【摘要】：外周神經(jīng)是哺乳動(dòng)物體內(nèi)少數(shù)可以在損傷后實(shí)現(xiàn)再生修復(fù)的組織,其中包裹外周神經(jīng)軸突的膠質(zhì)細(xì)胞——施萬細(xì)胞自發(fā)去分化在這一過程中起到了重要作用。同時(shí),在神經(jīng)損傷修復(fù)過程中激活了一系列炎癥反應(yīng),雖然TNF-α已被證實(shí)是參與這一過程的核心炎癥因子,但是關(guān)于其對(duì)神經(jīng)再生的作用還存在爭(zhēng)議,尤其還未有研究關(guān)注TNF-α對(duì)施萬細(xì)胞去分化的影響。另一方面,成體細(xì)胞去分化與重編程兩個(gè)過程中都有不成熟細(xì)胞特征性基因的激活,且已有研究證實(shí)施萬細(xì)胞去分化過程中Sox2、Nanog等干細(xì)胞特征基因被自發(fā)激活。在2006年山中伸彌等通過轉(zhuǎn)染Oct4、Klf4、Sox2、c-Myc(OKSM)四個(gè)基因使小鼠皮膚成纖維細(xì)胞重編程為誘導(dǎo)多能干細(xì)胞(iPS)以后,科學(xué)家們一直致力于研究調(diào)控這四個(gè)因子表達(dá)的影響因素,但是iPS誘導(dǎo)生成率一直較低,且沒有找到一個(gè)很好的模型對(duì)內(nèi)源性O(shè)KSM基因表達(dá)的升高或降低進(jìn)行細(xì)致的觀察�！灸康摹�1、研究TNF-α對(duì)外周神經(jīng)再生和再生過程中施萬細(xì)胞去分化的影響。2、研究施萬細(xì)胞去分化過程中OKSM基因表達(dá)情況及TNF-α對(duì)OKSM基因表達(dá)的影響。3、研究TNF-α下游通路NF-κB、JNK、P38、ERK對(duì)施萬細(xì)胞去分化的影響。4、研究NF-κB、JNK、P38、ERK通路對(duì)OKSM基因表達(dá)的影響�！痉椒ā�1、利用神經(jīng)再生室模型,觀察TNF-α對(duì)大鼠坐骨神經(jīng)再生的影響,并通過免疫熒光染色檢測(cè)TNF-α對(duì)施萬細(xì)胞去分化標(biāo)志物P75表達(dá)的影響。2、離體培養(yǎng)大鼠坐骨神經(jīng)(離體時(shí)施萬細(xì)胞同樣發(fā)生自發(fā)去分化),利用TUNEL檢測(cè)、免疫熒光檢測(cè)和RT-PCR技術(shù),觀察TNF-α對(duì)施萬細(xì)胞去分化的影響。3、離體培養(yǎng)大鼠坐骨神經(jīng),利用RT-PCR技術(shù)觀察外周神經(jīng)中OKSM基因表達(dá)的變化及TNF-α對(duì)它們的影響。4、離體培養(yǎng)大鼠坐骨神經(jīng),利用Western-blot技術(shù)檢測(cè)NF-κB、JNK、P38、ERK四條通路激活情況。5、離體培養(yǎng)大鼠坐骨神經(jīng),利用免疫熒光染色和RT-PCR檢測(cè)NF-κB、JNK、P38、ERK對(duì)施萬細(xì)胞去分化和OKSM基因表達(dá)的影響。【結(jié)果】1、通過大體觀察坐骨神經(jīng)再生室內(nèi)神經(jīng)再生情況,我們發(fā)現(xiàn)注射TNF的一側(cè)坐骨神經(jīng)再生率低于注射生理鹽水一側(cè),同時(shí)免疫熒光染色顯示注射TNF-α一側(cè)再生的神經(jīng)中施萬細(xì)胞去分化標(biāo)志物P75表達(dá)低于注射生理鹽水一側(cè),提示TNF-α抑制外周神經(jīng)再生和施萬細(xì)胞去分化。2、通過western-blot檢測(cè),證實(shí)TNF-α在離體外周神經(jīng)中激活了NF-κB、JNK、P38、ERK通路。3、加入0-80 ng/ml的TNF-α均未引起離體神經(jīng)組織細(xì)胞凋亡,但是,隨著TNF-α濃度增加,免疫熒光染色和RT-PCR均檢測(cè)到施萬細(xì)胞去分化標(biāo)志物P75表達(dá)降低。通過阻斷劑實(shí)驗(yàn),我們發(fā)現(xiàn)NF-κB阻斷劑可阻斷TNF-α的抑制作用,JNK、P38、ERK阻斷劑則沒有明顯改變,提示TNF-α通過NF-κB通路抑制施萬細(xì)胞去分化。4、通過RT-PCR檢測(cè),我們發(fā)現(xiàn)離體培養(yǎng)的外周神經(jīng)中OKSM基因轉(zhuǎn)錄增加并受TNF-α抑制。同時(shí),通過阻斷劑實(shí)驗(yàn),我們發(fā)現(xiàn)分別加入NF-κB、JNK、P38、ERK阻斷劑后,OKSM基因中的一個(gè)或多個(gè)基因轉(zhuǎn)錄增加�！窘Y(jié)論】本研究發(fā)現(xiàn),持續(xù)的TNF-α作用對(duì)外周神經(jīng)再生有不利影響,至少部分與其抑制再生過程中施萬細(xì)胞去分化有關(guān)。此外,在外周神經(jīng)離體培養(yǎng)過程中,干細(xì)胞特征性基因OKSM的轉(zhuǎn)錄被自發(fā)激活,提示可以用其作為模型研究影響OKSM基因表達(dá)的因素,但是需進(jìn)一步研究它們?cè)谵D(zhuǎn)錄后水平的變化,以評(píng)估此模型的價(jià)值。本研究同時(shí)揭示,TNF-α抑制OKSM轉(zhuǎn)錄,且其下游通路NF-κB、JNK、P38、ERK均對(duì)OKSM基因的一個(gè)或多個(gè)基因轉(zhuǎn)錄有抑制作用。
[Abstract]:The peripheral nerve is a small number of tissues in mammals that can be regenerated after injury. The spontaneous dedifferentiation of Schwann cells, which encapsulates the peripheral neuroglial cells, plays an important role in this process. At the same time, a series of inflammatory reactions have been activated during the repair of nerve damage, although TNF- alpha has been proved to be The core inflammatory factors involved in this process are controversial, but there is still a dispute about its effect on nerve regeneration. Especially, there is no concern about the effect of TNF- alpha on the dedifferentiation of Schwann cells. On the other hand, the two processes of adult cell dedifferentiation and reprogramming have the activation of the immature cell specific genes, and the existing research evidence has been implemented. In the process of cell dedifferentiation, Sox2, Nanog and other stem cell characteristic genes were activated spontaneously. In 2006, after Yamanaka Nobuya and other four genes transfected Oct4, Klf4, Sox2, c-Myc (OKSM) genes to reprogramming mouse skin fibroblasts as induced pluripotent stem cells (iPS), scientists have been working to study the factors that regulate the expression of these four factors, But the induction rate of iPS has been low, and no good model has been found to make a careful observation of the increase or decrease of endogenous OKSM gene expression. [Objective] 1, to study the effect of.2 on the dedifferentiation of Schwann cells in the process of peripheral nerve regeneration and regeneration of TNF- a, and to study the expression of OKSM gene during the dedifferentiation of Schwann cells. And the effect of TNF- alpha on the expression of OKSM gene.3, the influence of TNF- alpha downstream pathway NF- kappa B, JNK, P38, ERK on the dedifferentiation of Schwann cells was studied. [method] 1, the effects of the neural regeneration chamber model on the regeneration of sciatic nerve in rats were observed and immunofluorescence staining was used. The effect of TNF- a on the expression of P75 expression of Schwann cell dedifferentiation marker,.2, in vitro culture of rat sciatic nerve (as in vitro Schwann cells also spontaneous dedifferentiation), TUNEL detection, immunofluorescence detection and RT-PCR technology were used to observe the effect of TNF- a on the dedifferentiation of Schwann cells.3, in vitro culture of rat sciatic nerve, and the use of RT-PCR technology to observe. The changes in the expression of OKSM gene in peripheral nerve and the effect of TNF- alpha on them,.4, cultured rat sciatic nerve in vitro, and using Western-blot technique to detect NF- kappa B, JNK, P38, ERK four pathway activation.5, in vitro culture rat sciatic nerve, immunofluorescence staining and RT-PCR detection of NF- kappa [results] [results] 1, by observing the regeneration of the sciatic nerve, we found that the regeneration rate of the sciatic nerve at one side of the injected TNF was lower than that of the injected physiological saline. At the same time, the immunofluorescent staining showed that the P75 expression of the Schwann cell dedifferentiation marker in the nerve of the injected TNF- a side was lower than that of the injection physiology. It is suggested that TNF- alpha inhibits peripheral nerve regeneration and Schwann cells dedifferentiated.2. Through Western-blot detection, TNF- alpha activates NF- kappa B, JNK, P38, ERK pathway.3, and the addition of 0-80 ng/ml TNF- alpha does not induce apoptosis in the isolated nerve tissue. R detected the decrease in the expression of Schwann cell dedifferentiation marker P75. Through the blocker experiment, we found that NF- kappa B blockers blocked the inhibition of TNF- alpha, JNK, P38, and ERK blockers did not change significantly, suggesting that TNF- alpha inhibited Schwann cell dedifferentiation.4 through NF- kappa B pathway, and we found the peripheral nerve cultured in vitro. OKSM gene transcription increased and was inhibited by TNF- alpha. At the same time, we found that one or more genes in the OKSM gene were added to the NF- kappa B, JNK, P38 and ERK blockers by the blocker experiment. [Conclusion] this study found that the continuous TNF- alpha action has adverse effects on the regeneration of the peripheral God, at least partly by its inhibition of regeneration. In addition, the transcription of the characteristic gene OKSM of stem cells is activated spontaneously during the culture of peripheral nerve in vitro, suggesting that it can be used as a model to study the factors affecting the expression of OKSM genes, but the changes in post transcriptional levels should be further studied to evaluate the value of the model. TNF-a inhibited the transcription of OKSM, and its downstream pathways, such as NF-kappa B, JNK, P38 and ERK, inhibited the transcription of one or more genes of OKSM.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R329.2

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