K、CL离子通道阻滞对体外培养兔关节软骨细胞基质合成代谢的影响
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摘要
目的：探讨阻断体外培养兔关节软骨细胞膜上的K、CL离子通道对其糖胺多糖(glycosaminoglycan, GAG)、Ⅱ型胶原基质合成代谢的影响。
方法：2月龄新西兰白兔,无菌条件下切取双膝关节软骨,酶解法分离软骨细胞,以5×104/孔接种于24孔板中。正常培养2天细胞贴壁后换液,并以12个孔为一组随机分为三组,对照组：用DMEM/F12正常培养；K离子通道阻滞组(简称4-AP组)：利用含lmmol/L4-氨基吡啶(4-aminopyridine,4-AP)的DMEM/F12培养,特异性阻断电压门控型K离子通道；CL离子通道阻滞组(简称SITS组)：利用含lmmol/L 4-乙酰氨基-4‘-异硫氰基-2,2’-乙拌磷酸均二苯乙烯(4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene, SITS)的DMEM/F12培养,阻断阴离子通道,主要是CL离子通道。分别在换液后第3、6、9天留取各孔上清液并分为两份,一份以阿尔新蓝法检测其GAG的含量,同时另一份以ELISA法检测Ⅱ型胶原的含量(n=12)。
结果：与对照组比较,4-AP组在第3天时GAG含量明显下降(P<0.05),但第6天和9天并无显著差别(p>0.05),同时Ⅱ型胶原在第3天和6天含量显著增加(P<0.05),第9天时有下降趋势,但统计学检验无明显差异；SITS组在第3、6、9天GAG含量都显著增加(P<0.05),同时Ⅱ型胶原在第3天和6天含量显著增加(P<0.05),第9天仍然有增加的趋势,但统计学检验无明显差异。
结论：阻断K、CL离子通道后显著促进软骨细胞GAG、Ⅱ型胶原的合成,尤其是阻断CL离子通道后,GAG的合成增加尤为明显。
Objective:To investgate the effects of potassium and chloride channel blockers on The glycosaminoglycan(GAG) and collagen type II synthesis of rabbit articular chondrocytes in vitro.
Methods:2 months New Zealand rabbits were killed and their knee joint were taken out under aseptic condition. The chondrocytes were isolated by enzymolysis method and cultivated in 24-well plates (seeded 5×104 cells per well).Then the chondrocytes were divided into three groups randomly after cultured 2 days when the cells were adherent. The control group was cultured by DMEM/F12 while the potassium channel blockon group(4-AP group)was cultured in the media contained 1mmol/L 4-aminopyridine (4-AP), which can lead to the blockon of the potassium channel,and the chloride channel blockon group(SITS group)was cultured in the media contained 1mmol/L 4-acetamido-4-isothiocyano-2,2-disulfonic acid stilbene(SITS), which can lead to the blockon of the chloride channel.The GAG synthesis were measured by Alician Blue method and collagen typeⅡsynthesis were estimated by the ELISA in the media when medium was change after3-,6-and 9-day (n=12)
Result:Compared with the control group.the GAG content of 4-AP group has significant decrease at 3th day(P<0.05),but has no significant difference at 6th and 9th day (p>0.05),while the collagen typeⅡcontent has significant increased at 3th and 6th day (P<0.05), and has decrease tendency at 9th day. but have no significant difference.The GAG content of SITS group has significant increased at 3th,6th and 9th day (P<0.05).while the collagen typeⅡcontent has significant increased at 3th and 6th day (P<0.05),and has increased tendency at 9th day, but have no significant difference.
Conclusion:The blockon of potassium and chloride channel can increase the GAG and collagen typeⅡsynthesis of chondrocytes in vitro, especially when block chloride channel,the GAG synthesis was significant.
方法：2月龄新西兰白兔,无菌条件下切取双膝关节软骨,酶解法分离软骨细胞,以5×104/孔接种于24孔板中。正常培养2天细胞贴壁后换液,并以12个孔为一组随机分为三组,对照组：用DMEM/F12正常培养；K离子通道阻滞组(简称4-AP组)：利用含lmmol/L4-氨基吡啶(4-aminopyridine,4-AP)的DMEM/F12培养,特异性阻断电压门控型K离子通道；CL离子通道阻滞组(简称SITS组)：利用含lmmol/L 4-乙酰氨基-4‘-异硫氰基-2,2’-乙拌磷酸均二苯乙烯(4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene, SITS)的DMEM/F12培养,阻断阴离子通道,主要是CL离子通道。分别在换液后第3、6、9天留取各孔上清液并分为两份,一份以阿尔新蓝法检测其GAG的含量,同时另一份以ELISA法检测Ⅱ型胶原的含量(n=12)。
结果：与对照组比较,4-AP组在第3天时GAG含量明显下降(P<0.05),但第6天和9天并无显著差别(p>0.05),同时Ⅱ型胶原在第3天和6天含量显著增加(P<0.05),第9天时有下降趋势,但统计学检验无明显差异；SITS组在第3、6、9天GAG含量都显著增加(P<0.05),同时Ⅱ型胶原在第3天和6天含量显著增加(P<0.05),第9天仍然有增加的趋势,但统计学检验无明显差异。
结论：阻断K、CL离子通道后显著促进软骨细胞GAG、Ⅱ型胶原的合成,尤其是阻断CL离子通道后,GAG的合成增加尤为明显。
Objective:To investgate the effects of potassium and chloride channel blockers on The glycosaminoglycan(GAG) and collagen type II synthesis of rabbit articular chondrocytes in vitro.
Methods:2 months New Zealand rabbits were killed and their knee joint were taken out under aseptic condition. The chondrocytes were isolated by enzymolysis method and cultivated in 24-well plates (seeded 5×104 cells per well).Then the chondrocytes were divided into three groups randomly after cultured 2 days when the cells were adherent. The control group was cultured by DMEM/F12 while the potassium channel blockon group(4-AP group)was cultured in the media contained 1mmol/L 4-aminopyridine (4-AP), which can lead to the blockon of the potassium channel,and the chloride channel blockon group(SITS group)was cultured in the media contained 1mmol/L 4-acetamido-4-isothiocyano-2,2-disulfonic acid stilbene(SITS), which can lead to the blockon of the chloride channel.The GAG synthesis were measured by Alician Blue method and collagen typeⅡsynthesis were estimated by the ELISA in the media when medium was change after3-,6-and 9-day (n=12)
Result:Compared with the control group.the GAG content of 4-AP group has significant decrease at 3th day(P<0.05),but has no significant difference at 6th and 9th day (p>0.05),while the collagen typeⅡcontent has significant increased at 3th and 6th day (P<0.05), and has decrease tendency at 9th day. but have no significant difference.The GAG content of SITS group has significant increased at 3th,6th and 9th day (P<0.05).while the collagen typeⅡcontent has significant increased at 3th and 6th day (P<0.05),and has increased tendency at 9th day, but have no significant difference.
Conclusion:The blockon of potassium and chloride channel can increase the GAG and collagen typeⅡsynthesis of chondrocytes in vitro, especially when block chloride channel,the GAG synthesis was significant.
引文
[1]Isoya E, Toyoda F, Imai S, Okumura N, Kumagai K, Omatsu-Kanbe M, Kubo M, Matsuura H, Matsusue Y. Swelling-activated Cl(-) current in isolate d rabbit articular chondrocytes:inhibition by arachidonic Acid. J Pharmacol Sci.2009 Feb; 109(2):293-304.
[2]Mobasheri A, Gent TC, Nash Al, Womack MD, Moskaluk CA, Barrett-Jolley R. Evidence for functional ATP-sensitive (K(ATP)) potassium channels in human and equine articular chondrocytes. Osteoarthritis Cartilage. 2007 Jan;15(1):1-8.
[3]Ponce A. Expression of voltage dependent potassium currents in freshly dissociated rat articular chondrocytes. Cell Physiol Biochem.2006; 18(1-3):35-46.
[4]Sanchez JC, Powell T, Staines HM, Wilkins RJ. Electrophysiological demonstration of voltage-activated H+channels in bovine articular chondrocytes. Cell Physiol Biochem.2006; 18(1-3):85-90.
[5]Tanaka N,Ohno S,Honda K,et al.Cyclic mechanical strain regulates the PTHrP expression in cultured chondrocytes via activation of the Ca2+chan nel.J Dent Res,2005,84:64-68.
[6]Salter,Millward-Sadler S,Nuki,et al.MBChB+integrin-interleukin-4 mechanotransduction pathways in human chondrocytes.Clin Orthop Relat Res, 2001,49-60.
[7]Yao X, Kwan HY. Activity of voltage-gated K channels is associated with cell proliferation and Ca2 influx in carcinoma cells of colon cancer. Life Sci 1999;65:55-62.
[8]Wohlrab D, Wohlrab J, Reichel H, et al. Is the proliferation of human chondrocytes regulated by ionic channels? J Orthop Sci 2001;6:155-9.
[9]David Wohlrab, Michaela Vocke, Thomas Klapperstuck, and Werner Hein. Effects of potassium and anion channel blockers on the cellular response of human osteoarthritic chondrocytes.J Orthop Sci (2004) 9:364-371
[10]Bojrnsson S. Simultaneous preparation and quantitation of proteoglycans by precipitation with alcian blue. Anal Biochem.1993,210:282-291.
[11]Wohlrab D, Wohlrab J, Reichel H, et al. Is the proliferation of human chondrocytes regulated by ionic channels? J Orthop Sci 2001;6:155-9.
[12]Hille B. Ionic channels of excitable membranes.2nd ed. Sunderland, Massachusetts:Sinauer Associates; 1992.
[13]Gryphon L. Perkins. Assia Derfoul. Allison Ast.David J. Hall An inhibitor of the stretch-activated cation receptor exerts a potent effect on chondrocyte phenotype. Differentiation.2005 Jun;73(5):199-211
[1]Blanco FJ,Ochs RL, Schwarz H,et al. Chondrocyte apoptosis induce by nitric oxide. Am J Pathol,1995,146 (1):75-85.
[2]Blanco FJ,Lotz M. IL-1-induced nitric oxide inhibits chondrocyte proliferation via PGE-2. Exp Cell RES,1995,218 (1):319-325.
[3]Pelletier J P,Jovanovic DV,Lascau-Coman V. Selective inhibition of inducible nitric oxide synthase reduces progression of esperimental osteoarthritis in vivo possible linkwith the reduction in chondrocyte apoptosis and caspase 3 level. Arthritis Rheum,2000,43 (6):1290-1299.
[4]Studer R,Jaffurs D,Stefanovic-Racic M. Nitric oxide in osteoarthritis. Osteoarthritis Cartilage,1999,7 (4):377-379.
[5]Delcarlo M J r,Loeser RF. Nitric oxide-mediated chondrocytecell death requires the generation of additional reactive oxygen species[J]. Arthritis Rheum,2002,46 (2):394-403.
[6]Miwa M,Saura R,Hirata S,et al. Induction of apoptosis in bovine articular chondrocyte by prostaglandin E(2) through cAMP-dependent pathway. Osteoarthritis Cartilage,2000,8 (1):17-24.
[7]Hashimoto S, Setareh M, Roseen F, et al. Fas/Fas ligand expression and induction of apoptosis in chondrocytes. Arthritis Rheum,1997,40(10):1749-1755.
[8]Masuko-Hongo K,Sakata M, Yuan GH,et al. Expression of Fas-associated death domain-like interleukin-1 beta-converting enzyme (FL ICE) inhibitory protein (FL IP) in human articular chondrocytes:possible contribution to the resistance to Fas-mediated death of in vitro cultured human articular chondrocytes [J] Rheumatol Int,2001,21 (3):112—121.
[9]Lisignoli G,Grassi F,Zini N,et al. Anti-Fas-induced apoptosis in chondrocytes reduced by hyaluronan:evidence for CD44 and CD54 (intercellular adhesion molecule 1) invovement [J].Arthritis Rheum,2001,44 (8):1800—1807.
[10]Hashimoto S,Ochs RL,Komiya S,et al. Linkage of Chondrocyte apoptosis and cartilage degradation in human osteoarthritis. Arthritis Rheum,1998,41 (9):1632-1638.
[11]Blanco FJ,Guitian R,Vazquez-Martul E,et al. Osteoarthritis chondrocyte die by apoptosis. A possible pathway for Osteoarthritis pathology. Arthritis Rheum,1998,41 (2):284-289.
[12]Adams SC,JR Horton WE. Chondrocyte apoptosis increases with age in the articular cartilage of adult animals. Anat Record,1998,250:418-425.
[13]Aigner T,Hemmel M,Neureiter D,et al. Apoptotic cell death is not a widespread phenomenon in normal aging and osteoarthritis human articular knee cartilage [J]. Arthritis Rheum,2001,44 (6):1304—1312.
[14]Hashimoto S,Ochs RL.Rosen F,et al. Chondrocyte-derived apoptotic bodies and calcification of articular cartilage. Proc Natl Acad Sci USA.1998,95:3094-3099.
[15]GoldringMB, Birkhead J, Sandell LJ,et al. [J]. J Clin Invest,1998,82(6):2026.
[16]Miwa M, Saura R,Hirata S, et al. [J]. Osteo Cart,2002,8 (1):17-24.
[17]Wong M, Carter DR. Articular cart ilage functional histomorphology and mechanobiology:a research perspective [J]. Bone,2003,33:1-13.
[18]Loening AM, James IE, Levenston ME, Badger AM, Frank EH, Kurz B, Nuttall ME, Hung HH, Blake SM, Grodzinsky AJ, Lark MW.Injurious mechanical compression of bovine articular cartilage induces chondrocyte apoptosis.Arch Biochem Biophys.2000 Sep 15;381(2):205-12.
[19]D'Lima DD, Hashimoto S, Chen PC, Colwell CW Jr, Lotz MK.Human chondrocyte apoptosis in response to mechanical injury.Osteoarthritis Cartilage. 2001 Nov;9(8):712-9.
[20]Borrelli J Jr, Tinsley K, Ricci WM, Burns M, Karl IE, HotchkissR.Induction of chondrocyte apoptosis following impact load. J Orthop Trauma.2003 Oct;17(9):635-41.
[21]Healy ZR, Lee NH, Gao X, Goldring MB, Talalay P, Kensler TW, Konstantopoulos K.Divergent responses of chondrocytes and endothelial cells to shear stress:cross-talk among COX-2, the phase 2 response, and apoptosis.Proc Natl Acad Sci U S A.2005 Sep 27;102(39):14010-5.
[22]David Wohlrab, Michaela Vocke, Thomas Klapperstuck, and Werner Hein. Effects of potassium and anion channel blockers on the cellular response of human osteoarthritic chondrocytes.J Orthop Sci (2004) 9:364-371
[23]Wohlrab D, Vocke M, Klapperstiick T, Hein W.The influence of lidocaine and verapamil on the proliferation, CD44 expression and apoptosis behavior of human chondrocytes. Int J Mol Med.2005 Jul;16(1):149-57.
[24]Mancilla EE, Galindo M, Fertilio B, Herrera M, Salas K, Gatica H, Goecke A.L-type calcium channels in growth plate chondrocytes participate in endochondral ossification.J Cell Biochem.2007 May 15;101(2):389-98.
[25]Kim HA, Suh DI, Song YW.Relationship between chondrocyte apoptosis and matrix depletion in human articular cartilage. J Rheumatol.2001 Sep;28(9):2038-45.
[26]Lotz M,Hashimoto S, Kuhn K. [J]. Osteo Cart,1999,7 (4):389-391.
[27]Zhao H, Qiu GX, Guan J, Zhao Y, Zhou X. [Correlation of apoptosis of articular chondrocytes in osteoarthritis with degree of cartilage destruction and expression of apoptosis-related proteins:surviving, caspase-3, and Bcl-xl]. Zhonghua Yi Xue Za Zhi.2008 May 20;88(19):1339-41.
[2]Mobasheri A, Gent TC, Nash Al, Womack MD, Moskaluk CA, Barrett-Jolley R. Evidence for functional ATP-sensitive (K(ATP)) potassium channels in human and equine articular chondrocytes. Osteoarthritis Cartilage. 2007 Jan;15(1):1-8.
[3]Ponce A. Expression of voltage dependent potassium currents in freshly dissociated rat articular chondrocytes. Cell Physiol Biochem.2006; 18(1-3):35-46.
[4]Sanchez JC, Powell T, Staines HM, Wilkins RJ. Electrophysiological demonstration of voltage-activated H+channels in bovine articular chondrocytes. Cell Physiol Biochem.2006; 18(1-3):85-90.
[5]Tanaka N,Ohno S,Honda K,et al.Cyclic mechanical strain regulates the PTHrP expression in cultured chondrocytes via activation of the Ca2+chan nel.J Dent Res,2005,84:64-68.
[6]Salter,Millward-Sadler S,Nuki,et al.MBChB+integrin-interleukin-4 mechanotransduction pathways in human chondrocytes.Clin Orthop Relat Res, 2001,49-60.
[7]Yao X, Kwan HY. Activity of voltage-gated K channels is associated with cell proliferation and Ca2 influx in carcinoma cells of colon cancer. Life Sci 1999;65:55-62.
[8]Wohlrab D, Wohlrab J, Reichel H, et al. Is the proliferation of human chondrocytes regulated by ionic channels? J Orthop Sci 2001;6:155-9.
[9]David Wohlrab, Michaela Vocke, Thomas Klapperstuck, and Werner Hein. Effects of potassium and anion channel blockers on the cellular response of human osteoarthritic chondrocytes.J Orthop Sci (2004) 9:364-371
[10]Bojrnsson S. Simultaneous preparation and quantitation of proteoglycans by precipitation with alcian blue. Anal Biochem.1993,210:282-291.
[11]Wohlrab D, Wohlrab J, Reichel H, et al. Is the proliferation of human chondrocytes regulated by ionic channels? J Orthop Sci 2001;6:155-9.
[12]Hille B. Ionic channels of excitable membranes.2nd ed. Sunderland, Massachusetts:Sinauer Associates; 1992.
[13]Gryphon L. Perkins. Assia Derfoul. Allison Ast.David J. Hall An inhibitor of the stretch-activated cation receptor exerts a potent effect on chondrocyte phenotype. Differentiation.2005 Jun;73(5):199-211
[1]Blanco FJ,Ochs RL, Schwarz H,et al. Chondrocyte apoptosis induce by nitric oxide. Am J Pathol,1995,146 (1):75-85.
[2]Blanco FJ,Lotz M. IL-1-induced nitric oxide inhibits chondrocyte proliferation via PGE-2. Exp Cell RES,1995,218 (1):319-325.
[3]Pelletier J P,Jovanovic DV,Lascau-Coman V. Selective inhibition of inducible nitric oxide synthase reduces progression of esperimental osteoarthritis in vivo possible linkwith the reduction in chondrocyte apoptosis and caspase 3 level. Arthritis Rheum,2000,43 (6):1290-1299.
[4]Studer R,Jaffurs D,Stefanovic-Racic M. Nitric oxide in osteoarthritis. Osteoarthritis Cartilage,1999,7 (4):377-379.
[5]Delcarlo M J r,Loeser RF. Nitric oxide-mediated chondrocytecell death requires the generation of additional reactive oxygen species[J]. Arthritis Rheum,2002,46 (2):394-403.
[6]Miwa M,Saura R,Hirata S,et al. Induction of apoptosis in bovine articular chondrocyte by prostaglandin E(2) through cAMP-dependent pathway. Osteoarthritis Cartilage,2000,8 (1):17-24.
[7]Hashimoto S, Setareh M, Roseen F, et al. Fas/Fas ligand expression and induction of apoptosis in chondrocytes. Arthritis Rheum,1997,40(10):1749-1755.
[8]Masuko-Hongo K,Sakata M, Yuan GH,et al. Expression of Fas-associated death domain-like interleukin-1 beta-converting enzyme (FL ICE) inhibitory protein (FL IP) in human articular chondrocytes:possible contribution to the resistance to Fas-mediated death of in vitro cultured human articular chondrocytes [J] Rheumatol Int,2001,21 (3):112—121.
[9]Lisignoli G,Grassi F,Zini N,et al. Anti-Fas-induced apoptosis in chondrocytes reduced by hyaluronan:evidence for CD44 and CD54 (intercellular adhesion molecule 1) invovement [J].Arthritis Rheum,2001,44 (8):1800—1807.
[10]Hashimoto S,Ochs RL,Komiya S,et al. Linkage of Chondrocyte apoptosis and cartilage degradation in human osteoarthritis. Arthritis Rheum,1998,41 (9):1632-1638.
[11]Blanco FJ,Guitian R,Vazquez-Martul E,et al. Osteoarthritis chondrocyte die by apoptosis. A possible pathway for Osteoarthritis pathology. Arthritis Rheum,1998,41 (2):284-289.
[12]Adams SC,JR Horton WE. Chondrocyte apoptosis increases with age in the articular cartilage of adult animals. Anat Record,1998,250:418-425.
[13]Aigner T,Hemmel M,Neureiter D,et al. Apoptotic cell death is not a widespread phenomenon in normal aging and osteoarthritis human articular knee cartilage [J]. Arthritis Rheum,2001,44 (6):1304—1312.
[14]Hashimoto S,Ochs RL.Rosen F,et al. Chondrocyte-derived apoptotic bodies and calcification of articular cartilage. Proc Natl Acad Sci USA.1998,95:3094-3099.
[15]GoldringMB, Birkhead J, Sandell LJ,et al. [J]. J Clin Invest,1998,82(6):2026.
[16]Miwa M, Saura R,Hirata S, et al. [J]. Osteo Cart,2002,8 (1):17-24.
[17]Wong M, Carter DR. Articular cart ilage functional histomorphology and mechanobiology:a research perspective [J]. Bone,2003,33:1-13.
[18]Loening AM, James IE, Levenston ME, Badger AM, Frank EH, Kurz B, Nuttall ME, Hung HH, Blake SM, Grodzinsky AJ, Lark MW.Injurious mechanical compression of bovine articular cartilage induces chondrocyte apoptosis.Arch Biochem Biophys.2000 Sep 15;381(2):205-12.
[19]D'Lima DD, Hashimoto S, Chen PC, Colwell CW Jr, Lotz MK.Human chondrocyte apoptosis in response to mechanical injury.Osteoarthritis Cartilage. 2001 Nov;9(8):712-9.
[20]Borrelli J Jr, Tinsley K, Ricci WM, Burns M, Karl IE, HotchkissR.Induction of chondrocyte apoptosis following impact load. J Orthop Trauma.2003 Oct;17(9):635-41.
[21]Healy ZR, Lee NH, Gao X, Goldring MB, Talalay P, Kensler TW, Konstantopoulos K.Divergent responses of chondrocytes and endothelial cells to shear stress:cross-talk among COX-2, the phase 2 response, and apoptosis.Proc Natl Acad Sci U S A.2005 Sep 27;102(39):14010-5.
[22]David Wohlrab, Michaela Vocke, Thomas Klapperstuck, and Werner Hein. Effects of potassium and anion channel blockers on the cellular response of human osteoarthritic chondrocytes.J Orthop Sci (2004) 9:364-371
[23]Wohlrab D, Vocke M, Klapperstiick T, Hein W.The influence of lidocaine and verapamil on the proliferation, CD44 expression and apoptosis behavior of human chondrocytes. Int J Mol Med.2005 Jul;16(1):149-57.
[24]Mancilla EE, Galindo M, Fertilio B, Herrera M, Salas K, Gatica H, Goecke A.L-type calcium channels in growth plate chondrocytes participate in endochondral ossification.J Cell Biochem.2007 May 15;101(2):389-98.
[25]Kim HA, Suh DI, Song YW.Relationship between chondrocyte apoptosis and matrix depletion in human articular cartilage. J Rheumatol.2001 Sep;28(9):2038-45.
[26]Lotz M,Hashimoto S, Kuhn K. [J]. Osteo Cart,1999,7 (4):389-391.
[27]Zhao H, Qiu GX, Guan J, Zhao Y, Zhou X. [Correlation of apoptosis of articular chondrocytes in osteoarthritis with degree of cartilage destruction and expression of apoptosis-related proteins:surviving, caspase-3, and Bcl-xl]. Zhonghua Yi Xue Za Zhi.2008 May 20;88(19):1339-41.