水通道蛋白-1在角膜内皮细胞液体转运中的作用研究

目的 研究水通道蛋白-1在牛角膜内皮细胞中的表达及在角膜内皮液体转运中的作用.方法 用免疫组织化学方法,检测水通道蛋白-1在培养的牛角膜内皮细胞中的表达,并用水通道蛋白阻滞剂对氯汞苯磺酸酯（pCMBS）处理培养的牛角膜内皮细胞,观察处理前后角膜内皮细胞的渗透性水通透率（Pf）改变.结果 培养的牛角膜内皮细胞中有水通道蛋白-1的表达,主要位于细胞膜上.汞剂处理前后的角膜内皮细胞Pf分别为（0.044±0.005）cm/s（n=15）和（0.017±0.003）cm/s（n=15）,其差异有统计学意义（P＜0.01）.结论 水通道蛋白-1在牛角膜内皮细胞液体转运中起着重要作用,其表达的改变能够导致角膜水肿和功能改变.     

水通道蛋白-1在培养的牛角膜内皮细胞中的表达研究

目的研究水通道蛋白-1在培养的牛角膜内皮细胞中的表达情况及意义。方法用免疫组织化学方法,检测水通道蛋白-1在培养的牛角膜内皮细胞中的表达。结果培养的牛角膜内皮细胞中有水通道蛋白-1的表达,且主要位于细胞膜上。结论牛角膜内皮细胞中的水通道蛋白-1在角膜内皮液体转运中起着重要作用,其表达改变可能导致角膜水肿和功能改变。     

乙酰唑胺对培养的牛角膜内皮细胞液体转运功能的影响

目的 研究乙酰唑胺对培养的牛角膜内皮细胞液体转运功能的影响．方法 观察并记录给予不同浓度乙酰唑胺（0．1μmol／L，1μmol／L，10μmol／L，100μmol／L）角膜内皮细胞在低渗溶液中的体积改变．并计算其渗透水通透性（osmoilc water permeability，Pf）的变化。结果 培养的正常牛角膜内皮细胞的Pf值为（0．0446±0．0089）cm／s，在0．1-100μmol／L乙酰唑胺孵育72h后，培养的牛角膜内皮细胞的渗透性水通透性逐渐减少，其Pf值分别为（0．0406±0．0014）cm／s，（0．0364±0．0015）cm／s，（0．0300±0．0022）cm／s，（0．0226±0．0019）cm／s。结论 乙酰唑胺呈剂量依赖性地减低角膜内皮渗透性水通透性．抑制角膜内皮细胞液体转运功能。     

水通道蛋白1在人角膜内皮细胞的表达

目的 研究水通道蛋白1(water channel protein1,AQP1)抗体在人角膜中的表达及水转运机制。方法 运用免疫组织化学法测定人角膜中AQP1的表达。结果 在角膜内皮细胞和基质细胞中有AQP1的表达，而角膜上皮细胞不表达AQP1。结论 本实验证实了AQP1在角膜内皮的表达，为角膜的水转运及角膜水肿机制的研究提供了分子生物学基础。     

眼与水通道蛋白的研究进展

水的跨膜转运有 2条基本途径 :跨越脂质双层的简单扩散和通道介导的水转运。不同组织扩散水通透性 (diffusionalwater permeability,Pd)不同 ,如果 Pf/ Pd≈ 1(渗透水通透性 :osm otic water perm eability,Pf,是反映水在跨膜梯度存在下的渗透情况 )表明水的跨膜运动是通过简单扩散完成的。而通道介导的水转运 ,不能仅用简单扩散来解释。其 Pf较高 ,而活化能较低 (Ea<5 kal· mol- 1 ) ,且 Pf/ Pd>1,可特异地介导大量的水向高渗方向移动。AQPs介导这一类水转运。1　水通道蛋白家族的结构、分布与功能目前的水通道蛋白家族包括 10个成员…     

水通道蛋白1与角膜内皮

水通道蛋白(aquaporins，AQPs)是存在于动植物及微生物细胞膜上的一组与水通透有关的转运蛋白，参与许多生理过程，维持机体的正常状态。第一个水通道蛋白AQPl的cDNA序列是在1991年完成鉴定的，它存在于包括眼在内的多种组织和细胞中。角膜内皮细胞上也发现有AQPl的表达，随着对AQPl结构及功能认识的逐步深入，发现其跨膜水转运功能及其他的可能功能对角膜病变的阐明与角膜透明的维持至关重要。     

地塞米松对牛眼小梁水通道蛋白表达的影响

目的观察地塞米松对牛眼小梁细胞的损伤使其水通道蛋白-1(aquaporin-1,AQP-1)表达的影响。方法体外培养牛眼小梁细胞,取第4代细胞鉴定后用于实验。应用浓度为5,25,50,250μg/L地塞米松的培养液培养7天,通过WesternBlot方法检测培养的牛眼小梁细胞在不同浓度地塞米松作用下AQP-1的蛋白表达水平。结果牛眼小梁细胞可见AQP-1蛋白条带表达,未经地塞米松作用的AQP-1蛋白条带表达灰度值为102.87±11.73。在浓度为5,25,50,250μg/L地塞米松的培养液培养7天后AQP-1蛋白条带表达灰度值分别为111.64±13.32,115.31±9.41,121.39±9.81,129.42±13.52。当地塞米松浓度≥25μg/L时,AQP-1表达出现抑制作用(P<0.05)。结论地塞米松使培养的牛眼小梁细胞AQP-1的表达减少,这可能是皮质类固醇性青光眼房水流出阻力增加的原因之一。     

无血清器官培养法保存大鼠角膜内皮细胞的活性

目的:研究两种新型培养基-内皮细胞培养基(endothelial cell medium,ECM)和无动物成分培养基(animal compoundfree medium,ACF)在无血清器官培养法保存角膜内皮细胞活性方面的应用效果。方法:将大鼠角膜密闭保存于ECM和ACF培养基中4wk,再经葡聚糖T500脱水2d。以含低浓度牛血清的MEM基础培养基作为对照。计数保存前后角膜内皮细胞密度,检测保存结束大鼠角膜内皮层中胞质紧密粘连蛋白1(zonula occludens-1,ZO-1)的表达水平,评估内皮细胞层的屏障功能。结果:保存结束后,MEM+20mL/L FBS对照组大鼠的角膜内皮细胞密度值最低,ECM+20mL/L FBS组则高达2250±202个/mm2,ECM和ACF组结果与ECM+20mL/LFBS组接近,细胞死亡率也较低,组间差异有统计学意义(F=28.965,P=0.000)。冰冻切片显示ZO-1在保存后大鼠角膜内皮层成功表达。RT-PCR检测表明ZO-1mRNA在各组的表达趋势与内皮细胞密度结果一致(F=592.751,P=0.000)。结论:无血清ECM和ACF培养基可以很好地保持器官培养法保存角膜内皮细胞的活性和紧密连接的屏障功能。     

高压力培养下角膜内皮细胞凋亡的启动机制

目的 探讨高压力培养下角膜内皮细胞凋亡的启动机制.方法 原代兔角膜内皮细胞经免疫组织化学鉴定后,在50mmHg(1 kPa =7.5 mmHg)压力条件下,培养lh、2h、24 h,另设正常压力(15 mmHg)作为正常压力组,培养24 h.第一代兔角膜内皮细胞融合达70％～80％后,细胞在加压前lh分别使用浓度均为10-6 mol·L-l抗Caspase-8和抗Caspase-9预处理,然后置于压力装置中,压力设定为50 mmHg,培养24h后检测蛋白Bcl-2和P53表达,并且以无抑制剂处理的50 mmHg压力培养的细胞为对照组.各组培养的细胞均经Western blot检测Bcl-2和P53的表达.免疫荧光染色检测兔角膜内皮细胞胞浆细胞色素C的含量.结果 50 mmHg压力组角膜内皮细胞,加压lh、2h、24 h P53的表达量分别为0.651±0.007、0.805±0.006、0.839±0.011,较正常压力组(0.033±0.004)升高,差异均有统计学意义(均为P＜0.01);50 mmHg压力组随着加压时间的延长,角膜内皮细胞中P53蛋白表达量逐渐增加,各时间两两比较,差异均有统计学意义(均为P＜0.01).50 mmHg压力组中加压lh、2h、24 h Bcl-2的表达量分别为0.590±0.009、0.724±0.005、0.314±0.016,较正常压力组(0.081±0.013)反应性升高,差异均有统计学意义(均为P＜0.01);50 mmHg压力组各时间两两比较差异均有统计学意义(均为P＜0.01).实验发现正常压力组培养24h后,细胞核呈蓝色,胞浆中无细胞色素C释放;50 mmHg压力组培养lh可见部分细胞胞浆呈红色,提示细胞色素C释放进入到胞浆内,随着加压时间延长荧光强度增加,加压24 h见胞浆内出现广泛红色强荧光.抗Caspase-9和抗Caspase-8预处理组的角膜内皮细胞P53表达量分别为0.535±0.007、0.703±0.010,较对照组(0.727±0.021)下调,差异均有统计学意义(均为P＜0.0I);抗Caspase-9和抗Caspase-8预处理组中Bcl-2的表达量呈抑制性下降,分别为0.312±0.003、0.442±0.011,较对照组(0.501±0.011)下降,差异均有统计学意义(均为P＜0.01).抗Caspase-9预处理组的角膜内皮细胞P53和Bcl-2表达量较抗Caspase-8预处理组下降,差异亦均有统计学意义(均为P＜0.01),表明抗Caspase-9对高压下角膜内皮细胞凋亡的抑制作用更显著.结论 Caspase-9抑制剂可有效阻断高压力对角膜内皮细胞的促凋亡作用,高压力对角膜内皮细胞的损伤主要是触发了线粒体细胞色素C的释放,激活了Caspase-9参与的内源性酶联反应性凋亡途径.     

大泡性角膜病变水通道蛋白和Na+K+-ATP酶表达变化

目的 研究水通道蛋白1(Aquaporin-1,AQP1)、水通道蛋白4(Aquaporin-4,AQP4)和Na+,K+-ATP酶在人大泡性角膜病变角膜中的表达变化,探讨其在大泡性角膜病变发生机制中的作用.方法 运用免疫荧光技术和免疫印迹方法分别测定人大泡性角膜病变角膜和正常角膜中AQP1、AQP4和Na+,K+-ATP酶的表达变化.结果 大泡性角膜病变角膜AQP1表达量升高而AQP4和Na+,K+-ATP酶表达量下降.结论 本实验结果表明,AQP1、AQP4和Na+,K+-ATP酶在角膜组织水容量调节中具有重要意义,它们的异常表达可能与大泡性角膜病变的病理改变有一定关联.     

Role of aquaporins -1 in corneal endothelial fluid transport

AIM: To investigate the expression of aquaporins-1 (AQP-1) in cultured bovine corneal endothelial cells and to explore the role of AQP-1 in corneal endothelial fluid transport. METHODS: The bovine corneal cells were cultured in DMEM containing 200mL/L neonate bovine serum. AQP-1 expression in the bovine corneal endothelial cells was detected with immunohistochemistry method before and after treatment of the cells with aquaporin inhibitor,p -chloromercuribenzene sulfonate. The osmotic water permeability was determined by monitoring volume changes of cultured bovine corneal endothelial cells. RESULTS: Positive staining was used to reveal the AQP-1 expression in the membrane of cultured bovine (in brown color). The reading of osmotic water permeability of the cultured bovine corneal endothelial cells before treatment with p -chloromercuribenzene sulfonate was 0.044±0.005cm/s, which significantly decreased to 0.017±003cm/s after treatment (n =15). CONCLUSION: AQP-1 expressed in the membrane of cultured bovine corneal endothelial cells may play an important role in fluid transport of corneal endothelial cells. Alteration of the AQP-1 expression may cause abnormal corneal function and corneal edema.     

Fluid transport across cultured layers of corneal endothelium from aquaporin-1 null mice

We explored the role of AQP1, the only known aquaporin in corneal endothelium, on active fluid transport and passive osmotic water movements across corneal endothelial layers cultured from AQP1 null mice and wildtype mice. AQP1 null mice had grossly transparent corneas, just as wildtype mice. Endothelial cell layers grown on permeable supports transported fluid at rates of (in microl h(-1) cm(-2), n = 9 mean+/-s.e.): 4.3+/-0.6, wildtype mice (MCE); 3.5+/-0.6, AQP1 null mice (KMCE; difference not significant). The osmotic water flow (also in microl h(-1) cm(-2)) induced by a 100 mOsm sucrose gradient across MCE cell layers (8.7+/-0.6, n = 8) was significantly greater than that across KMCE (5.7+/-0.7, n = 6, p = 0.007). When plated on glass coverslips, plasma membrane osmotic water permeability determined by light scattering was significantly higher for cells from wildtype vs. AQP1 null mice (in microm sec(-1): 74+/-4, n = 19 vs. 44+/-4 microm sec(-1), n = 11, p < 0.001). Unexpectedly, after 10% hypo-osmotic challenge, the extent of the regulatory volume recovery was significantly reduced for AQP1 null mice cells (in%: MCE controls, 99+/-1, n = 19 vs. KMCE: 64+/-5, n = 11, p < 0.001). Thus, as in other 'low rate' fluid transporting epithelia, deletion of AQP1 in mice corneal endothelium reduces osmotic water permeability but not active transendothelial fluid transport. However, that deletion impaired the extent of regulatory volume decrease after a hypo-osmotic challenge, suggesting a novel role for AQP1 in corneal endothelium.     

Effect of AQP1 expression level on Co(2) permeability in bovine corneal endothelium

PURPOSE: Corneal endothelial fluid transport is dependent on HCO(3)(-) and CO(2) fluxes. CO(2) permeability (P:CO(2)) measurements in an oocyte expression system and in reconstituted proteoliposomes have suggested that the water channel AQP1 can transport CO(2). An AQP1 knockout mouse model, however, showed no evidence for CO(2) transport through AQP1 in erythrocytes or lung. Because HCO(3)(-) and CO(2) fluxes are essential to endothelial function, the current study was conducted to determine whether AQP1 expression levels in confluent cultures of bovine corneal endothelial cells (BCECs) affects membrane PCO(2). METHODS: BCEC endogenous AQP1 expression was reduced by antisense oligonucleotide (AO) transfection or adenoviral antisense-AQP1 (AV) infection. AQP1 was overexpressed by adenoviral sense-AQP1 (SV) infection, which directs expression of recombinant AQP1. RESULTS: Expression of AQP1 and osmotic water permeability (control P(f) = 0.046 +/- 0.005 cm/sec) were reduced 45% and 36.5%, respectively, by AO transfection and reduced 67% and 49%, respectively, by AV infection. SV infection induced a more than threefold overexpression of AQP1 but showed only a 37% increase in P(f). Adenoviral empty virus (EV) infection did not change AQP1 expression or P(f). PCO(2) was determined by measuring the rate of intracellular pH decrease after exposure to CO(2)/HCO(3)(-)-rich solutions, as measured by the pH-sensitive fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Apparent PCO(2) of BCEC (0.0036 +/- 0.00023 cm/sec) was not different among control, oligonucleotide-transfected, and adenoviral-infected cells. P(f) could also be reduced more than 50% by 3 to 5 minutes' exposure of control cells to 0.5 mM p-chloromercuriphenylsulfonic acid (pCMBS), but this had no effect on rates of intracellular pH decrease. CONCLUSIONS: AQP1 does not contribute to PCO(2) in corneal endothelial cells.     

Basolateral Na(+)-K(+)-2Cl(-) cotransport in cultured and fresh bovine corneal endothelium

PURPOSE: To examine whether Na(+)-K(+)-2Cl(-) cotransport has the potential to contribute to corneal endothelial ion and fluid transport in cultured and fresh bovine corneal endothelial cells. METHODS: Cl- and Na+ sensitive fluorescent dyes were used to measure furosemide-dependent ion fluxes in cultured and fresh endothelial cells. Immunoblot analysis and immunofluorescence were used to determine expression and location of the Na(+)-K(+)-2Cl(-)cotransporter (NKCC1). RESULTS: Application of furosemide (50-100 microM) reduced Cl- and Na+ influx in approximately 50% of trials using cultured cells and only 10% of trials with fresh cells; however, in all cases pretreatment with furosemide slowed Cl- efflux when cells were bathed in Cl(-)-free Ringer's. Double-sided perfusion of cultured cells indicated that furosemide-sensitive Cl- fluxes were located on the basolateral side. Immunoblot analysis revealed 174-kDa bands in both fresh and cultured cells, but the bands were denser in fresh endothelial cells. Immunofluorescence showed distinct lateral membrane staining in addition to significant amounts of perinuclear staining. CONCLUSIONS: The Na(+)-K(+)-2Cl(-) cotransporter is present in both fresh and cultured bovine corneal endothelium, and the expression is apparently higher in the fresh cells. The cotransporter is present on the lateral membrane consistent with a role in loading endothelial cells with Cl-, thereby possibly contributing to a transendothelial Cl- flux. However, in the resting cell, net flux through the transporter is often not apparent.     

Studies on the expression of mRNA for anion transport related proteins in corneal endothelial cells

PURPOSE: Chloride and bicarbonate are necessary for maintenance of fluid transport by the corneal endothelium, however there is little information on the identity of anion transport proteins that could serve as anion efflux mechanisms in endothelial cells. Therefore, we ask whether mRNA for the anion transport related proteins, CFTR, CLC-2, ClC-3, ClC-5 and AE2, are expressed in human, bovine or rabbit corneal endothelium. METHODS: RT-PCR was performed for CFTR, CLC-2, ClC-3, ClC-5 and AE2 using total RNA from fresh human, bovine and rabbit corneal endothelium as well as cultured bovine corneal endothelial cells (CBCEC). Specificity of PCR products was confirmed by sequencing. RESULTS: RT-PCR analysis gave positive bands at the predicted size for CLC-3 and CLC-5 from fresh human, rabbit and bovine as well as CBCEC. However, for CLC-2, no band was apparent around the predicted size from fresh and cultured corneal endothelium. A band at the predicted size was obtained for CFTR from fresh human, rabbit and bovine endothelium, as well as from CBCEC. RT-PCR analysis for AE2 produced specific bands from fresh human, rabbit and bovine corneal endothelium, but no positive band was obtained from CBCEC. Sequencing analysis further confirmed the identities of CLC-3, CLC-5, CFTR and AE2 in corneal endothelium. CONCLUSIONS: CFTR, CLC-3 and ClC-5 are expressed in fresh and cultured corneal endothelial cells. However, consistent with previous immunoblots studies, AE2 is only expressed in fresh corneal endothelium. These results have implications for modeling possible apical anion efflux mechanisms in corneal endothelium.     

RNA干扰抑制水通道蛋白1对血管内皮细胞迁移影响的研究

目的 探讨RNA干扰抑制水通道蛋白1(AQP-1)对血管内皮细胞迁移的影响以及角膜新生血管的形成机制.方法 实验研究.体外培养人血管内皮细胞,设计并合成特异性针对人AQP-1的小RNA干扰片段,用脂质体转染入血管内皮细胞,逆转录聚合酶链反应(RT-PCR)法检测RNA干扰效果,Transwell实验和损伤愈合实验观察血管内皮细胞迁移能力的改变.AQP-1 mRMA表达的组间比较采用单因素方差分析.Transwell细胞迁移实验和损伤愈合实验的结果采用独立样本的t检验.结果 转染24、48及72 h后AQP-1 mRNA表达下降,分别为正常对照组的11.3%、17.4%及29.7%,与正常对照组相比差异有统计学意义(P值分别为0.004、0.007、0.002).Transwell实验和损伤愈合实验提示转染AQP-1 siRNA后血管内皮细胞的迁移能力有明显下降,与正常对照组相比差异有统计学意义(t=10.813,P=0.016和t=22.431,P=0.027).结论 AQP-1特异性siRNA能有效抑制AQP-1的表达并明显降低血管内皮细胞的迁移能力,从而抑制角膜新生血管的形成.(中华眼科杂志,2008,44:741-744)     

Modulation of tight junction properties relevant to fluid transport across rabbit corneal endothelium

Paracellular junctions could play an important role in corneal endothelial fluid transport. In this study we explored the effects of different reagents on the tight junctional barrier by assessing the translayer specific electrical resistance (TER) across rabbit corneal endothelial preparations and cultured rabbit corneal endothelial cells' (CRCEC) monolayers, the paracellular permeability (Papp) for fluorescein isothiocyanate (FITC) dextrans across CRCEC, and fluid transport across de-epithelialized rabbit corneal endothelial preparations. Palmitoyl carnitine (PC), poly-L-lysine (PLL), adenosine triphosphate (ATP), and dibutyryl adenosine 3',5'-cyclic monophosphate (dB-cAMP) were used to modulate corneal endothelial fluid transport and tight junctions (TJs). After seeding, the TER across CRCEC reached maximal values (29.2+/-1.0 Omega cm2) only after the 10th day. PC (0.1 mM) caused decreases both in TER (by 40%) and fluid transport (swelling rate: 18.5+/-0.3 microm/h), and an increase in Papp. PLL resulted in increased TER rose and Papp but decreased fluid transport (swelling rate: 10+/-0.3 microm/h). dB-cAMP (0.1 mM) and ATP (0.1 mM) decreased TER by 16% and 6%, increased Papp slightly, and stimulated fluid transport; the rates of de-swelling (in microm/h) were -5.4+/-0.3 and -12.1+/-0.4, respectively. PC might cause the junctions to open up unspecifically and thus increase passive leak. PLL is a known junctional charge modifier that may be adding steric hindrance to the tight junctions. The results with dB-cAMP and ATP are consistent with fluid transport via the paracellular route.     

Dependence of cAMP meditated increases in Cl- and HCO(3)- permeability on CFTR in bovine corneal endothelial cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is present on the apical membrane of corneal endothelial cells. Increasing intracellular [cAMP] with forskolin stimulates an NPPB and glibenclamide-inhibitable apical Cl(-) and HCO(3)(-) permeability [Sun, X.C., Bonanno, J.A., 2002. Expression, localization, and functional evaluation of CFTR in bovine corneal endothelial cells. Am. J. Physiol. Cell Physiol. 282, C673-C683]. To definitively determine that the increased permeability is dependent on CFTR, we used an siRNA knockdown approach. Apical Cl(-) and HCO(3)(-) permeability and steady-state HCO(3)(-) flux were measured in the presence or absence of forskolin using cultured bovine corneal endothelial cells that were transfected with CFTR siRNA or a scrambled sequence control. CFTR protein expression was reduced by approximately 80% in CFTR siRNA treated cultures. Forskolin (10 microM) increased apical chloride permeability by 7-fold, which was reduced to control level in siRNA treated cells. CFTR siRNA treatment had no effect on baseline apical chloride permeability. Apical HCO(3)(-) permeability was increased 2-fold by 10 microM forskolin, which was reduced to control level in siRNA treated cultures. Similarly, there was no effect on baseline apical HCO(3)(-) permeability by knocking down CFTR expression. The steady-state apical-basolateral pH gradient (DeltapH) at 4h in control cultures was increased approximately 2.5-fold by forskolin. In CFTR siRNA treated cells, the baseline DeltapH was similar to control, however forskolin did not have a significant effect. We conclude that forskolin induced increases in apical HCO(3)(-) permeability in bovine corneal endothelium requires CFTR. However, CFTR does not have a major role in determining baseline apical chloride or HCO(3)(-) permeability.