TNF-α对施万细胞去分化和干细胞标志物表达的影响

发布时间：2018-08-04 10:53

【摘要】：外周神经是哺乳动物体内少数可以在损伤后实现再生修复的组织,其中包裹外周神经轴突的胶质细胞——施万细胞自发去分化在这一过程中起到了重要作用。同时,在神经损伤修复过程中激活了一系列炎症反应,虽然TNF-α已被证实是参与这一过程的核心炎症因子,但是关于其对神经再生的作用还存在争议,尤其还未有研究关注TNF-α对施万细胞去分化的影响。另一方面,成体细胞去分化与重编程两个过程中都有不成熟细胞特征性基因的激活,且已有研究证实施万细胞去分化过程中Sox2、Nanog等干细胞特征基因被自发激活。在2006年山中伸弥等通过转染Oct4、Klf4、Sox2、c-Myc(OKSM)四个基因使小鼠皮肤成纤维细胞重编程为诱导多能干细胞(iPS)以后,科学家们一直致力于研究调控这四个因子表达的影响因素,但是iPS诱导生成率一直较低,且没有找到一个很好的模型对内源性OKSM基因表达的升高或降低进行细致的观察。【目的】1、研究TNF-α对外周神经再生和再生过程中施万细胞去分化的影响。2、研究施万细胞去分化过程中OKSM基因表达情况及TNF-α对OKSM基因表达的影响。3、研究TNF-α下游通路NF-κB、JNK、P38、ERK对施万细胞去分化的影响。4、研究NF-κB、JNK、P38、ERK通路对OKSM基因表达的影响。【方法】1、利用神经再生室模型,观察TNF-α对大鼠坐骨神经再生的影响,并通过免疫荧光染色检测TNF-α对施万细胞去分化标志物P75表达的影响。2、离体培养大鼠坐骨神经(离体时施万细胞同样发生自发去分化),利用TUNEL检测、免疫荧光检测和RT-PCR技术,观察TNF-α对施万细胞去分化的影响。3、离体培养大鼠坐骨神经,利用RT-PCR技术观察外周神经中OKSM基因表达的变化及TNF-α对它们的影响。4、离体培养大鼠坐骨神经,利用Western-blot技术检测NF-κB、JNK、P38、ERK四条通路激活情况。5、离体培养大鼠坐骨神经,利用免疫荧光染色和RT-PCR检测NF-κB、JNK、P38、ERK对施万细胞去分化和OKSM基因表达的影响。【结果】1、通过大体观察坐骨神经再生室内神经再生情况,我们发现注射TNF的一侧坐骨神经再生率低于注射生理盐水一侧,同时免疫荧光染色显示注射TNF-α一侧再生的神经中施万细胞去分化标志物P75表达低于注射生理盐水一侧,提示TNF-α抑制外周神经再生和施万细胞去分化。2、通过western-blot检测,证实TNF-α在离体外周神经中激活了NF-κB、JNK、P38、ERK通路。3、加入0-80 ng/ml的TNF-α均未引起离体神经组织细胞凋亡,但是,随着TNF-α浓度增加,免疫荧光染色和RT-PCR均检测到施万细胞去分化标志物P75表达降低。通过阻断剂实验,我们发现NF-κB阻断剂可阻断TNF-α的抑制作用,JNK、P38、ERK阻断剂则没有明显改变,提示TNF-α通过NF-κB通路抑制施万细胞去分化。4、通过RT-PCR检测,我们发现离体培养的外周神经中OKSM基因转录增加并受TNF-α抑制。同时,通过阻断剂实验,我们发现分别加入NF-κB、JNK、P38、ERK阻断剂后,OKSM基因中的一个或多个基因转录增加。【结论】本研究发现,持续的TNF-α作用对外周神经再生有不利影响,至少部分与其抑制再生过程中施万细胞去分化有关。此外,在外周神经离体培养过程中,干细胞特征性基因OKSM的转录被自发激活,提示可以用其作为模型研究影响OKSM基因表达的因素,但是需进一步研究它们在转录后水平的变化,以评估此模型的价值。本研究同时揭示,TNF-α抑制OKSM转录,且其下游通路NF-κB、JNK、P38、ERK均对OKSM基因的一个或多个基因转录有抑制作用。
[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.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R329.2

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