白细胞介素1β诱导软骨细胞中雷帕霉素靶蛋白信号通路  

Expression of signaling pathway of mammalian target of rapamycin in articular cartilage cell induced by interleukin 1β

作　　者：熊寿良[1] 徐宏光[2] 王强[1] 王剑[1] 贺华正[1] 杨昕[1] 董利军[1] 赵泉来[2] 黄德刚[1] 宣华兵[1] 

机构地区：[1]皖南医学院附属弋矶山医院关节外科,安徽芜湖241011 [2]皖南医学院附属弋矶山医院脊柱外科,安徽芜湖241011

出　　处：《中华医学杂志》2017年第7期540-544,共5页National Medical Journal of China

基　　金：国家自然科学基金（81272048）;皖南医学院中青年科研基金（WK2015F17）

摘　　要：目的检测白细胞介素-1β（IL-1β）诱导下哺乳动物雷帕霉素靶蛋白（mTOR）信号通路在大鼠关节软骨细胞中表达变化。方法无菌术下分离出大鼠关节软骨，采用胰蛋白酶及Ⅱ型胶原酶连续消化法提取细胞，二代软骨细胞经过3 d预培养后，加入不同浓度IL-1作用24 h，通过甲苯胺蓝染色进行细胞表型鉴定后；用倒置相差显微镜和HE染色观察细胞形态学变化；逆转录聚合酶链反应（PCR）检测软骨细胞中Ⅱ型胶原、蛋白聚糖、mTOR以及核糖体40S小亚基S6蛋白激酶（P70S6K）基因表达变化；酶联免疫测定mTOR信号通路相关蛋白表达变化。结果随着IL-1β浓度的增加，软骨细胞表型出现由多角形变成梭形，PCR检测Ⅱ型胶原在各组中的相对表达量（对照组：0.821±0.014；1 ng/ml组：0.614±0.014；10 ng/ml组：0.549±0.009；100 ng/ml组：0.520±0.008）、蛋白聚糖（0.867±0.005；0.857±0.001；0.554±0.008；0.538±0.004）以及mTOR（0.845±0.015；0.785±0.009；0.569±0.025；0.518±0.014）的表达量减少，且各基因组组内表达差异均有统计学意义（均P〈0.05）；而P70S6K基因表达量（0.465±0.024；0.566±0.022；0.663±0.022；0.896±0.015）增加，各组表达差异均有统计学意义（均P〈0.05）；免疫印迹检测mTOR蛋白以及P70S6K蛋白表达量与PCR检测趋势一致。结论在软骨细退变过程中mTOR信号通路可能起着重要作用，通过调控mTOR信号通路可能延缓关节软骨退变。Objective To observe effect of intedeukin （IL）-1β on the expression of signaling pathway of mammalian target of rapamycin （roTOR） of articular cartilage. Methods Articular cartilage of rats was isolated under sterile technique,cells were digested by type Ⅱ collagenase and trypsin and cultured in vitro, pre-culture the Ⅱ cells for three days, different concentrations of IL-1β were added for 24 hours. The cells were stained with toluidine blue and HE, to observe morphological changes of cells. RT-PCR was used to detect the mRNA expression of type 11 collagen gene, aggrecan gene, mTOR gene and P70S6K gene, Western blotting was used to detect the expression of protein related to mTOR. Results With increasing concentrations of IL-1β, the phenotype of cells appeared polygon into a spindle, the mRNA expression of gene of type Ⅱ collagen （ the control group ： 0. 821 ± 0. 014 ; 1 ng/ml ： 0. 614 ± 0. 014 ; 10 ng/ml ： 0. 549 ± 0. 009 ; 100 ng/ml ： 0. 520 ± 0. 008 ）, aggrecan （ 0. 867 ± 0. 005 ; 0. 857 ± 0. 001 ; 0. 554 ± 0. 008 ; 0. 538 ± 0. 004） and roTOR（0. 845 ±0. 015; 0. 785 ±0. 009;0. 569 ±0. 025;0. 518 ±0. 014） reduced, but PTOS6K （0. 465 ±0. 024;0. 566 ±0. 022; 0. 663 ±0. 022;0. 896 ±0. 015） increased by PCR . Expression of protein detected by Western blotting was similar to the trend of PCR. Conclusion mTOR signaling pathway may play an important role on the degeneration of articular cartilage, regulating mTOR signaling pathway may provides a new idea of delaying the degeneration process of cells.

关 键 词：软骨 关节 细胞衰老 白细胞介素1 蛋白激酶类 雷帕霉素 

分 类 号：R684[医药卫生—骨科学]