【摘要】： 多發(fā)性硬化癥(MS)患者中樞神經(jīng)系統(tǒng)(CNS)髓鞘表現(xiàn)為進(jìn)行性缺失,損傷的髓鞘無(wú)法再生。髓鞘的形成包括一系列的過(guò)程：干細(xì)胞定向分化為少突膠質(zhì)前體細(xì)胞(OPCs)、OPCs增殖、遷移、分化成熟,進(jìn)而包繞軸突,緊縮形成髓鞘。OPCs分化是髓鞘再生過(guò)程中的關(guān)鍵環(huán)節(jié)之一,最新觀點(diǎn)認(rèn)為,OPCs分化受阻是髓鞘難以再生的主要原因之一。然而,OPCs分化的相關(guān)調(diào)節(jié)機(jī)制尚不十分清楚。 OLs的分化受到很多生長(zhǎng)因子的調(diào)節(jié),如CNTF、NT3和ErBb2等。生長(zhǎng)因子作用于OLs要通過(guò)下游信號(hào)傳導(dǎo)完成,信號(hào)分子磷酸化是這些信號(hào)傳導(dǎo)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細(xì)胞分化和髓鞘形成過(guò)程中發(fā)揮重要作用。細(xì)胞內(nèi)信號(hào)分子磷酸化水平受磷酸激酶和磷酸酶的共同調(diào)節(jié),目前,有關(guān)磷酸激酶與髓鞘化之間的關(guān)系已成熱點(diǎn),但磷酸酶與髓鞘化之間的研究還很少。 釩酸鈉(SOV),是一種磷酸酶的廣譜抑制劑,可以廣泛地抑制磷酸酶活性,可以通過(guò)抑制酪氨酸位點(diǎn)的去磷酸化上調(diào)酪氨酸激酶相關(guān)生長(zhǎng)因子受體信號(hào)。SHP2是一種胞漿的非受體型酪氨酸磷酸酶,廣泛表達(dá)于神經(jīng)系統(tǒng)。研究發(fā)現(xiàn),SHP2敲除后神經(jīng)干細(xì)胞分化為少突膠質(zhì)細(xì)胞的數(shù)量顯著減少,SHP2在腦缺血性損傷后的表達(dá)顯著增加。然而,SHP2是否參與調(diào)節(jié)OLs細(xì)胞分化尚無(wú)報(bào)道。 本研究包括五個(gè)部分：1.通過(guò)SOV喂服E18孕大鼠,給予圍產(chǎn)期大鼠SOV處理,免疫組織化學(xué)技術(shù)檢測(cè)新生第7天(P7)SD大鼠腦內(nèi)胼胝體區(qū)MBP表達(dá)水平,觀察到MBP信號(hào)明顯減少；2.體外培養(yǎng)純化OPCs,加入不同濃度的SOV處理,發(fā)現(xiàn)25gM及以上濃度均可顯著抑制OLs分化；3.采用酪氨酸磷酸酶特異性抑制劑(PTP inhibitorⅣ)處理,發(fā)現(xiàn)2μM濃度以上顯著抑制OLs細(xì)胞分化；4.體外培養(yǎng)純化OPCs,檢測(cè)到SHP2在少突膠質(zhì)細(xì)胞的前體和成熟階段均有表達(dá)。免疫組織化學(xué)染色,進(jìn)一步確證SHP2在大鼠腦內(nèi)少突膠質(zhì)細(xì)胞系中存在表達(dá)；5.應(yīng)用RNA i干擾、過(guò)表達(dá)方法轉(zhuǎn)染OPCs,發(fā)現(xiàn)SHP2促進(jìn)OLs細(xì)胞突起分支和生長(zhǎng),同時(shí)還可以促進(jìn)MBP表達(dá)。SHP2促進(jìn)OLs細(xì)胞分化作用受細(xì)胞外T3刺激的影響。 總之,我們的研究表明,SHP2作為一種新的分化調(diào)節(jié)因子,參與調(diào)節(jié)OLs的分化過(guò)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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