巴戟天多糖对体外培养兔软骨细胞增殖和凋亡的影响
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摘要
目的：建立体外培养兔软骨细胞的体系，应用体外培养的兔软骨细胞，在细胞和分子水平观察巴戟天多糖对软骨细胞增殖和凋亡的影响，探讨巴戟天多糖治疗骨性关节炎的机理。方法：分离大耳白兔的软骨细胞，通过体外培养，进行形态学和超微结构观察、应用MTT法进行生长曲线描绘和对原代软骨细胞Ⅱ型胶原鉴定，建立兔软骨细胞的体外培养体系，为实验提供应用细胞；培养后的软骨细胞应用MTT法、流式细胞仪和5-BrdU掺入法测定巴戟天多糖对软骨细胞增殖的影响，应用免疫组化检测PCNA的表达情况，对巴戟天多糖促进软骨细胞增殖的机理进行初步探讨；通过形态学、细胞超微结构和生化特征的改变来验证由全反式维甲酸(RA)诱导的软骨细胞凋亡模型，流式细胞术检测凋亡软骨细胞凋亡率、应用Realtime-PCR对凋亡相关基因bcl-2、bax的表达等进行观察，对巴戟天多糖在细胞凋亡过程中的作用进行初步评价。结果：体外培养的兔软骨细胞有Ⅱ型胶原表达，说明培养的细胞为软骨细胞；一定浓度的巴戟天多糖对体外培养的兔软骨细胞增殖有显著促进作用，且这种作用在第5天时较第3天明显，巴戟天多糖对兔软骨细胞增殖的影响与最适浓度的维骨力无显著性差异，其促进软骨细胞增殖与增殖核抗原(PCNA)的表达相关，巴戟天多糖可以使软骨细胞PCNA的表达增加；巴戟天多糖的应用可以显著降低由于应用全反式维甲酸(RA)而引起的软骨细胞凋亡率，最适浓度的巴戟天多糖组与维骨力组对软骨细胞凋亡的影响无显著性差异，诱导凋亡的软骨细胞在巴戟天多糖组bcl-2表达明显高于诱导组，而bax表达明显低于诱导组，巴戟天多糖对bcl-2和bax表达情况的改变与维骨力相似。结论：巴戟天多糖治疗骨性关节炎机理有三：其一，巴戟天多糖中可能直接刺激兔软骨细胞增殖，使其数量不断增加；其二，巴戟天多糖可以使软骨细胞中的细胞增殖核抗原的表达增加，进而促使软骨细胞增殖；其三，巴戟天多糖的应用可以使全反式维甲酸诱导的凋亡软骨细胞减少，从而促进细胞有丝分裂，抑制细胞凋亡；可以使凋亡抑制基因bcl-2的表达增加，凋亡促进基因bax的表达下降，从而使bcl-2在与bax形成的同源二聚体中占据优势来阻止线粒体膜上的PT孔道开放，达到抑制凋亡的目的。
Objective: To establish a model of cultivating the rabbit chondral cells outside the body,observe the effect of Morida officinalis How polysaccharide on the proliferation andapoptosis on the level of cell and molecule and explore the mechanism of curing osteoarthritisby using Morinda officinalis How polysaccharide.
Method: Separate the chondral cells of white rabbits for observations of morphologyand ultrastructure, take advantage of MTT method to depict the growth curvature and identifytypeⅡcollangen of the primary chondral cells, establish the cultivating system of the rabbitchondral cells outside the body to supply applicable cells for the experiments; use thecultivated chondral cells for MTT method, flow cytometer and incorporating 5-BrdU to detectthe effect of Morida officinalis How polysaccharide on the proliferation of chondral cells, andapply the expressions of immunohistochemical detecting PCNA to make preliminaryexplorations of the mechanism of Morida officinalis How polysaccharide boosting theproliferation of chondral cells; employ the changes in morphology, ultrastructure andbiochemical characters to validate the model of apoptosis of chondral cells induced byRA,use flow cytometer to detect the apoptosis rates of chondral cells, use Realtime-PCR toconduct observations of the expressions of the relevant factors such as bcl-2 and bax, andmake preliminary appraisals of the role of Morida officinalis How polysaccharide in thecourse of cell apoptosis.
Findings: the rabbit chondral cells cultivated outside the body have the expression oftypeⅡcollangen, which means the cultivated cells are chondral cells; Morida officinalis Howpolysaccharide of a certain density acts as a remarkable stimulus to the proliferation of therabbit chondral cells cultivated outside the body and the effect becomes more obvious on the5th day than on the 3rd day. There is no significant difference between the effect of Moridaofficinalis How polysaccharide on the proliferation of the rabbit chondral cells and viartrils ofthe most suitable density, but it is related with PCNA; in other words, Morida officinalis Howpolysaccharide enhances the expression of PCNA of chondral cells. The application ofMorida officinalis How polysaccharide can remarkably reduce the rate of apoptosis ofchondral cells caused by the use of RA. There is no significant difference between Moridaofficinalis How polysaccharide group of the most suitable density and the effect of viartrilsgroup on the apoptosis of chondral cells. The expression of the chondral cells inducingapoptosis in Morida officinalis how polysaccharide group bcl-2 is obviously higher than thatin the inducing group while the expression of bax is much lower than the contrast group. Thechanges in the expressions of Morida officinalis how polysaccharide in bcl-2 and bax aresimilar to that in viartrils.
Conclusion: There are 3 mechanisms that Morida officinalis How polysaccharide can beused to treat osteoarthritis: first, Morida officinalis How polysaccharide may directlystimulate the proliferation of rabbit chondral cell and boost up its number; second, Moridaofficinalis How polysaccharide increases the expression of Proliferating Cell Nuclear Antigenin chondral cells, and further promote the proliferation of chondral cell; third, the applicationof Morida officinalis How polysaccharide reduce the number of extinct chondral cell inducedby all-trans-retinoic acid, which promote the mitosis of the cells and hold up the apoptosis ofthe cells. Besides, Morida officinalis how polysaccharide can increase the expression of bcl-2but lower the expression of bax, thus opening the PT channel to check the process ofapoptosis.
Objective: To establish a model of cultivating the rabbit chondral cells outside the body,observe the effect of Morida officinalis How polysaccharide on the proliferation andapoptosis on the level of cell and molecule and explore the mechanism of curing osteoarthritisby using Morinda officinalis How polysaccharide.
Method: Separate the chondral cells of white rabbits for observations of morphologyand ultrastructure, take advantage of MTT method to depict the growth curvature and identifytypeⅡcollangen of the primary chondral cells, establish the cultivating system of the rabbitchondral cells outside the body to supply applicable cells for the experiments; use thecultivated chondral cells for MTT method, flow cytometer and incorporating 5-BrdU to detectthe effect of Morida officinalis How polysaccharide on the proliferation of chondral cells, andapply the expressions of immunohistochemical detecting PCNA to make preliminaryexplorations of the mechanism of Morida officinalis How polysaccharide boosting theproliferation of chondral cells; employ the changes in morphology, ultrastructure andbiochemical characters to validate the model of apoptosis of chondral cells induced byRA,use flow cytometer to detect the apoptosis rates of chondral cells, use Realtime-PCR toconduct observations of the expressions of the relevant factors such as bcl-2 and bax, andmake preliminary appraisals of the role of Morida officinalis How polysaccharide in thecourse of cell apoptosis.
Findings: the rabbit chondral cells cultivated outside the body have the expression oftypeⅡcollangen, which means the cultivated cells are chondral cells; Morida officinalis Howpolysaccharide of a certain density acts as a remarkable stimulus to the proliferation of therabbit chondral cells cultivated outside the body and the effect becomes more obvious on the5th day than on the 3rd day. There is no significant difference between the effect of Moridaofficinalis How polysaccharide on the proliferation of the rabbit chondral cells and viartrils ofthe most suitable density, but it is related with PCNA; in other words, Morida officinalis Howpolysaccharide enhances the expression of PCNA of chondral cells. The application ofMorida officinalis How polysaccharide can remarkably reduce the rate of apoptosis ofchondral cells caused by the use of RA. There is no significant difference between Moridaofficinalis How polysaccharide group of the most suitable density and the effect of viartrilsgroup on the apoptosis of chondral cells. The expression of the chondral cells inducingapoptosis in Morida officinalis how polysaccharide group bcl-2 is obviously higher than thatin the inducing group while the expression of bax is much lower than the contrast group. Thechanges in the expressions of Morida officinalis how polysaccharide in bcl-2 and bax aresimilar to that in viartrils.
Conclusion: There are 3 mechanisms that Morida officinalis How polysaccharide can beused to treat osteoarthritis: first, Morida officinalis How polysaccharide may directlystimulate the proliferation of rabbit chondral cell and boost up its number; second, Moridaofficinalis How polysaccharide increases the expression of Proliferating Cell Nuclear Antigenin chondral cells, and further promote the proliferation of chondral cell; third, the applicationof Morida officinalis How polysaccharide reduce the number of extinct chondral cell inducedby all-trans-retinoic acid, which promote the mitosis of the cells and hold up the apoptosis ofthe cells. Besides, Morida officinalis how polysaccharide can increase the expression of bcl-2but lower the expression of bax, thus opening the PT channel to check the process ofapoptosis.
引文
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[10] 舒谦，卿茂盛，曹业飞，等．六味地黄汤含药血清对软骨细胞增殖、凋亡影响的体外实验研究[J]．中国中医药科技，2006；13(4)：217—219．
[11] 邵敏，牛维，黄杰文．补肾活血中药促进体外培养软骨细胞增殖和蛋白质合成的作用[J]．中国临床康复，2006：10(19)：50—52．
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6．许学猛，王羽丰，邓晋丰，等．补肾活血胶囊影响兔膝关节退变性疾病骨内压变化的实验研究[J]．中国中医骨伤科杂志，2001，91(4)：24．
7．张德辉，薛刚，黄昌林．应用玻璃酸钠对关节镜术后膝骨关节炎功能恢复的影响[J]．中国临床康复，2002，6(12)：1730-1731．
8. Bukhardt GS,Wright GC,Sokoloff L.The effects of sodium selenite on chondrcvtes in monolayer culture.Arthritis Rheum[J]. 1986, 29:660.
9．许鹏，姚建锋，蔡乾坤，等．骨关节病患者病情程度与体内自由基含量变化分析[J]．中国矫形外科杂志，2001，8(5)：469．
10．张洁浩，许善锦．自由基在大骨节病病理过程中的作用．北京医科大学学报．1990，22(1)：50．
11. Largo R,Alvarez-Soria MA,Diez-Ortego I,et al.Glucosamine inhibits IL-1 beta-induced NfkappaB activation in human osteoarthritic chondrocytes[J].Osteoarthritis Cartilage,2003,11 (4):290-298.
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19．李德达，张凯，王毅，等．OA患者关节液与血清中金属蛋白酶及抑止剂的检测及，临床意义[J]．中国骨伤，2002，15(10)：580-585．
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[1] 吕宏斌，岳珍，王嘉芙．4种中药对体外培养兔软骨细胞代谢的影响[J]．中国运动医学杂志，1995；14(3)：135—137．
[2] 宴雪生，彭亚琴，明安萍．川芎嗪注射液对体外培养软骨细胞影响的实验研究[J]．中国中医骨伤科杂志，2002；10(1)：15—17．
[3] 吕宏斌，王嘉芙，岳珍。体外培养的兔软骨细胞对某些中药反应的超微结构观察．中国运动医学杂志[J]．1997；16(1)：56—58．
[4] 沈霖，沈耀，杨述华．丹参注射液对维甲酸诱导的软骨细胞凋亡的影响[J]．中国中医骨伤科杂志，2002；10(5)：1—3．
[5] 郭颖杰，周秋丽，刘萍，等．鹿茸多肽对骨、软骨细胞增殖的实验研究[J]．中国生化药物杂志，1998；19(2)：74—75．
[6] 岳珍，王嘉芙，吕宏斌．行气消肿类中药对体外培养的软骨细胞代谢的影响[J]．中国运动医学杂志，1998；17(1)：34—36．
[7] 冯秋珍，杨运东，胡继鹰，等．防风注射液对体外培养软骨细胞超氧化物歧化酶的影响[J]．时珍国医国药，2002；13(4)：205—207．
[8] 陈飞雁，顾湘杰，钟明康，等．威灵仙注射液对骨关节炎关节液与软骨白介素—1水平的影响[J]．中国矫形外科杂志，2004；12(7)：524—525．
[9] 唐勇，马民，曹勇，等．补肾益气活血方对家兔软骨细胞增殖与凋亡的影响[J]．安徽中医学院学报，2004；23(2)：42—46．
[10] 舒谦，卿茂盛，曹业飞，等．六味地黄汤含药血清对软骨细胞增殖、凋亡影响的体外实验研究[J]．中国中医药科技，2006；13(4)：217—219．
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[12] 黄枫，谢国平．补肾方含药血清对软骨细胞增殖的影响[J]．广州中医药大学学报，2005；22(2)：138—140．
[13] 郭玉海，林定坤，王昭佩，等．参麦注射液对体外培养软骨细胞增殖的影响[J]．广东中医，2006；27(4)：476—477．
[14] 冯伟，石印玉，沈培芝，等．中药对软骨细胞代谢的影响[J]．中国中医骨伤科杂志，2000；8(2)：9—11．
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[16] 董朝岚，朱萱萱．痛痹颗粒对兔软骨细胞及其分泌NO影响的实验研究[J]．江西中医学院学报，2006；18(1)：38—39．
1. Alon T, HEMO I,ITIN,et al.VEGF acts as a survival factor for newly formed retinal vessels and implications for retinophathy of prematurity[J].Nat Med, 1995:1024.
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4. Felson DT, Zhang Y, Anthony JM,et al.Weight loss reduce the risk for symptomatic knee osteoarthritis in women[J].Ann Intern Med, 1992,116:535-539.
5. Arnoldi CC,Lemperg RK, Linderholm H.Intrasseous hypertension and pain in the knee[J]. Bone Joint Surg(Br). 1975,57:360.
6．许学猛，王羽丰，邓晋丰，等．补肾活血胶囊影响兔膝关节退变性疾病骨内压变化的实验研究[J]．中国中医骨伤科杂志，2001，91(4)：24．
7．张德辉，薛刚，黄昌林．应用玻璃酸钠对关节镜术后膝骨关节炎功能恢复的影响[J]．中国临床康复，2002，6(12)：1730-1731．
8. Bukhardt GS,Wright GC,Sokoloff L.The effects of sodium selenite on chondrcvtes in monolayer culture.Arthritis Rheum[J]. 1986, 29:660.
9．许鹏，姚建锋，蔡乾坤，等．骨关节病患者病情程度与体内自由基含量变化分析[J]．中国矫形外科杂志，2001，8(5)：469．
10．张洁浩，许善锦．自由基在大骨节病病理过程中的作用．北京医科大学学报．1990，22(1)：50．
11. Largo R,Alvarez-Soria MA,Diez-Ortego I,et al.Glucosamine inhibits IL-1 beta-induced NfkappaB activation in human osteoarthritic chondrocytes[J].Osteoarthritis Cartilage,2003,11 (4):290-298.
12. Homandberg GA,Umadi V, Kang H,et al.High molecular weight hyaluronan promotes repair of IL-1beta-damaged cartilag explants from both young and oldbovines[J].Osteoarthritis Cartilage,2003,11 (3): 177-186.
13. Vuolteenaho K,Moilanen T, hamalainen M,et al.Regulation of nitric oxide production in osteoarthritis and rheumatoid cartilage[J].Scand J Rheumatol,2003,32(1): 19-24.
14. Deschner J,Hofman CR, Piesco NP, et al.Signal transduction by mechanical strain in chondrocytes[J].Curr Opin Nutr Metab Care,2003,6(3):289-293.
15. Ehrlich MG, Arinstrong AL,Treadwe BV, et al.The effects of degneradative enzyme systems in cartilage[J].Chin Orthop,2002,76:57-61.
16. Ohta S,Imai K,Yamashita K,et al.Expression of matrix metalloproteinase 7(matrilysin) in human osteoarthritis cartilage[J].Lab Invest, 1998,78:79-87.
17. Singer I,Kawka DW, Scott S,etal.The relation of matrix metalloproteinase and human osteoarthritis[J].Arthritis Rheum,2003,46(9):2642-2648.
18. Tain A,Nanchahal J,Troeberg L,et al.Production of cytokines,vascular endothelial growth factor, matrix metalloproteinases,and tissue inhibitor of metalloproteinases 1 by tenosynovium demonstrates its proteial for tendon destruction in rheumatoid arthritis[J].Arthritis Rheum.2001,44(8): 1754-1760.
19．李德达，张凯，王毅，等．OA患者关节液与血清中金属蛋白酶及抑止剂的检测及，临床意义[J]．中国骨伤，2002，15(10)：580-585．
20. Stremme S,Duerr S,Bau B,et al.MMP-8 is only a minor gene product of human adult articular chondrocytes of the knee[J].Clin Exp Rheumatol.2003,21 (2):205.
21. Apparailly F, Verwaerde C,Jacpuet C,et al.Adenovirus-mediatedbvtransfer of vial IL-10gene inhibits murine collagen-induced arthritis [J].J Immunol,2004,172: 6450-6456.
22. Haynes MK,Hume EL,Smith JB.Phenotypic characterization of inflammatory cells from osteoarthric synovium and synovical fluoids[J].Clin Immunal,2002,105(3):315-325.
23. Raimund w Kinne,Thomas Liehr, Volkmar Beensen,et al.Mosaic chromosomal aberrations in synovial fibroblasts of potients with rheumatoid arthritis,osteoarthritis and other inflammatory joint disease[J].Arthritis Res,2001,3(5):319-330.