroxide (H_2O_2), superoxide anion are postulated to contribute to this process. ROS causes a number of biochemical changes, which lead to an increase in water-insoluble lens proteins and the appearance of lens opacity.
The plasma membrane of the lens is extremely rich in cholesterol. This high level of cholesterol is proposed to preserve lens membrane
structure in the physiological state. Disruption of cholesterol has been shown to destroy subdomains of lipid rafts, and methyl-β-cyclodextrin (MpCD) is known to deplete cholesterol from cell membranes.
Caveolae are non-clathrin-coated plasma membrane microdomains rich in cholesterol and glycosphingolipid. They invaginate to form 50-100 nm vesicles in the plasma membrane that have been found in many cell types. Caveolae contain the signature marker proteins termed caveolins, which have been implicated in vesicular trafficking and signal transduction. The mammalian caveolin family members include three isoforms: caveolin-1, caveolin-2 and caveolin-3.
Some studies showed that caveolin and caveolae exits in lens and lens epithelial cells, and samples from cataract lens were not exhibited nonsedimenting bands which included abundant caveolin after sucrose linear density gradient centrifiigation. But the reports on study of human lens epithelial cells line are very rare, and no paper in China at present. Oxidative stress induced by H_2O_2 is a good model for study on cataract, so we select this model for our study on caveolin and cell stress and to further explore the mechanism of cataract.
Purpose: Oxidative stimulation induced by hydrogen peroxide on human lens epithelial cells (HLECs) was performed to observe the effects on cell proliferation, caveolin expression, caveolin-1 phosphorylation, and cholesterol depletion in HLECs caused by MβCD was also studied. The expression and distribution of caveolin in HLECs under H_2O_2 stimulation and cholesterol depletion were determined to assess the possible roles of caveolin in cell signal transduction and cholesterol trafficking. And the possible relationship between the caveolin and cataract formation are also explored.
Methods: SRA01/04 HLECs were exposed to H_2O_2 or MβCD of various concentrations and durations. We used a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrozolium bromide (MTT) assay to measure the effect of H_2O_2 on the proliferation of SRA01/04 HLECs. The distributions of caveolin after oxidative stimulation were probed by fluorescence microscopy and laser scanning confocal microscopy. Western blot was performed to analyze the alterations of caveolin expression and caveolin-1 phosphorylation. And also, we observed the caveolae distribution in HLECs by electron microscopy.
Results: We observed that the proliferation of SRA01/04 HLECs under 0.1 mM H_2O_2 for 10 min or longer, or 1.0 mM for 5 min or longer was significantly reduced (p<0.05, F=11.63; or p<0.05,F=185.984, respectively). Laser scanning microscopy showed immunofluorescent caveolin in SRA01/04 HLECs under 1.0 mM H_2O_2 for 10 min or longer, caveolin were largely confined to intracellular domains. SRA01/04 HLECs under 10 mM MβCD for 5 min or longer became remarkably smaller in size under fluorescence microscopy. Western blot showed both membrane and total caveolin protein (22 kDa) levels in SRA01/04 HLECs treated with 0.1, 0.2, 0.5 or 1.0 mM H_2O_2 for 30 min were significantly reduced, compared with the untreated (p<0.05, F=6.149; or p<0.05, F=14.489, respectively). In addition, the membrane and total caveolin protein level after treated with 0.1mM (p<0.05, F=6.843; or p<0.05, F=7.944, respectively) and 1.0 mM (p<0.05, F=6.242; p<0.05, F=5.457, respectively) H_2O_2 for different durations also down regulated. Western blot showed that the membrane and total caveolin protein level down regulated when treated with 10mM MβCD. The membrane caveolin under 10mM M(3CD for 15min or longer showed significant down regulation (p<0.05, F=9.890), whereas the total caveolin showed no significant down regulation (p>0.05, F=1.480). Western blot also showed
caveolin-1 was phosphorylated on tyrosine 14 in SRA01/04 HLECs after stimulated with 1.0 mM H_2O_2 for 60 min. Fluorescence microscopy also showed that phosphorylated caveolin-1 was distributed near the focal adhesions of the cells. On electron microscopy, the surface of untreated HLECs exhibited plasma membrane invaginated to form omega- or flask-shaped caveolae in a small amount.
Conclusions: This study concludes that the responses of HLECs to oxidative stress may include the suppression of the proliferation of HLECs, down regulation of caveolin and tyrosine 14 phosphorylation of caveolin-Land MβCD also down regulates caveolin while depleting cholesterol in HLECs. These results show that HLECs contain caveolae and caveolin/caveolin-1. Abundant cholesterols are necessary for maintenance the function of caveolae. Caveolin might involve in the signal transduction of HLECs. And tyrosine 14 phosphorylation of caveolin-1 is a key factor that caveolin participate in the signal transduction. Oxidative stress down regulating caveolin might be one of the many mechanisms, which result in the formation of cataract.
引文
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