本文選題：蛋白磷酸酶2A(PP2A) + 新生死亡�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：蛋白磷酸酶2A (Protein Phosphatase 2A, PP2A)是真核生物蛋白磷酸酶家族中表達(dá)最為豐富的成員之一,它通過(guò)對(duì)蛋白質(zhì)絲氨酸及蘇氨酸殘基的去磷酸化作用進(jìn)而調(diào)控多種細(xì)胞進(jìn)程,包括細(xì)胞增殖、分化、遷移及凋亡,細(xì)胞周期,DNA損傷應(yīng)答甚至腫瘤的發(fā)生。PP2A全酶是由一個(gè)催化亞單位C,一個(gè)結(jié)構(gòu)亞單位A和一個(gè)調(diào)節(jié)亞單位B所組成的異三聚體復(fù)合物。由于其調(diào)節(jié)亞單位B的底物特異性,PP2A能識(shí)別多種靶蛋白而發(fā)揮不同的生物學(xué)功能。其與相應(yīng)的蛋白激酶共同調(diào)控生物體內(nèi)重要蛋白質(zhì)磷酸化水平與去磷酸化水平的動(dòng)態(tài)平衡,這對(duì)于維持機(jī)體內(nèi)正常信號(hào)轉(zhuǎn)導(dǎo)是至關(guān)重要的。南京模式動(dòng)物所首先建立了Ppp2cα基因全身敲除的小鼠模型,發(fā)現(xiàn)其約在胚胎第6.5天發(fā)生死亡。為了進(jìn)一步研究其生物學(xué)功能,建立了Ppp2cα條件性敲除的小鼠模型,并發(fā)現(xiàn)在巨噬細(xì)胞、肝臟及心肌細(xì)胞中特異性敲除Ppp2cα后可分別導(dǎo)致機(jī)體免疫系統(tǒng)中Ⅰ型IFN信號(hào)通路應(yīng)答增強(qiáng)；胚肝內(nèi)紅細(xì)胞生成障礙以及肝臟葡萄糖代謝異常；心肌肥大,甚至心功能不全。在此研究基礎(chǔ)上,本課題組建立了PP2ACα中樞神經(jīng)系統(tǒng)特異性敲除的小鼠模型(PP2ACαCNS-del)。首先,我們觀察發(fā)現(xiàn)PP2ACαCNS-del小鼠在出生時(shí)即發(fā)生死亡,取其全腦稱腦重發(fā)現(xiàn)較對(duì)照組相比有明顯降低。隨后對(duì)其全腦組織進(jìn)行一系列形態(tài)學(xué)檢測(cè)發(fā)現(xiàn)在胚胎發(fā)育期其大腦皮質(zhì)中神經(jīng)元存在凋亡增加以及增殖降低的現(xiàn)象。同時(shí)我們?cè)隗w外對(duì)原代培養(yǎng)的大腦皮質(zhì)神經(jīng)元也進(jìn)行了一系列增殖以及凋亡的檢測(cè),發(fā)現(xiàn)其結(jié)果與體內(nèi)實(shí)驗(yàn)一致。由此我們推斷,PP2ACα缺失確實(shí)可以引起皮質(zhì)神經(jīng)元的凋亡增加并伴有增殖降低。探究其原因,我們發(fā)現(xiàn)在PP2ACα缺失的皮質(zhì)神經(jīng)元中存在內(nèi)源性DNA損傷應(yīng)答ATR/Chk1信號(hào)通路的激活,并且在給予皮質(zhì)神經(jīng)元DNA單鏈損傷藥物處理后,觀察發(fā)現(xiàn)參與損傷應(yīng)答的sensor-ATR與PP2AC有共定位現(xiàn)象。于是進(jìn)一步利用免疫共沉淀的方法證實(shí)了PP2ACα缺失確實(shí)可以直接引起ATR磷酸化持續(xù)激活,進(jìn)而激活下游Chk1等效應(yīng)分子,從而導(dǎo)致內(nèi)源性DNA損傷應(yīng)答的激活。其過(guò)度激活進(jìn)一步引起p53蛋白的表達(dá)上調(diào),一方面通過(guò)p21靶蛋白導(dǎo)致細(xì)胞周期阻滯；另一方面可誘導(dǎo)促進(jìn)凋亡的Bax表達(dá)上調(diào),而抑制凋亡的Bcl-2表達(dá)下調(diào)。這種表達(dá)失衡最終導(dǎo)致了皮質(zhì)神經(jīng)元的異常凋亡增加。為了更全面地研究PP2ACα的生物學(xué)功能,我們又建立了在背側(cè)皮質(zhì)以及海馬特異性敲除PP2ACα的小鼠模型(PP2ACαD6-del),我們發(fā)現(xiàn)其小鼠出生后能存活并伴有明顯頭小畸形(microcephaly)的表型,PP2ACα缺失的皮質(zhì)神經(jīng)元中也存在由于內(nèi)源性DNA損傷應(yīng)答激活而引起的凋亡增加現(xiàn)象。另外,由于海馬及其對(duì)應(yīng)皮質(zhì)區(qū)域是調(diào)控學(xué)習(xí)和認(rèn)知功能的高級(jí)中樞,我們采用以Morris水迷宮為主的行為學(xué)實(shí)驗(yàn)指標(biāo)來(lái)評(píng)價(jià)其學(xué)習(xí)和記憶功能狀態(tài),發(fā)現(xiàn)PP2ACαD6-del小鼠與對(duì)照相比伴有明顯的學(xué)習(xí)和記憶功能的降低,這與臨床上microcephaly病人的臨床體征相符合。綜上所述,PP2ACα缺失可引起內(nèi)源性DNA損傷應(yīng)答缺陷,進(jìn)而導(dǎo)致皮質(zhì)神經(jīng)元的凋亡增加并伴有增殖降低,最終表現(xiàn)為以皮質(zhì)變薄及海馬萎縮等為特征的頭小畸形表型,這為探究microcephaly致病機(jī)制以及臨床治療提供了新的線索和理論依據(jù)。
[Abstract]:Protein phosphatase 2A (Protein Phosphatase 2A, PP2A) is one of the most abundant members of the eukaryotic protein phosphatase family. It regulates a variety of cell processes by dephosphorylation of protein serine and threonine residues, including cell proliferation, differentiation, migration and apoptosis, cell cycle, DNA damage response and even response. The.PP2A total enzyme is a heterogeneous tripolymer complex composed of a catalytic subunit, C, a structural subunit A and a regulatory subunit B. Because of its substrate specificity of modulating the subunit B, PP2A can recognize a variety of target proteins and play different biological functions. The dynamic balance of protein phosphorylation level and dephosphorylation level is essential to maintain normal signal transduction in the body. The Nanjing model animal first established a mouse model of Ppp2c alpha gene knockout, and found that it died on the 6.5 day of the embryo. In order to further study its biological function, Ppp2c alpha was established. The conditioned knockout mouse model found that the specific knockout of Ppp2c alpha in macrophages, liver and cardiac myocytes could lead to enhanced response of type I IFN signaling pathway in the immune system, dysfunction of erythrocyte in the liver and abnormal glucose metabolism in the liver, myocardial hypertrophy and even cardiac insufficiency. We have established a mouse model of PP2AC alpha central nervous system specific knockout (PP2AC alpha CNS-del). First of all, we observed that PP2AC alpha CNS-del mice died at birth, and the brain weight found in the whole brain was significantly lower than that of the control group. Then a series of morphological tests on the whole brain tissue of the mice were found to be found in the embryo. The apoptosis and proliferation of neurons in the cortex of the cerebral cortex were increased and the proliferation decreased. We also detected a series of proliferation and apoptosis in the primary cultured cortical neurons in vitro. We found that the results were consistent with the experiment in the body. Therefore, we infer that the deletion of PP2AC alpha does cause cortical neurons to wither. To explore the reason, we found that the activation of endogenous DNA damage response ATR/Chk1 signaling pathway was found in the cortical neurons of PP2AC - alpha deletion, and after the treatment of DNA single strand damage in cortical neurons, it was observed that the sensor-ATR and PP2AC involved in the injury response were Co located. The method of immunoprecipitation proved that PP2AC alpha deletion did directly cause the continuous activation of ATR phosphorylation, and then activated the downstream Chk1 and other effector molecules, which led to the activation of endogenous DNA damage response. Its overactivation further caused the up regulation of p53 protein expression, and one side through the p21 target protein resulted in cell cycle arrest. On the other hand, the expression of Bax, which promotes apoptosis, is up-regulated, and the inhibition of apoptosis is down down. This imbalance eventually leads to the abnormal apoptosis of cortical neurons. In order to study the biological function of PP2AC alpha more comprehensively, we also established a mouse model (PP2AC alpha D) in the dorsal cortex and the sea Matt heterosexual knockout of PP2AC a. 6-del), we found that the mice were able to survive and have a distinct head small deformity (microcephaly) phenotype after birth. There was also an increase in apoptosis caused by the activation of endogenous DNA damage responses in the cortical neurons of PP2AC alpha deletion. In addition, the hippocampus and its corresponding cortex area are the advanced Centers for regulating learning and cognitive function. We evaluated the learning and memory function of the Morris water maze, and found that the PP2AC alpha D6-del mice were associated with a significant decrease in learning and memory function compared with the control, which was consistent with the clinical signs of the clinical microcephaly patients. To sum up, the deletion of PP2AC alpha could cause endogenous DNA damage. The answer to the defect leads to the increase of cortical neuron apoptosis and the decrease of proliferation. Finally, the phenotype of the head small deformity characterized by the thinning of the cortex and the atrophy of the hippocampus, provides a new clue and theoretical basis for exploring the pathogenesis and clinical treatment of microcephaly.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R741
，

本文編號(hào)：2034882