| 不同血清浓度对大鼠软骨细胞增殖、形态和基因表达的影响 |
| 投稿时间：2017-11-23 修订日期：2017-12-22 点此下载全文 |
| 引用本文：刘丽芳,薛艳,郑昱新,曹月龙,丁道芳.不同血清浓度对大鼠软骨细胞增殖、形态和基因表达的影响[J].医学研究杂志,2018,47(9):53-57,33 |
| DOI：
10.11969/j.issn.1673-548X.2018.09.013 |
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| 基金项目:国家自然科学基金资助项目（81073114，81072830） |
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| 中文摘要:目的 观察不同血清浓度对大鼠软骨细胞体外培养的形态、增殖和基因表达的调控作用。方法 取出生24h SD大鼠关节处软骨，0.1%浓度的Ⅱ型胶原酶多次消化后获得软骨细胞。软骨细胞培养于含5%胎牛血清（fetal bovine serum，FBS），10% FBS和20% FBS的DMEM低糖培养基中，分别为5FBS组，10FBS组和20FBS组。24h后镜下观察细胞形态变化，CCK8方法检测24、48和72h软骨细胞增殖。Western blot法检测软骨指标Ⅱ型胶原A1（COL2A1）和MMP13（matrix metalloproteinases13）表达，定量PCR检测软骨特异性基因Sox9、A-can、ADAMTS5、MMP9和MMP3的表达及代谢相关基因Nme2和PnP表达。结果 5FBS组的软骨细胞呈由鹅卵石样排列，10FBS组和20FBS组细胞呈现长梭形改变，且血清浓度越大，形态改变越明显。和5FBS组比较，10FBS组和20FBS组细胞增殖明显，呈血清浓度依赖性增加。10FBS组和20FBS组的MMP13表达显著上调，Ⅱ型胶原表达显著下调。PCR结果表明，和5FSB组比较，10FBS组和20FBS组的A-can和Sox9表达下降，MMP9、MMP3和ADAMTS5表达上调，且基因表达变化和血清浓度改变呈正相关。同时随着血清浓度增加，Nme2和PnP表达显著上调。结论 血清浓度的增高促进软骨细胞代谢，引起软骨细胞退变。 |
| 中文关键词:血清浓度 软骨细胞 细胞增殖 形态改变 营养状态 |
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| Influences of Different Nutritional Status in Rat Chondrocytes |
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| Abstract:Objective To observe the effects of different concentrations of serum on the morphology, proliferation and gene expression in rat chondrocytes in vitro. Methods Chondrocytes were isolated from articular cartilages in newly-born (within 24 hours)SD rats, and incubated with 0.1% concentration type Ⅱ collagenase by several times.The chondrocytes were cultured in a low glucose DMEM containing 5% fetal bovine serum (FBS group), 10%FBS(10FBS group) and 20%FBS(20FBS group) to simulate the different nutritional status of chondrocytes in vitro, and were defined as 5FBS group, and 20FBS group.The morphological changes were observed under the microscope after 24 hours culturing, The proliferation of chondrocytes was detected by CCK8 method after chondrocytes were treated with different concentrations of serum for 24 hours, 48 hours and 72 hours. The expressions of collagen Ⅱ type A1 (COL2A1) and Matrix metallopeptidase 13 (MMP13) were analyzed by Western blot. The expression of Sox9, A-can, ADAMTS5,MMP9, MMP3 and metabolic genes(NME2,PnP) were assessed by quantitative PCR. Results The chondrocytes in 5FBS group exhibited the cobblestone arrangement, while the chondrocytes in 10FBS group and 20FBS group showed the change of the spindle,and higher the concentration of serum was, the more obvious the morphological changes were.Compared with 5FBS group, the chondrocytes' proliferation in 10FBS group and 20FBS group were obvious,which was dependent on the serum concentration.The expressions of MMP13 in 10 FBS group and 20FBS group significantly increased, while the expressions of COL2A1 significantly decreased. Compared with 5FSB group, the expression of A-can and Sox9 in 10FBS group and 20FBS group decreased, while MMP9, MMP3 and ADAMTS5 expression were up-regulated, moreover the changes of gene expressions and the concentrations of serum were positively correlated. With the increase of serum concentration, the expressions of Nme2 and PnP were also significantly increased. Conclusion The increased serum concentration promotes chondrocyte metabolism and causes chondrocyte degeneration. |
| keywords:Serum concentration Chondrocytes Cell proliferation Morphological change Nutritional status |
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