ERK5信号通路介导的TNF-α诱导髓核细胞退变的研究
发布时间：2019-05-26 18:22
【摘要】：目的:探讨ERK5信号转导通路在炎症因子TNF-α诱导的髓核细胞退变过程中的调控作用。 方法:1.运用免疫组织化学DAB染色的方法，检测ERK5在髓核细胞中的表达情况，再运用Western blot免疫印迹检测ERK5和磷酸化ERK5（p-ERK5）在正常和退变的髓核组织中表达的差异；2.用TNF-α诱导体外分离培养的髓核细胞发生退变，运用Western blot免疫印迹检测此过程中ERK5和p-ERK5的蛋白表达水平的变化情况；3.通过mRNA干扰序列转染正常髓核细胞使ERK5基因沉默，再用TNF-α诱导其退变，以无TNF-α刺激组为对照，RT-PCR检测ERK5和II型胶原，糖胺多糖，SOX9的基因转录水平。 结果:1.免疫组织化学染色见实验组髓核细胞胞浆呈棕黄色，对照组髓核细胞胞浆无染色；2.Western blot显示正常组和退变组髓核组织ERK5的表达无差异（n=11,P0.05），但退变组p-ERK5的表达比正常组低，差异有统计学意义（n=11,P0.05）；3.TNF-α作用于髓核细胞15分钟后，，p-ERK5信号开始下降，一直持续到60分钟；4.mRNA干扰序列沉默ERK5基因后，髓核细胞ERK5、II型胶原、糖胺多糖和SOX9的基因转录水平下降，与无沉默组相比较差异有统计学意义（n=5,P0.05），施加TNF-α刺激后髓核细胞的ERK5、II型胶原的基因转录水平下降更加明显，与无TNF-α刺激组相比较差异有统计学意义（n=5,P0.05）。 结论:1.ERK5在人髓核细胞中有表达，分子定位在胞浆；2.退变髓核组织p-ERK5的表达比正常髓核组织低,ERK5的表达与正常髓核组织相比无明显差异；3.ERK5信号通路参与TNF-α诱导的髓核细胞退变的过程中，TNF-α可抑制ERK5的活化；4.ERK5基因沉默后髓核细胞Ⅱ型胶原，糖胺多糖和SOX9的基因转录水平下降。
[Abstract]:Aim: to investigate the regulatory role of ERK5 signal transduction pathway in the degeneration of nucleus pulposus cells induced by inflammatory factor TNF- 伪. Method: 1. The expression of ERK5 in nucleus pulposus cells was detected by immunohistochemical DAB staining, and the expression of ERK5 and phosphorylation ERK5 (p-ERK5) in normal and degenerative nucleus pulposus tissues were detected by Western blot immunoblotting. 2. The degeneration of nucleus pulposus cells isolated and cultured in vitro was induced by TNF- 伪. The protein expression levels of ERK5 and p-ERK5 were detected by Western blot immunoblotting. Normal nucleus pulposus cells were silenced by mRNA interference sequence and induced by TNF- 伪. The gene transcription levels of ERK5 and type II collagen, glycosaminoglycan and SOX9 were detected by RT-PCR compared with those without TNF- 伪 stimulation. Result: 1. The cytoplasm of nucleus pulposus cells in the experimental group was brown and yellow, while the cytoplasm of the nucleus pulposus cells in the control group was not stained. 2.Western blot showed that there was no difference in the expression of ERK5 between the normal group and the degenerative group (n 鈮
本文编号：2485515
[Abstract]:Aim: to investigate the regulatory role of ERK5 signal transduction pathway in the degeneration of nucleus pulposus cells induced by inflammatory factor TNF- 伪. Method: 1. The expression of ERK5 in nucleus pulposus cells was detected by immunohistochemical DAB staining, and the expression of ERK5 and phosphorylation ERK5 (p-ERK5) in normal and degenerative nucleus pulposus tissues were detected by Western blot immunoblotting. 2. The degeneration of nucleus pulposus cells isolated and cultured in vitro was induced by TNF- 伪. The protein expression levels of ERK5 and p-ERK5 were detected by Western blot immunoblotting. Normal nucleus pulposus cells were silenced by mRNA interference sequence and induced by TNF- 伪. The gene transcription levels of ERK5 and type II collagen, glycosaminoglycan and SOX9 were detected by RT-PCR compared with those without TNF- 伪 stimulation. Result: 1. The cytoplasm of nucleus pulposus cells in the experimental group was brown and yellow, while the cytoplasm of the nucleus pulposus cells in the control group was not stained. 2.Western blot showed that there was no difference in the expression of ERK5 between the normal group and the degenerative group (n 鈮
本文编号：2485515
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