SHP2调控少突胶质细胞分化研究

发布时间：2018-07-17 05:36

【摘要】： 多发性硬化症(MS)患者中枢神经系统(CNS)髓鞘表现为进行性缺失,损伤的髓鞘无法再生。髓鞘的形成包括一系列的过程：干细胞定向分化为少突胶质前体细胞(OPCs)、OPCs增殖、迁移、分化成熟,进而包绕轴突,紧缩形成髓鞘。OPCs分化是髓鞘再生过程中的关键环节之一,最新观点认为,OPCs分化受阻是髓鞘难以再生的主要原因之一。然而,OPCs分化的相关调节机制尚不十分清楚。 OLs的分化受到很多生长因子的调节,如CNTF、NT3和ErBb2等。生长因子作用于OLs要通过下游信号传导完成,信号分子磷酸化是这些信号传导中的主要事件。已有研究表明,PI3K/Akt, Fyn/Rho, MAPK等分子磷酸化在OLs细胞分化和髓鞘形成过程中发挥重要作用。细胞内信号分子磷酸化水平受磷酸激酶和磷酸酶的共同调节,目前,有关磷酸激酶与髓鞘化之间的关系已成热点,但磷酸酶与髓鞘化之间的研究还很少。 钒酸钠(SOV),是一种磷酸酶的广谱抑制剂,可以广泛地抑制磷酸酶活性,可以通过抑制酪氨酸位点的去磷酸化上调酪氨酸激酶相关生长因子受体信号。SHP2是一种胞浆的非受体型酪氨酸磷酸酶,广泛表达于神经系统。研究发现,SHP2敲除后神经干细胞分化为少突胶质细胞的数量显著减少,SHP2在脑缺血性损伤后的表达显著增加。然而,SHP2是否参与调节OLs细胞分化尚无报道。 本研究包括五个部分：1.通过SOV喂服E18孕大鼠,给予围产期大鼠SOV处理,免疫组织化学技术检测新生第7天(P7)SD大鼠脑内胼胝体区MBP表达水平,观察到MBP信号明显减少；2.体外培养纯化OPCs,加入不同浓度的SOV处理,发现25gM及以上浓度均可显著抑制OLs分化；3.采用酪氨酸磷酸酶特异性抑制剂(PTP inhibitorⅣ)处理,发现2μM浓度以上显著抑制OLs细胞分化；4.体外培养纯化OPCs,检测到SHP2在少突胶质细胞的前体和成熟阶段均有表达。免疫组织化学染色,进一步确证SHP2在大鼠脑内少突胶质细胞系中存在表达；5.应用RNA i干扰、过表达方法转染OPCs,发现SHP2促进OLs细胞突起分支和生长,同时还可以促进MBP表达。SHP2促进OLs细胞分化作用受细胞外T3刺激的影响。 总之,我们的研究表明,SHP2作为一种新的分化调节因子,参与调节OLs的分化过程。
[Abstract]:The myelin sheath of the central nervous system (CNS) in patients with multiple sclerosis (MS) showed progressive absence, and the damaged myelin sheath could not be regenerated. The formation of myelin sheath includes a series of processes: the differentiation of stem cells into oligodendrocyte progenitor cells (OPCs) proliferates, migrates, differentiates and matures, then encircles axons, and compacts to form myelin. OPCs differentiation is one of the key links in the process of myelin regeneration. According to the latest view, one of the main reasons why myelin sheath is difficult to regenerate is that the differentiation of OPCs is blocked. However, the mechanism of OPCs differentiation is not well understood. The differentiation of OLs is regulated by many growth factors, such as CNTFTF-NT3 and ErBb2. Growth factors act on OLs through downstream signal transduction, and phosphorylation of signal molecules is the main event in these signal transduction. It has been shown that phosphorylation of PI3K / Akt, Fyn-r Rhoand MAPK plays an important role in the differentiation and myelin formation of OLs cells. The phosphorylation level of intracellular signaling molecules is regulated by phosphokinase and phosphatase. At present, the relationship between phosphokinase and myelinization has become a hot topic, but little research has been done on the relationship between phosphatase and myelinization. Sodium vanadate (SOV), a broad-spectrum inhibitor of phosphatase, can extensively inhibit the activity of phosphatase. Tyrosine kinase associated growth factor receptor signal. SHP2 is a cytoplasmic tyrosine phosphatase which is widely expressed in the nervous system by inhibiting the dephosphorylation of tyrosine site. It was found that the number of neural stem cells differentiated into oligodendrocytes after SHP2 knockout significantly decreased the expression of SHP2 in ischemic brain injury. However, whether SHP2 is involved in the regulation of OLs cell differentiation has not been reported. This research includes five parts: 1. E18 pregnant rats were treated with SOV. The expression of MBP in corpus callosum was detected by immunohistochemistry on the 7th day of birth (P7) SD rats, and the signal of MBP was significantly decreased. Purified OPCs were cultured in vitro and treated with different concentrations of SOV. It was found that 25gM and above could significantly inhibit the differentiation of OLs. Treated with tyrosine phosphatase specific inhibitor (PTP inhibitor 鈪，

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