[利用大鼠血清建立体外软骨细胞退变模型]

[Establishment of chondrocyte degeneration model in vitro by rat serum].

作者信息

Wang Xiao-jun, Zhang Hao, Zhan Hong-sheng, Ding Dao-fang

机构信息

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.

出版信息

Zhejiang Da Xue Xue Bao Yi Xue Ban. 2015 May;44(3):308-14. doi: 10.3785/j.issn.1008-9292.2015.05.11.

DOI:10.3785/j.issn.1008-9292.2015.05.11

PMID:26350012

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10396886/

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.

摘要

目的

建立体外软骨细胞退变模型。

方法

采用Ⅱ型胶原酶消化法从新生SD大鼠关节软骨中分离软骨细胞。分别用含10%胎牛血清（FBS）的高糖杜氏改良Eagle培养基（H-DMEM）、含50 ng/mL白细胞介素-1β（IL-1β）+10%FBS、2.5%大鼠血清和5%大鼠血清培养软骨细胞；实验采用第1代软骨细胞。用大鼠血清和IL-1β处理软骨细胞后，在相差显微镜下观察细胞形态变化。采用MTT法检测软骨细胞增殖情况。通过蛋白质印迹法检测增殖细胞核抗原（PCNA）、Ⅱ型胶原和基质金属蛋白酶-13（MMP-13）的蛋白表达水平。通过实时荧光定量PCR检测含血小板凝血酶敏感蛋白基序的解聚素样金属蛋白酶5（ADAMTS5）、MMP-9、聚集蛋白聚糖和SRY-box转录因子9（SOX-9）mRNA水平。