转化生长因子β1对大鼠髓核细胞凋亡影响的实验研究

Influence of transforming growth factor-β1 on nucleus pulposus cell apoptosis

中文关键词:  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

英文关键词: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

中文摘要:

  【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 factor α，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，RT-PCR）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（Collagen Ⅱ）mRNA相对表达量，Western blot检测各组细胞中MMP3、Bax、ACAN、Collagen Ⅱ、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）、Collagen Ⅱ（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）、Collagen Ⅱ（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通路来拮抗TNF-α诱导的大鼠髓核细胞凋亡。

英文摘要:

  【Abstract】 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 with 50ng/ml TNF-α; group D, cultured in DMEM with 100ng/ml TNF-α; group E, cultured in DMEM with 100ng/ml TNF-α and 10ng/ml TGF-β1; group F, cultured in DMEM with 100ng/ml TNF-α, 10ng/ml TGF-β1 and 10μmol/L SB431542. The cell counting Kit-8(CCK-8) was used to detect the cell proliferation activity after culturing for 12h, 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 statistically significant(P<0.05). Conclusions: TGF-β1 can activate smad/Runx2 pathway and antagonize TNF-α induced nucleus pulposus cell apoptosis.