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、Agg...

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Bibliographic Details
Published in浙江大学学报（医学版） no. 3; pp. 308 - 314
Main Author 王小军 张浩 詹红生 丁道芳
Format Journal Article
LanguageChinese
Published 浙江中医药大学附属湖州市中医院骨科,浙江湖州,313000%上海中医药大学附属曙光医院石氏伤科医学中心,上海,201203 2015
Subjects
Wistar
培养的
大鼠
白细胞介素1β
细胞
细胞增殖
血清
软骨疾病
软骨细胞
Cell proliferation
软骨疾病
Interleukin-1beta
Cells,cultured
细胞,培养的
白细胞介素1β
血清
软骨细胞
Chondrocytes
Serum
大鼠,Wistar
Rats,wistar
细胞增殖
Cartilage diseases
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ISSN1008-9292
DOI10.3785/j.issn.1008-9292.2015.05.11

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Summary: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。
Bibliography:33-1248/R
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-1βgroup, and the proliferation of chondrocytes in these groups was much higher than that in control group .The results showed that the expression le
ISSN:1008-9292
DOI:10.3785/j.issn.1008-9292.2015.05.11