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Journal of ZheJiang University(Medical Science)  2015, Vol. 44 Issue (3): 308-314    DOI: 10.3785/j.issn.1008-9292.2015.05.11
    
Establishment of chondrocyte degeneration model in vitro by rat serum
WANG Xiao-jun1, ZHANG Hao1, ZHAN Hong-sheng2, DING Dao-fang2
1. Huzhou Hospital Affiliated to Zhejiang Chinese Medical University, Huzhou 313000, China;
2. Shi's Center of Orthopedics and Traumatology, Shuguang Hospital Affiliated to Shanghai TCM University, Shanghai 201203, China
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Abstract  

Objective: To establish a model of chondrocyte degeneration in vitro. Methods: Chondrocytes were isolated from articular cartilages of newly born SD rats by digestion with typeⅡ collagenase. The chondrocytes were cultured with H-DMEM medium containing 10%FBS, 50 ng/mL IL-1β+10%FBS, 2.5% rat serum and 5% rat serum, respectively; and the chondrocytes at passage one were used in the experiments. The morphology changes were investigated under phase contrast microscope after chondrocytes were treated with rat serum and IL-1β. Proliferation of chondrocytes was detected by MTT method. The protein expression levels of PCNA, typeⅡ collagen and MMP-13 were examined by Western blotting. The levels of ADAMTS5, MMP-9, Aggrecan and SOX-9 mRNA were detected by real-time PCR. Results: The cell morphology was changed from polygon to spindle in both rat serum groups and IL-1β group, and the proliferation of chondrocytes in these groups was much higher than that in control group. The results showed that the expression levels of typeⅡ collagen, Aggrecan and SOX-9 decreased while the expression levels of MMP-13, MMP-9 and ADMATS5 were up-regulated in rat serum and IL-1β-treated groups compared with control group. Conclusion: The results indicate that rat serum can induce chondrocyte degeneration and may be used for osteoarthritis model in vitro.



Key wordsInterleukin-1beta      Chondrocytes      Rats, wistar      Serum      Cell proliferation      Cartilage diseases      Cells, cultured     
Received: 31 January 2015      Published: 25 May 2015
CLC:  R68  
Cite this article:

WANG Xiao-jun, ZHANG Hao, ZHAN Hong-sheng, DING Dao-fang. Establishment of chondrocyte degeneration model in vitro by rat serum. Journal of ZheJiang University(Medical Science), 2015, 44(3): 308-314.

URL:

http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2015.05.11     OR     http://www.zjujournals.com/med/Y2015/V44/I3/308


应用大鼠血清建立体外软骨细胞退变模型

目的:应用大鼠血清建立大鼠体外软骨细胞退变模型。方法:取出生24 h SD大鼠关节处软骨,Ⅱ型胶原酶多次消化后获得原代软骨细胞,取原代细胞进行实验。软骨细胞分别用含10%胎牛血清的DMEM(对照组)、含50 ng/mL 白细胞介素(IL)-1β+10%胎牛血清的DMEM(IL-1β组)、含2.5%大鼠血清的DMEM(2.5%血清组)及含5.0%大鼠血清的DMEM(5.0%血清组)中培养。培养24 h后,观察细胞形态变化,MTT法检测各组细胞的增殖情况,蛋白质印迹法检测增殖细胞核抗原的表达和Ⅱ型胶原及MMP-13的表达,实时定量PCR检测退变相关基因ADAMTS5、MMP-9、Aggrecan和SOX-9的表达。结果:两血清组和IL-1β组的软骨形态均由原来的多角形变成长梭形,且两血清组和IL-1β组均促进软骨细胞的增殖,下调转录因子SOX-9和上调基质降解酶MMP-13、MMP-9、ADAMTS5的表达。结论:大鼠血清具有促进软骨退变的作用,可用于建立体外软骨退变模型。


关键词: 白细胞介素1&beta,  软骨细胞,  大鼠,  Wistar,  血清,  细胞增殖,  软骨疾病,  细胞,  培养的 

[1] TROEBERG L, NAGASE H. Proteases involved in cartilage matrix degradation in osteoarthritis [J]. Biochim Biophys Acta, 2012, 1824(1):133-145.
[2] LIN E A, LIU C J. The role of ADAMTSs in arthritis [J]. Protein Cell, 2010, 1(1):33-47.
[3] WU C W, TCHETINA E V, MWALE F, et al. Proteolysis involving matrix metalloproteinase 13(collagenase-3) is required for chondrocyte differentiation that is associated with matrix mineralization[J]. J Bone Miner Res, 2002, 17(4):639-651.
[4] DAHLBERG L, BILLINGHURST R C, MANNER P, et al. Selective enhancement of collagenase-mediated cleavage of resident type II collagen in cultured osteoarthritic cartilage and arrest with a synthetic inhibitor that spares collagenase 1(matrix metalloproteinase 1) [J]. Arthritis Rheum, 2000, 43(3):673-682.
[5] KOBAYASHI M, SQUIRES G R, MOUSA A, et al. Role of interleukin-1 and tumor necrosis factor alpha in matrix degradation of human osteoarthritic cartilage[J]. Arthritis Rheum, 2005, 52(1):128-135.
[6] KELLY T A, FISHER M B, OSWALD E S, et al. Low-serum media and dynamic deformational loading in tissue engineering of articular cartilage[J]. Ann Biomed Eng, 2008,36(5):769-79.
[7] MALPELI M, RANDAZZO N, CANCEDDA R, et al. Serum-free growth medium sustains commitment of human articular chondrocyte through maintenance of SOX9 expression [J]. Tissue Eng, 2004, 10(1-2):145-155.
[8] SHAO X X, DUNCAN N A, LIN L, et al. Serum-free media for articular chondrocytes in vitro expansion [J]. Chin Med J (Engl), 2013, 126(13):2523-2529.
[9] 丁道芳, 李玲慧, 宋 奕, 等. MAPK-ERK1/2信号通路调控成骨性基因表达和细胞增殖[J].南方医科大学学报, 2013, 33(10):1432-1436. DING Dao-fang, LI Ling-hui, SONG Yi, et al. MAPK-ERK1/2 signaling pathway regulates cell proliferation and osteogenic gene expression in rat osteoblasts in vitro[J]. Journal of Southern Medical University, 2013, 33(10): 1432-1436. (in Chinese)
[10] 丁道芳, 韦宋谱,李晓锋,等. 蛇床子素对大鼠原代软骨细胞增殖的抑制作用[J].中西医结合学报, 2012, 10(12):1413-1418. DING Dao-fang, WEI Song-pu, LI xiao-feng, et al. Inhibition effect of osthole on proliferation of rat chondrocytes [J]. Journal of Chinese Integrative Medicine, 2012, 10(12):1413-1418. (in Chinese)
[11] PESESSE L, SANCHEZ C, HENROTIN Y. Osteochondral plate angiogenesis: a new treatment target in osteoarthritis [J]. Joint Bone Spine, 2011, 78(2):144-149.
[12] NAGAI T, SATO M, KOBAYASHI M, et al. Bevacizumab, an anti-vascular endothelial growth factor antibody, inhibits osteoarthritis [J]. Arthritis Res Ther, 2014, 16(5):427.
[13] AIGNER T, DUDHIA J. Phenotypic modulation of chondrocytes as a potential therapeutic target in osteoarthritis: a hypothesis [J]. Ann Rheum Dis, 1997, 56(5):287-291.
[14] HO S T, YANG Z, HUI H P, et al. A serum free approach towards the conservation of chondrogenic phenotype during in vitro cell expansion [J]. Growth Factors, 2009, 27(5):321-333.
[15] PARRA-TORRES N M, C ZARES-RAGA F E, KOURI J B. Proteomic analysis of rat cartilage: the identification of differentially expressed proteins in the early stages of osteoarthritis [J]. Proteome Sci, 2014, 12(1):55.
[16] SURI S, GILL S E, MASSENA DE CAMIN S, et al. Neurovascular invasion at the osteochondral junction and in osteophytes in osteoarthritis [J]. Ann Rheum Dis, 2007, 66(11):1423-1428.
[17] AIGNER T, SACHSE A, GEBHARD P M, et al. Osteoarthritis: pathobiology-targets and ways for therapeutic intervention [J]. Adv Drug Deliv Rev, 2006, 58(2):128-149.
[18] GOLDRING M B. Update on the biology of the chondrocyte and new approaches to treating cartilage diseases [J]. Best Pract Res Clin Rheumatol, 2006, 20(5):1003-1025.
[19] USHIJIMA T, OKAZAKI K, TSUSHIMA H, et al. CCAAT/enhancer-binding protein β regulates the repression of type II collagen expression during the differentiation from proliferative to hypertrophic chondrocytes[J]. J Biol Chem, 2014, 289(5):2852-2863.
[20] SEKIYA I, TSUJI K, KOOPMAN P, et al. SOX9 enhances aggrecan gene promoter/enhancer activity and is up-regulated by retinoic acid in a cartilage-derived cell line, TC6 [J]. J Biol Chem, 2000, 275(15): 10738-10744.
[21] OKUBO Y, REDDI A H. Thyroxine downregulates SOX9 and promotes chondrocyte hypertrophy [J]. Biochem Biophys Res Commun, 2003,306(1): 186-190.
[22] RUTGERS M, SARIS D B, DHERT W J, et al. Cytokine profile of autologous conditioned serum for treatment of osteoarthritis, in vitro effects on cartilage metabolism and intra-articular levels after injection [J]. Arthritis Res Ther, 2010, 12(3):R114.

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