【摘要】：目的:探討ERK5信號轉(zhuǎn)導(dǎo)通路在炎癥因子TNF-α誘導(dǎo)的髓核細(xì)胞退變過程中的調(diào)控作用。 方法:1.運用免疫組織化學(xué)DAB染色的方法，檢測ERK5在髓核細(xì)胞中的表達(dá)情況，再運用Western blot免疫印跡檢測ERK5和磷酸化ERK5（p-ERK5）在正常和退變的髓核組織中表達(dá)的差異；2.用TNF-α誘導(dǎo)體外分離培養(yǎng)的髓核細(xì)胞發(fā)生退變，運用Western blot免疫印跡檢測此過程中ERK5和p-ERK5的蛋白表達(dá)水平的變化情況；3.通過mRNA干擾序列轉(zhuǎn)染正常髓核細(xì)胞使ERK5基因沉默，再用TNF-α誘導(dǎo)其退變，以無TNF-α刺激組為對照，RT-PCR檢測ERK5和II型膠原，糖胺多糖，SOX9的基因轉(zhuǎn)錄水平。 結(jié)果:1.免疫組織化學(xué)染色見實驗組髓核細(xì)胞胞漿呈棕黃色，對照組髓核細(xì)胞胞漿無染色；2.Western blot顯示正常組和退變組髓核組織ERK5的表達(dá)無差異（n=11,P0.05），但退變組p-ERK5的表達(dá)比正常組低，差異有統(tǒng)計學(xué)意義（n=11,P0.05）；3.TNF-α作用于髓核細(xì)胞15分鐘后，，p-ERK5信號開始下降，一直持續(xù)到60分鐘；4.mRNA干擾序列沉默ERK5基因后，髓核細(xì)胞ERK5、II型膠原、糖胺多糖和SOX9的基因轉(zhuǎn)錄水平下降，與無沉默組相比較差異有統(tǒng)計學(xué)意義（n=5,P0.05），施加TNF-α刺激后髓核細(xì)胞的ERK5、II型膠原的基因轉(zhuǎn)錄水平下降更加明顯，與無TNF-α刺激組相比較差異有統(tǒng)計學(xué)意義（n=5,P0.05）。 結(jié)論:1.ERK5在人髓核細(xì)胞中有表達(dá)，分子定位在胞漿；2.退變髓核組織p-ERK5的表達(dá)比正常髓核組織低,ERK5的表達(dá)與正常髓核組織相比無明顯差異；3.ERK5信號通路參與TNF-α誘導(dǎo)的髓核細(xì)胞退變的過程中，TNF-α可抑制ERK5的活化；4.ERK5基因沉默后髓核細(xì)胞Ⅱ型膠原，糖胺多糖和SOX9的基因轉(zhuǎn)錄水平下降。
[Abstract]:Aim: to investigate the regulatory role of ERK5 signal transduction pathway in the degeneration of nucleus pulposus cells induced by inflammatory factor TNF- 偽. 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- 偽. 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- 偽. The gene transcription levels of ERK5 and type II collagen, glycosaminoglycan and SOX9 were detected by RT-PCR compared with those without TNF- 偽 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

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