摘要
水通道蛋白(aquaporins, AQP),又称水孔蛋白,是一种疏水跨膜蛋白,是细胞内外水分流通的主要方式。至今已经在哺乳动物体内发现13种水通道蛋白。不同的AQP成员具有特异的组织分布,AQP家族成员已经在人的泌尿、呼吸、神经和消化系统等组织定位和生理功能研究已经取得了重要进展。AQP的特异性分布特点,说明其参与了特定的生理功能。有研究证实,水通道蛋白主要参与尿浓缩机制和腺体的分泌作用。
前列腺是雄性泌尿生殖系统的重要部分,分泌的前列腺液具有重要作用,是精液的重要组成成分,为精子提供养分和适宜的环境,对精子的运输和营养具有促进作用。水通道蛋白必然参与了前列腺腺体的分泌作用。Yu等报道,睾丸的输出小管、精囊腺和前列腺上皮也存在AQP1。吕丹瑜等报道,AQP1在雄性小鼠精囊腺、前列腺的表达水平均随年龄增长而增加,且AQP1蛋白广泛表达于发育成熟的精囊腺和前列腺上皮细胞。AQP3在结肠、肾脏、肝、胰腺、肺、气管、前列腺、皮肤等均有表达,分布于上皮细胞基底膜。AQP3在肾脏集合管细胞的基底膜有高表达,是重吸收水的重要通道。
现有报道证实,AQPs成员间有一定的协同作用。目前有关前列腺AQP1、3的共同研究较少。
本实验采用RT-PCR技术、western Blot和免疫组织化学方法研究小鼠前列腺组织的AQP1、3的表达与分布,借此深入探讨AQPs在正常小鼠前列腺及疾病状态下的表达及生理作用。AQP1、AQP3在小鼠前列腺组织中mRNA和蛋白质均有较强表达,提示,AQP1、AQP3在促进前列腺液分泌过程中可能发挥着重要的生理作用。
Research background
Aquaporins (AQPs) is the specific water channel protein, widespread in the nuclear and real life、animal、plants and microorganism generally. It is the main way that water is transported through biomembrane by aquaporins.
The discovery channel, technology and medicine to open another very important area of research for biochemical and medical domain. In water circulation, the ability through AQPs of the cells double the capacity of five to fifty times than water penetration.
As yet there are thirteen forms in mammal cloned by scientist(AQP0～AQP12), their genetic structure、gene expression and regulation、chromosomal localization、protein structure、tissue deposition and physiological function have been deeply reached in last decade.
Different AQPs member of the organization of the distribution and participated in the physical features. AQPs in mammals participate in an enrichment mechanisms and glands secretion. AQPs family members have been fouded in the urological, breathing and the digestive system organizations, and other physical location, and functional study has made important progress. The gene mutations of AQP 1 will lead to fluid retention concentrated function defect. Yu and others reports, AQP1 express in testosterone and, pouch, and prostate gland. Lv reported such reports, aqp1 in the male every mouse and prostate gland are expressed level with age increasing, and AQP 1 proteins widely express to mature pouch and prostate gland of epithelial cells. AQP3 of the colon, liver, kidneys, pancreas, lungs, prostate, skin, are expressions of the epithelial Cell basement membrane. AQP3 kidney collecting pipe in the basement membrane cells have high expression, is the important channel heavy absorption water.
Research of Dan Zhao etc shows that AQP3 in human prostate tissue and prostate cancer cell lines have strong expression, In the normal prostate tissue and peripheral zone transition zone are AQP3 expression, and AQP3 transition zone of protein expression peak above peripheral zone, presumably, AQP3 high expression when BPH may play an important role. Similarly, in normal prostate tissue AQP1 endothelial also have high expression, presumably by promoting cells AQP may participate in the penetration of water secretion of the prostatic secretion.
At present, AQP1,3 knockout mice model has been established, and through mutual mating got different combination of double and triple knockout mice, we are also discussed in the prostate tissue for AQP1,3 cooperative mechanism provides reference.
objective:The experiment based on AQP3 in normal mice AQP1, of expressing with prostate tissue to study the distribution of the probe in normal mice AQP prostate and disease states expression regulation and physiological function lay the foundation.
Methods:In order to AQP1, AQP3 described in mice prostate ventral leaves distribution and its possible physiological function. First, we applied for PCR technology 12-RT ventral prostate mice did AQP0, leaf organization 1,2, 3,4 mRNA express research. In order to further confirmed AQP3 AQP1, the existence of prostate tissue in mice and physiological function, we adopted Western Blot and immunohistochemical method in the protein level for research.
Results:RT-the result showed that in the prostate PCR appeared a treaty of 130bp AQP1,110-AQP3 mRNA express band, and did not see AQP0, 2,4 mRNA expressions. Speculation in prostate tissue glands secretes and water metabolism process, AQP1, AQP3 may plays an important role. Western Blot the result showed that in the 28KDa place have peculiar patterns AQP1 expression but AQP3 respectively in 35KDa and 27KDa place express glycosyl is changed and the glycosyl turn belt. This suggests that in prostate tissue AQP1, AQP3 in the antigens are its antibody recognition, and plays a certain physiological function. By immunohistochemical results show that has not been immune rabbit serum instead of one group for negative resistance, the group not seen specific positive dyeing.
Conclusion:The results demonstrate, prostate gland secretion epithelial cells express AQP1 and 3, tip AQP1 and AQP3 through to stimulate prostate epithelial cells in the penetration of water during the prostatic secretion secretion of physiological function may play an important.
引文
1. Papadopoulos MC, Saadoun S, Verkman AS. Aquaporins and cell migration. Pflugers Arch,2008,456 (4):693-700.
2. Verkman AS. Physiological importance of aquaporin waterchannels[J]. AnnMed,2002,34(3):192-200.
3.杨宝学,赵雪俭.水通道蛋白研究进展[J].中国病理生理杂志,2005,21(8)：1619-1622.
4.郑跃英,兰允平,祝胜美.大鼠脑缺血再灌注损伤后梗死灶周围水通蛋白4与血脑屏障通透性的动态变化[J].中国病理生理杂志,2008,24(8)：1647-1649.
5. Verkman AS. Mammalian aquaporins:diverse physiological roles and potential clinical significance. Expert Rev Mol Med,2008,16 (10):1-18.
1. Verkman AS. Aquaporins:translating bench research to human disease. J Exp Biol,2009,212(11):1707-1715.
2. Marina Z,Sergey Z,Anita A.Water channels (Aquaporins) and their role for postnatal adaptation [J].Pediatric Res,2005,57:47-53.
3. David A,James B,Russell D, et al. Expression localization and regulation of aquaporin-1 to-3 in rat urotheria [J]. Am J Physiol Renal Physiol,2002,282 (6):1034-1042.
4. Verkman AS. More than just water channels:unexpected cellular roles of aquaporins. J Cell Sci,2005,118 (15):3225-3232.
5. Preston GM, Carroll TP, GugginoWB, et al. Appearance of water channels in xenopus oocytes exp ressing red cell CH IP28 p rotein [J].Science,1992,256 (5055):385-387.
6. Nielsen S, Smith BL, Christensen El, et al. Distribution of the aquaporin CH IP in secretory are resorp tive ep ithelia and cap illary endothelia [J]. Proc Natl Acad Sci USA,1993,90(15):7275-7279.
7. Hasegawa H,L ian SC, FinkbeinerWE, et al. Extrarenal tissue distribution of CH IP28 water channels by in situ hybridization and antibody staining[J]. Am J Physiol,1994,266 (41):C893-C903.
8. Mobasheri A,Wray S, Marp les D. Distribution of AQP2 and AQP3 water channels in human tissue microarrays [J]. Mol Hist,2005,36 (1):1-14.
9. Verkman AS. Physiological importance of aquaporin water channels. Ann Med,2002,34 (3):192-200.
10. Saadoun S, Papadopoulos MC, Hara-Chikuma M, Verkman AS. Impairment of angiogenesis and cell migration by targeted aquaporin-1 gene disruption. Nature,2005,434 (7034):786-792.
11. Hara-Chikuma M and Verkman AS. Aquaporin-1 facilitates epithelial cell migration in kidney proximal tubule. J Am Soc Nephrol,2006,17 (1): 39-45.
12. Hara-Chikuma M, Verkman AS. Aquaporin-3 facilitates epidermal cell migration and proliferation during wound healing. J Mol Med,2008,86 (2): 221-231.
13. Levin MH, Verkman AS. Aquaporin-3-dependent cell migration and proliferation during corneal re-epithelialization. Invest Ophthalmol Vis Sci, 2006,47(10):4365-4372.
14. Thiagarajah JR, Zhao D, Verkman AS. Impaired enterocyte proliferation in aquaporin-3 deficiency in mouse models of colitis. Gut,2007,56 (11): 1529-1535.
15.Verkman AS, Hara-Chikuma M, Papadopoulos MC. Aquaporins-new players in cancer biology. J Mol Med,2008,86 (5):523-529.
16.杨宝学,赵雪俭.水通道蛋白研究进展.中国病理生理杂志,2005,21(8)：1619-1622.
17. Hara-Chikuma M and Verkman AS. Prevention of skin tumorigenesis and impairment of epidermal cell proliferation by targeted aquaporin-3 gene disruption. Mol Cell Biol,2008,28 (1):326-332.
15.Royuela M, Arenas MI, Bechencourt FR, Sanchez-Chapado M, Fraile B, Paniagua R. Regulation of proliferation/apoptosis equilibrium by mitogen-activated protein kinases in normal, hyperplastic and carcinomatous human prostate. Hum pathol,2002,33 (3):299-306.
18. Evgenia V, Gerasimovskaya Doug A, Tucker Mary, Weiser-Evans, Janot M, Wenzlau Dwight J, Klemm,Mark Banks and Kurt R Stenmark. Extracellular ATP-induced proliferation of adventitial fibroblasts requires Phosphoinositide 3-kinase, Akt, Mammalian Target of Rapamycin and P70 S6 kinase signaling pathways. J Biol Chem,2005,280 (3):1838-1848.
19. Manley GT,Fujimuna M,MaT,et al.Aquaporin4 deletion in mice reduces brain edema after acute water intoxication and ischemic stroke[J]. Nat Med,2000,6(1):159-163.
20. TaniguchiM,Yamashita T,Kumura E,et al.Induction of aquaporin 4 water channel mRNA after focal cerebral ischemia in rat[J].B rain Res Mol Brain Res,2000,78(12):131-137.
21.Kiening KL,van Landeghem FK,Schreiber S,et al.Decreased hemispheric aquaporin4 is linked to evolving brain edema following controlled cortical impact injury in rats [J]. N eurosci Lett,2002,324(2):105-108.
22. Mao X,Enno TL,Del Bigio MR.Aquaporin 4 changes in rat brain with severe hydrocephalus[J]. Eur J Neurosci,2006,23(11):2929-2936.
23. Habib FK, Ross M, Bayne CW. Development of a newin vitro model for the study of benign prostatic hyperplasia. Prostate Suppl,2000,9 (7):15-20.
24. Herbert Lepor MD. Pathophysiology, epidemiology, and natural history of benign prostatic hyperplasia. Rev Urol,2004,6 (9):3-10.
25. Gong EM, Gerber GS. Saw palmetto and benign prostatic hyperplasia. Am J Chin Med,2004,32 (3):331-338.
26. Wang J, Tanji N, Kikugawa T, Shudou M, Song X, and Yokoyama M. Expression of aquaporin-3 in the human prostate. Int J Urol,2007,14 (12): 1088-1092.
27.赵丹,朱建国,王伟华,杨宝学,赵雪俭.水通道蛋白3在人前列腺组织中的表达及意义.中国病理生理杂志,2009,25(10)：2000-2003.
28. Ittmann M, Mansukhani A. Expression of fibrolast growth factors(FGFs) and FGF receptors in human prostate. J Urol,1997,157 (1):351-356.
29. Huang YS, Zeng J, Huang YP, Qui F, Ye HY, Wang SR. Antagomistic effect of 3'-daidzein sulfonate sodium on prostatic hyperplasia in mice. Zhonghua Nan Ke Yue,2007,13 (5):387-390.
30. Ishibachi K,Kuw ahara M,Gu Y,et al.Cloning and functional expression of a new water channel abundantly expressed in the test is permeable to water, glycerol and urea [J].J Biol Chem,1997,272:20782-20786.
31. Su zuki TF,Ish ibashi K,Yu as a S. Immunohistochemical localization of a water channel, aquaporin 7(AQP7), in the mouse test [J].Cell Tissue Res,1999,295(2):279-285.
32. Mobasheri A, Airley R, Hewitt SM, et al.Heterogeneous expression of the aquaporin 1 (AQP 1) water channel in tumors of the prostate, breast, ovary colon and lung:a study using high density multiple human tumor tissue microarrays [J].Inter J of Onc,2005,26(5):1149-1158.
1.吕丹瑜,李英,毕振武等,水通道蛋白1在生后小鼠精囊腺与前列腺发育过程中的表达与分布[J].解剖学报,2007,38(6)：722-726.
2. Papadopoulos MC, Saadoun S, Verkman AS. Aquaporins and cell migration. Pflugers Arch,2008,456 (4):693-700.
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4. Yu HM, Sun BM, Bai Q, et al. Influence of acetazolamide on AQP1 gene expression in testis and on sperm count/ motility in epididymis of rats[J] Arch Androl,2002,48(4):281-294.
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7. Marina Z,Sergey Z,Anita A.Water channels (Aquaporins) and their role for postnatal adaptation [J].Pediatric Res,2005,57:47-53.
8. David A,James B,Russell D, et al. Expression localization and regulation of aquaporin-1 to-3 in rat urotheria [J]. Am J Physiol Renal Physiol,2002, 282(6):1034-1042.
9. Verkman AS. More than just water channels:unexpected cellular roles of aquaporins. J Cell Sci,2005,118 (15):3225-3232.
10. Preston GM, Carroll TP, GugginoWB, et al. Appearance of water channels in xenopus oocytes exp ressing red cell CH IP28 p rotein [J].Science,1992,256 (5055):385-387.
11. Verkman AS. Physiological importance of aquaporin water channels[J]。 Ann Med.2002; 34(3):192～200.
12. Hatakeyama S, Yoshida Y, Tani T, Koyama Y et al. Choning of a new aquaporin (AQP10)abundantly expressed in duodenum and jejunum. Biochem Biophys Res Commun.2001;287(4):814-819.
13. Ishibashi K, Kuwahara M, Gu Y, et al. Cloning and functional expression of a new water channel abundantly expressed in the testis permeable to water, glycerol and urea. J Biol Chem.1997;272(33):20782-20786.
14. Agre P, Sasaki S, and Chrispeels MJ. Aquaporins:a family of water channel proteins. Am. J. Physiol.1993;265(3pt2):F461.
15. Preston GM, Carroll TP, Guggino WB, Agre P. Appearance of water channels in xenopus oocytes expressing red cell CHIP28 protein Science.1992 Apr 17;256(5055):385～387.
16. Nielsen S, Smith BL, Christensen El, et al. Distribution of the aquaporin CHIP in secretory ane resorptive epithelia and capillary endothelia. Proc. Natl. Acad. Sci.1993;90(15):7275～7279.
17. Hasegawa H, Lian SC, Finkbeiner WE, et al. Extrarenal tissue distribution of CHIP28 water channels by in situ hybridization and antibody staining. Am. J. Physiol.1994;266(4pt1):893～903.
18. Mobasheri A, Wray S and Marples D. Distribution of AQP2 and AQP3 water channels in human tissue microarrays. J Mol Hist.2005; 36:1～14
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22. Papadopoulos MC, Saadoun S, Verkman AS. Aquaporins and cell migration. Pflugers Arch,2008,456 (4):693-700.
23. Verkman AS. Mammalian aquaporins:diverse physiological roles and potential clinical significance. Expert Rev Mol Med,2008,16 (10):1-18.
24. Verkman AS. Aquaporins:translating bench research to human disease. J Exp Biol,2009,212(11):1707-1715.
25. Verkman AS. More than just water channels:unexpected cellular roles of aquaporins. J Cell Sci,2005,118 (15):3225-3232.
26. Verkman AS. Physiological importance of aquaporin water channels. Ann Med,2002,34 (3):192-200.
27. Saadoun S, Papadopoulos MC, Hara-Chikuma M, Verkman AS. Impairment of angiogenesis and cell migration by targeted aquaporin-1 gene disruption. Nature,2005,434 (7034):786-792.
28. Hara-Chikuma M and Verkman AS. Aquaporin-1 facilitates epithelial cell migration in kidney proximal tubule. J Am Soc Nephrol,2006,17 (1): 39-45.
29. Hara-Chikuma M, Verkman AS. Aquaporin-3 facilitates epidermal cell migration and proliferation during wound healing. J Mol Med,2008,86 (2): 221-231.
30. Manley GT,Fujimuna M,MaT,et al.Aquaporin4 deletion in mice reduces brain edema after acute water intoxication and ischemic stroke[J]. Nat Med,2000,6(1):159-163.
31. TaniguchiM,Yamashita T,Kumura E,et al.Induction of aquaporin 4 water channel mRNA after focal cerebral ischemia in rat[J].B rain Res Mol Brain Res,2000,78(12):131-137.
32. Kiening KL,van Landeghem FK,Schreiber S,et al.Decreased hemispheric aquaporin4 is linked to evolving brain edema following controlled cortical impact injury in rats [J]. N eurosci Lett,2002,324(2):105-108.
33. Mao X,Enno TL,Del Bigio MR.Aquaporin 4 changes in rat brain with severe hydrocephalus[J]. Eur J Neurosci,2006,23(11):2929-2936.
34. Levin MH, Verkman AS. Aquaporin-3-dependent cell migration and proliferation during corneal re-epithelialization. Invest Ophthalmol Vis Sci, 2006,47(10):4365-4372.
35. Thiagarajah JR, Zhao D, Verkman AS. Impaired enterocyte proliferation in aquaporin-3 deficiency in mouse models of colitis. Gut,2007,56 (11): 1529-1535.
36. Verkman AS, Hara-Chikuma M, Papadopoulos MC. Aquaporins-new players in cancer biology. J Mol Med,2008,86 (5):523-529.
37.杨宝学,赵雪俭.水通道蛋白研究进展.中国病理生理杂志,2005,21(8)：1619-1622.
38. Hara-Chikuma M and Verkman AS. Prevention of skin tumorigenesis and impairment of epidermal cell proliferation by targeted aquaporin-3 gene disruption. Mol Cell Biol,2008,28 (1):326-332.
39. Royuela M, Arenas MI, Bechencourt FR, Sanchez-Chapado M, Fraile B, Paniagua R. Regulation of proliferation/apoptosis equilibrium by mitogen-activated protein kinases in normal, hyperplastic and carcinomatous human prostate. Hum pathol,2002,33 (3):299-306.
40. Evgenia V, Gerasimovskaya Doug A, Tucker Mary, Weiser-Evans, Janot M, Wenzlau Dwight J, Klemm,Mark Banks and Kurt R Stenmark. Extracellular ATP-induced proliferation of adventitial fibroblasts requires Phosphoinositide 3-kinase, Akt, Mammalian Target of Rapamycin and P70 S6 kinase signaling pathways. J Biol Chem,2005,280 (3):1838-1848.
41. Habib FK, Ross M, Bayne CW. Development of a newin vitro model for the study of benign prostatic hyperplasia. Prostate Suppl,2000,9 (7): 15-20.
42. Herbert Lepor MD. Pathophysiology, epidemiology, and natural history of benign prostatic hyperplasia. Rev Urol,2004,6 (9):3-10.
43. Gong EM, Gerber GS. Saw palmetto and benign prostatic hyperplasia. Am J Chin Med,2004,32 (3):331-338.
44. Wang J, Tanji N, Kikugawa T, Shudou M, Song X, and Yokoyama M. Expression of aquaporin-3 in the human prostate. Int J Urol,2007,14 (12): 1088-1092.
45.赵丹,朱建国,王伟华,杨宝学,赵雪俭.水通道蛋白3在人前列腺组织中的表达及意义.中国病理生理杂志,2009,25(10)：2000-2003.
46. Ittmann M, Mansukhani A. Expression of fibrolast growth factors(FGFs) and FGF receptors in human prostate. J Urol,1997,157 (1):351-356.
47. Huang YS, Zeng J, Huang YP, Qui F, Ye HY, Wang SR. Antagomistic effect of 3'-daidzein sulfonate sodium on prostatic hyperplasia in mice. Zhonghua Nan Ke Yue,2007,13 (5):387-390.
48. Ishibachi K,Kuw ahara M,Gu Y,et al.Cloning and functional expression of a new water channel abundantly expressed in the test is permeable to water, glycerol and urea [J].J Biol Chem,1997,272:20782-20786.
49. Su zuki TF,Ish ibashi K,Yu as a S. Immunohistochemical localization of a water channel, aquaporin 7(AQP7), in the mouse test [J].Cell Tissue Res,1999,295(2):279-285.
50. Mobasheri A, Airley R, Hewitt SM, et al.Heterogeneous expression of the aquaporin 1(AQP1) water channel in tumors of the prostate, breast, ovary, colon and lung:a study using high density multiple human tumor tissue microarrays [J].Inter J of Onc,2005,26(5):1149-1158.
