摘要
目的研究大鼠糖尿病性白内障(diabetic cataract, DC)晶状体上皮细胞(lens epithelial cells, LECs)水通道蛋白-1(aquaporin-1 , AQP1)表达水平的变化及其与晶状体上皮细胞钙蛋白酶II(Calpain -2)表达水平、晶状体中Ca2+含量变化的关系。
方法给Wistar大鼠注射链脲佐菌素(streptozotocin, STZ)建立糖尿病模型,根据晶状体混浊程度将白内障分为0~IV期。每周在裂隙灯显微镜下观察并记录拍照晶状体的混浊情况;将各期白内障大鼠的一侧眼球制成石蜡切片,光镜下观察晶状体的组织学变化;应用免疫组织化学方法和计算机图像分析技术对LECs表达的AQP1、Calpain-2进行检测。摘取另一侧眼球晶状体,用SpectrAA–40型原子吸收光谱仪测定Ca2+浓度。
结果组织学观察,正常对照组晶状体结构正常,随着实验时间延长,实验组晶状体混浊逐渐加重,白内障I~IV期晶状体结构破坏逐渐加重;随着糖尿病发展和晶状体混浊程度的加重,LECs胞膜表达AQP1逐渐减少,而LECs胞浆表达的Calpain-2及晶状体Ca2+浓度逐渐增加。
结论糖尿病大鼠晶状体上皮细胞膜AQP1的活性表达减少与晶状体上皮细胞胞浆Calpain-2的活性表达增加及晶状体Ca2+浓度增加关系密切。
Objective To investigate the expression change of aquaporin-1 ( AQP1 ) in the membrane of lens epithelial cells (LECs) and the relation between this change and the expression of Calpain-2 in the cytoplasm of lens LECs and the total Ca2+ level in lenses of rat diabetic cataract( DC ).
Methods An animal model of diabetes was induced by a single intravenous dose of streptozotocin (STZ, 60mg/kg body weight) in 2-month-old male Wistar rats. The transparency of all lens were examined under microscope and photographied every week. The eyeballs of both sides were extracted periodically from each experimental groups and control group. The eyeballs of one side were made into paraffin sections. The histological changes were examined under light microscope. Immunohistochemical techniques and computer-imaging analysis were used to localize and semi-quantify the expression of AQP1 and Calpain-2 in LECs. The lenses of other side were isolated and the concentration of Ca2+ was determined by a SpectrAA–40 atomic absorption spectrophotometer.
Results All lenses in control group remained transparency and all lenses in experiment groups developed cataract of different stages. The breakage of the lens structure was gradually aggravated from stage I to stage IV. The expression of AQP1 on the cell membrane decreased significantly before the DC occurred and decreased gradually with the diabetes development. The elevated level of Calpain-2 in the cytoplasm of LECs and the increased ion concentration of Ca2+ in lenses were observed before lens opacification began. The expression of Calpain-2 increased gradually with the diabetes development and the concentration of Ca2+ did so.
Conclusion Decreased expression of Aquaporin-1 in the membrane of LECs of rat diabetic cataract were closely correlated with elevated expression of Calpain-2 in the cytoplasm of LECs and increased ion concentration of Ca2+ in lens.
引文
[1]Bloemendal H. Molecular Biology of the Eye Lens. Wiley, New York, 1981; 1-47.
[2]Diatigorsky J. Differentiation. 1981;19: 134.
[3]Hightower KR, Reddan JR, McCready JP, Dziedzic DC. Lens epithelium: a primary target of UVB irradiation. Exp Eye Res 1994 Nov; 59 (5): 557-564.
[4]Francois J, Victoria-Troncoso V, Cansu K. Normal and pathological cultured lens epithelial cells histochemical study (author's transl).J Fr Ophtalmol 1980; 3(10):561-569.
[5]李风鸣. 中华眼科学.第二版.北京:人民卫生出版社. 2005; 1471-1472.
[6] Harris JE, Hauschild JD, Nordquist LT. Transport of glucose across the lens surface [J]. Am J Ophthalmol, 1995; 39:161-169.
[7]Fischbarg J, Diecke FP, Kuang K, et al. Transport of fluid by lensepithelium. Am J physiol, 1999, 276(3 Pt 1): C548-557.
[8]Beitz E, Schultz JE. The mammalian aquaporin water channel family: A promising new drug target. Curr Med Chem, 1999, 6(6): 457-467.
[9] 张虹,彭洁,胡维琨, 等. 透明晶状体和老年性白内障晶状体上皮细胞水通道蛋白1的表达. 华中科技大学学报(医学版),2004,33(3): 360-362.
[10]何守志,伊素云,宋琛,等. 糖性白内障晶状体水肿的实验观察. 眼科研究,1988 ,6(2)∶94-97.
[11]Yoshida H, Murachi T, Tsukahara I. Distribution of calpain I,calpain II , and calpastatin in bovine lens. Invest Ophthalmol Vis Sci, 1985, 26: 953-956.
[12] David LL, Shearer TR. Purification of calpain II from rat lens and determination of endogenous substrates. Exp Eye Res, 1986, 42:227-238.
[13] Biswas S, Harris F, Singh J, at al. Role of calpains in diabetes mellitus-induced cataractogenesis: a mini review. Mol Cell Biochem, 2004, 261(1-2): 151-159.
[14] Beaulieu CF, Clark J I. 31P Nuclear magnetic resonance and laser spectroscopic analysis lens transparency during calcium-induced opacification. Invest Ophthalmol Vis Sci,1990,31(7)∶1339.
[15] Azuma M, Shearer T R, Matsumoto T. Calpain II in two in vivo models of sugar cataract. Exp Eye Res, 1990; 351-393.
[16] Cekic O, Bardak Y. Lenticular calcium, magnesium, and iron levels in diabetic rats and verapamil effect. Ophthalmic Res, 1998, 30(2): 107-112.
[17]刘爱琴,廖品正,郑燕林,等.芪明颗粒对糖尿病大鼠晶体抗氧化反应的影响.成都中医药大学学报.2004,27(1):9-10.
[18] 何花,张虹,罗爱珍. 糖尿病性白内障晶状体上皮细胞凋亡与增殖特性的实验研究.眼科新进展,2003, 23(5): 323-327.
[19] Kojima M, Sasaki K. Reinvestigation of streptozotocin induceddiabetic cataract as a standard experimental model.Nippon Ganka Gakkai Zasshi 1993; 97 (3):324-332.
[20] Andallu B, Varadacharyulu NCh. Control of hyperglycemia and retardation of cataract by mulberry (Morus indica L.) leaves in streptozotocin diabetic rats. Indian J Exp Biol 2002 Jul; 40 (7):791-795.
[21]Gajdosik A, Gajdosikova A, Stefek M,et al. Streptozotocin-induced experimental diabetes in male Wistar rats. Gen Physiol Biophys. 1999, 18:54-62.
[22]宋旭东,陈翠真. 药物防治链脲佐菌素-糖尿病性白内障的研究进展.国外医学眼科学分册.2000,24(4):201~206.
[23] Bhat SP. The ocular lens epithelium[J]. Biosci Rep, 2001; 21(4): 537-563.
[24] 赵桂秋,孙为荣,牛膺筠,等. 晶状体前囊膜下上皮细胞增殖性病变的临床病理分析[J].中华眼科杂志, 2001;37(3): 215-218.
[25] Hegde KR, Henein MG, Varma SD. Establishment of mouse as an animal model for study of diabetic cataracts: biochemical studies. Diabetes Obes Metab, 2003, 5(2): 113-119.
[26] Eshagian J. Human posterior subcapsular cataracts[J]. Trans Ophthalmol Soc UK, 1982; 102: 364-368.
[27] Mathai JC, Mori S, Smith BL, et al. Functional analysis of aquaporin-1 deficient red cells. The Colton-null phenotype. J Biol Chem, 1996, 271(3): 1309-1313.
[28] Preston GM, Carroll TP, Guggino WB, et al. Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein[J]. Science, 1992; 256(5055): 385-387.
[29] Jung JS, Preston GM, Smith BL, et al. Molecular structure of the water channel through aquaporin CHIP. The hourglass model. J Biol Chem, 1994;269 (20): 14648–14654.
[30] Kuang K, Yiming M, Wen Q, et al. Fluid transport across cultured layers of corneal endothelium from aquaporin-1 null mice[J]. Exp Eye Res, 2004; 78(4): 791-798.
[31] Sakurada T, Kuboshima S, Ogimoto G, et al. Aquaporin-1 is recruited to the plasma membrane by hyperosmotic stimuli via a protein kinase A-dependent pathway in rat peritoneal mesothelial cells[J]. Adv Perit Dial, 2004; 20: 37-42.
[32] Han Z, Patil RV. Protein kinase A-dependent phosphorylation of aquaporin-1. Biochem Biophys Res Commun,2000, 273(1): 328-332.
[33] Kyselova Z, Stefek M, Bauer V. Pharmacological prevention of diabetic cataract. J Diabetes Complications, 2004, 18(2): 129-140.
[34] Daxin Tang, Douglas Borchman, Marta C. Yappert, et al. Influence of Age,Diabetes, and Cataract on Calcium, Lipid-Calcium, and Protein-Calcium Relationships in Human Lenses. Invest Ophthalmol Vis Sci, 2003, 44(5): 2059-2066.
[35] Schey KL, Fowler JG, Shearer TR, et al. Modifications to rat lens major intrinsic protein in selenite-induced cataract. Invest Ophthalmol Vis Sci, 1999, 40(3): 657-667.
[36] 徐雯,姚克,王凯军,等.卡配因Ⅱ在过氧化氢诱发大鼠白内障的晶状体上皮细胞中的表达.中华眼科杂志,2002, 38(5): 282-286.
[37] Sakamoto-Mizutani K, Fukiage C, Tamada Y, et al. Contribution of ubiquitous calpains to cataractogenesis in the spontaneous diabetic WBN/Kob rat. Exp Eye Res, 2002, 75(5): 611-617.
[1] SφREN NIELSEN, BARBARAL.SMITH, ERIK ILSφCHRIS T ENSEN, et al. Distribution of the aquaporin CHIP in secretory and resorptive epithelia and capillary endothelia [J]. Proc Natl Acad Sci USA,1993; 90:7275-7279.
[2] Stamer WD, Synder RW, Smith BL, et al. Localization of aquaporin in the human eye: implications in the pathogenesis of glaucoma and other disorders of ocular fluid balance [J]. Invest Ophthalmol Vis Sci, 1994;35(11):3867-3872.
[3] Hasegawa H, lian SC, Finkbeiner WE, et al. Extrarenal tissue distribution of CHIP 28 water channels by in situ hybridization and antibody staining [J]. Am J Physiol, 1994; 266(4 Pt 1):893-903.
[4] Bok D, Dockstader J, Horwitz J. Immunocytochemical localization of the lens main intrinsic polypetide (MIP26) in communicating junctions [J]. J Cell Biol, 1982; 92(1): 213-220.
[5] Sabine M.Mulders, Gregory M.Preston, Peter M.t.Deen, et al. Water Channel Properties of Major Intrinsic Protein of Lens [J]. J Biol Chem, 1995; 15(270): 9010-9016.
[6] Nicholas A.Kent, Alan Shiels. Nucleotide and derived amino-acid sequence of the major intrinsic protein of rat eye-lens[J]. Nucleic Acid Research, 1990; 14(18): 4256.
[7] Jun Han, Mark Little, Larry L, et al. Sequence and peptide map of guinea pig aquaporin 0[J]. Molecular Vision, 2004; 10:215-222.
[8] Harris JE, Hauschild JD, Nordquist LT. Transport of glucose across the lens surface [J]. Am J Ophthalmol, 1995; 39:161-169.
[9] Rae J.L., Bartling C, Rae J, et al. Dye transfer between cells of the lens [J]. J Membr Biol, 1996; 150:89-103.
[10] F.Le Cahrec, P.Bron, J.M. Verbavatz, et al. Incorporation of protein into (Xenopus) oocytes by proteoliposome microinjection: functional characterization of a novel aquaporin[J]. Journal of Cell Science, 1996; 109: 1285-1295.
[11] Chandy G, Zampighi GA, Kreman M, et al. Comparison of the transporting properties of MIP and AQP1 [J]. J Membr Biol, 1997; 159(1):29-39.
[12] William E.C. Harries, David Akhavan, Larry J.W. Miercke, et al. The channel architecture of aquaporin 0 at a 2.2-resolution[J]. PANS, 2004; 39(101): 14045-14050.
[13] Fischbarg J, Diecke FP, Kuang K, et al. Transport of fluid by lens epithelium[J]. Am J physiol, 1999; 276(3 Pt 1): C548-557.
[14] Sas DF, Sas MJ, Johnson KR, et al. Junctions between lens fiber cells are labeled with a monoclonal antibody shown to be specific for MP26[J]. J Cell Biol, 1985; 100(1): 216-225.
[15] Gruijters WT.A non-connexon protein (MIP) is involved in eye gap-junction formation [J]. J CellSci, 1989; 93(Pt3): 509-513.
[16] Peracchia C, Girsch SJ, Bernardini G, et al. Lens junctions are communicating junctions [J]. Curr Eye Res, 1985; 4(11): 1155-1169.
[17] Gonen T, Sliz P, Kistler J, et al. Aquaporin-0 membrane junctions reveal the structure of a closed water pore [J]. Nature, 2004; 429(6988): 193-197.
[18] Kinoshita JH, Merola LO. Hydration of the lens during the development of potassium transport[J]. Invest Ophthalmol, 1964; 47:577-584.
[19] Fotiadis D, Hasler L, Muller DJ, et al. Surface tongue-and-groove contours on lens MIP faciliate cell-to-cell adherence[J].J Mol Biol, 2000;300(4):779-789.
[20] Michea LF, de la Fuente M, Lagos N. Lens major intrinsic pritein (MIP) promotes adhesion when reconstituted into large unilamellar liposomes[J]. Biolchemistry,1994;33(24):7663-7669.
[21] Michea LF, Andrinolo D, Ceppi H, et al. Biochemical evidence for adhension-promoting role of major intrinsic protein isolated from both normal and cataractous human lens[J]. Exp Eye Res, 1995; 61(3): 293-301.
[22] Al-Ghoul KJ, Kir-T, kuszak AJ, et al. Lens structure in MIP-deficient mice [J]. Anat Rec, 2003; 273A(2): 714-730.
[23] Karin L, Nemeth-Cahalam and James E, Hall JE. pH and Calcium Regulation the Water Permeability of Aquaporin-0[J]. Biol Chem, 1999; 275(10): 6777-6782.
[24] Nemth-Cahalan KL, Kalman K, Hall JE. Molecular basis of PH and Ca2+ regulation of aquaporin water permeability[J]. J Gen Physiol, 2004; 123(5): 573-580.
[25] Ball LE, Litte M, Nowak MW, et al. Water permeability of C-terminally truncated aquaporin 0 (AQP0 1-243) observed in the aging human lens[J]. Invest Ophthalmol Vis Sci, 2003; 44(11): 4820-4828.
[26] Ball LE, Garland DL, Crouch RK, et al. Post-translation modifications of aquaporin 0(AQP0) in the normal human lens: spatial and temporal occurrence[J]. Biochemistry, 2004; 43(30): 9856-9865.
[27] Swamy-Mruthinti S. Glycation decreases calmodulin binding to lens transmembrane protein, MIP[J]. Biochem Biophys Acta, 2001; 15369(1): 64-72.
[28] Schey KL, Little M, Fowler JG, et al. Characterization of human lens major intrinsic protein structure [J]. Invest Opthalmol Vis Sci, 2000; 41(1): 175-182.
[29] Takemoto LJ, Gorthy WC, Morin CL, et al. Changes in lens membrane major intrinsic polypeptide during cataractogenesis in aged Hannover Wistar rats[J]. Invest Opthalmol Vis Sci, 1991; 32(3): 556-561.
[30] Takemoto L, Kuck J, Kuck K. Changes in the major intrinsic polypeptide (MIP26k) during opacification of the Emory mouse lens[J].Exp Eye Res,1988;47(2):329-336.
[31] Granstrom D, Swamy M, Abraham E, et al. Covalent change in the major intrinsic polypeptide (MIP26k) during cataract development in the streptozocin-induced diabetic rat[J]. Curr Eye Res, 1989;8(6): 689-593.
[32] Okamura T, Miyoshi I, Takahashi K, et al. Bilateral congental cataracts result from a gain-of-function mutatioin the gene for aquaporin-0 in mice[J]. Genomics, 2003; 81(4):36-38.
[33] Peter Fraccis, Vanita Berry, Shomi Bhattacharya, et al. Congenital progressive polymorphic cataract caused by a mutation in the major intrinsic protein of the lens, MIP(AQP0)[J]. Br J Ophthalmol, 2000; 84:1376-1379.
[34] Francis P, Chung JJ, Yasui M, et al. Functional impairment of lens aquaporins in two families with dominantly inherited cataracts[J]. Hum Mol Genet, 2000; 9:2329-2334.
[35] Berry V, Francis P, Kaushal S, et al. Missense mutations in MIP underlie autosomal dominant ‘polymorphic’ and lamellar cataracts linked to 12q[J]. Nat Genet, 2000; 25(1):15-17.
[36] ALAN SHIELS, DONAMACKAY. Disruption of lens fiber cell architecture in mice expressing a chimeric AQP0-LTR protein[J]. FASEB J, 2000; 14: 2207-2212.
[37] Daniel L, Boyel and Larry J, Takemoto LJ. Localization of MIP 26 in nuclear fiber cells from aged normol and age-related nuclear cataractous human lenses[J]. Exp Eye Res, 1999; 68:41-49.
[38] Boyle DL, Takemoto LJ. Localization of MIP26 in unclear fiber cells from aged normal and age-related nuclear cataractous human lens[J]. Exp Eye Res, 1999; 68(1):41-49.
[39] Shi S, Bekhor I. Profile of messenger RNA decay in the Emory mouse lens in cataractogenesis and in aging[J]. Exp Eye Res, 1992; 55(4):629-636.
[40] Schey KL, Fowler JG, Shearer TR, et al. Modifications to rat lens major intrinsic protein in selenite-induced cataract[J]. Invest Ophthalmol Vis Sci, 1999; 40(3): 657-667.
[41] Padgaonkar VA, Lin LR, Leverenz VR,et al. Hyperbaric oxygen in vivo accelerates the loss of cytoskeletal proteins and MIP26 in guinea pig lens nucleus[J]. Exp Eye Res, 1999; 68(4): 493-504.
[42] 张虹,彭洁,胡维琨等. 透明晶状体和老年性白内障晶状体上皮细胞水通道蛋白-1的表达. 华中科技大学学报(医学版), 2004;33(3)360-363.
[43] Yang XIANG, BING MA, Tao LI, et al. Acetazolamide inhibits aquaporin-1 protein expression and angiogenesis[J]. Acta Pharmacol Sin, 2004; 25(6): 812-818.