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浙江大学学报(医学版)  2015, Vol. 44 Issue (3): 308-314    DOI: 10.3785/j.issn.1008-9292.2015.05.11
原著     
应用大鼠血清建立体外软骨细胞退变模型
王小军1, 张浩1, 詹红生2, 丁道芳2
1. 浙江中医药大学附属湖州市中医院骨科, 浙江 湖州 313000;
2. 上海中医药大学附属曙光医院石氏伤科医学中心, 上海 201203
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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摘要:

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    
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 words: Interleukin-1beta    Chondrocytes    Rats, wistar    Serum    Cell proliferation    Cartilage diseases    Cells, cultured
收稿日期: 2015-01-31 出版日期: 2015-05-25
:  R68  
基金资助:

国家自然科学基金 (81073114);上海市高校青年教师培养资助计划(ZZszy12017)

通讯作者: 丁道芳(1979-),女,博士,副研究员,主要从事中医药防治骨关节炎研究;E-mail:d.wilhel@qq.com;http://orcid.org/0000-0003-1754-8286     E-mail: d.wilhel@qq.com
作者简介: 王小军(1980-),男,学士,主治医师,从事中医骨伤科研究;E-mail:57052728@qq.com;http://orcid.org/0000-0001-8599-7898
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引用本文:

王小军, 张浩, 詹红生, 丁道芳. 应用大鼠血清建立体外软骨细胞退变模型[J]. 浙江大学学报(医学版), 2015, 44(3): 308-314.

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.

链接本文:

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

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