摘要
目的 :探讨转化生长因子β1(transforming growth factor-β1,TGF-β1)对大鼠髓核细胞凋亡的影响以及其作用机制。方法:采用序贯酶消化法分离培养SD大鼠髓核细胞(nucleus pulposus cells,NPCs),实验用第3代培养的NPCs,分为6组:A组,空白对照组,用DMEM培养基常规培养,不加任何处理;B组,用含有终浓度为20ng/ml肿瘤坏死因子α(tumor necrosis factorα,TNF-α)的DMEM培养基培养;C组,用含有终浓度为50ng/ml TNF-α的DMEM培养基培养;D组,用含有终浓度为100ng/ml TNF-α的DMEM培养基培养;E组,用同时含有终浓度为100ng/ml TNF-α和10ng/ml TGF-β1的DMEM培养基培养;F组,在E组培养基的基础上加TGF-β1/smad通路抑制剂SB431542(设置终浓度为10μmol/L)进行培养。培养12h后,Cell Counting Kit-8(CCK-8)法检测细胞增殖活性,逆转录聚合酶链式反应(reverse transcription-polymerase chain reaction,RTPCR)检测各组细胞中基质金属蛋白酶3(matrix metalloproteinases 3,MMP3)、凋亡相关蛋白(bcl-2-associated x protein,Bax)、蛋白聚糖(ACAN)、Ⅱ型胶原(CollagenⅡ)m RNA相对表达量,Western blot检测各组细胞中MMP3、Bax、ACAN、CollagenⅡ、磷酸化的smad3蛋白(phosphorylate drosophila mothers against decapentaplegic protein 3,p-smad3)和Runt相关转录因子2(Runt-related transcription factor 2,Runx2)蛋白表达量。结果 :与A组相比,不同剂量的TNF-α(B^D组)均能够促进MMP3(B^D组依次为0.652+0.015、0.899+0.018、1.026+0.023)和Bax(B^D组依次为0.725+0.058、0.928+0.018和1.138+0.019)的表达,诱导髓核细胞的凋亡,并且呈现剂量依赖性关系。与D组相比,E组MMP3(0.568+0.015)和Bax(0.626+0.024)表达下调,ACAN(1.056+0.014)、CollagenⅡ(1.098+0.032)、p-smad3和Runx2表达量增高,差异有统计意义(P<0.05)。与E组相比,F组MMP3(1.015+0.015)和Bax(1.126+0.024)表达上调,ACAN(0.314+0.023)、CollagenⅡ(0.299+0.0.19)、p-smad3和Runx2表达量下降,差异有统计意义(P<0.05)。结论 :TGF-β1可能是通过激活smad/Runx2通�
Objectives： To explore the effect of TGF-β1 on nucleus pulposus cell apoptosis and the potential mechanisms. Methods： Nucleus pulposus cells（NPCs） were isolated from Sprague Dawley rats and cultured by the sequential enzyme method. Cells at passage 3 were divided into six groups： group A, cultured in DMEM with no other treatment; group B, cultured in DMEM with 20ng/ml TNF-α; group C, cultured in DMEM with50ng/ml TNF-α; group D, cultured in DMEM with 100ng/ml TNF-α; group E, cultured in DMEM with100ng/ml TNF-α and 10ng/ml TGF-β1; group F, cultured in DMEM with 100ng/ml TNF-α, 10ng/ml TGF-β1and 10μmol/L SB431542. The cell counting Kit-8（CCK-8） was used to detect the cell proliferation activity after culturing for 12 h, transcription-polymerase chain reaction（RT-PCR） was reversed and Western blot were used to detect the expression of matrix metalloproteinases 3（MMP3）, bcl-2-associated x protein（Bax）, ACAN,Collagen Ⅱ, phosphorylate drosophila mothers against decapentaplegic protein 3（p-smad3） and runt-related transcription factor 2（Runx2）. Results： Compared with group A, the different concentrations of TNF-α groups（group B-D） promoted the expressions of MMP3（group B-D respectively： 0.652 ＋0.015, 0.899 ＋0.018, 1.026 ＋0.023 respectively） and Bax（0.725＋0.058, 0.928＋0.018, 1.138＋0.019 respectively）, and promoted apoptosis in a dose-dependent manner. Compared with group D, the expressions of MMP3（0.568 ＋0.015） and Bax（0.626 ＋0.024） were significantly inhibited in group E, while the expressions of ACAN（1.056＋0.014）, Collagen Ⅱ（1.098＋0.032）, p-smad3 and Runx2 significantly increased, the difference was statistically significant（P〈0.05）.Compared with group E, the expressions of MMP3（1.015＋0.015） and Bax（1.126＋0.024） significantly increased in group F, while the expression of ACAN（0.314 ＋0.023）, Collagen Ⅱ（0.299 ＋0.0.19）, p-smad3 and Runx2 were significantly inhibited, the difference was statisti
出处
《中国脊柱脊髓杂志》
CAS
CSCD
北大核心
2016年第2期156-161,共6页
Chinese Journal of Spine and Spinal Cord
关键词
转化生长因子Β1
信号转导蛋白smad/Runt相关转录因子
肿瘤坏死因子Α
髓核细胞
凋亡
大鼠
Transforming growth factor-β1（TGF-β1）
Drosophila mothers against decapentaplegic protein/ Runt-related transcription factor 2（Smad/Runx2）
Tumor necrosis factor-α（TNF-α）
Nucleus pulposus cells
Apoptosis
