中枢神经系统Nbn基因敲除小鼠大脑皮质胶质细胞的功能改变

发布时间：2018-06-28 12:18

本文选题：Nbs1 + 神经元变性　；参考：《北京协和医学院》2012年博士论文

【摘要】：在DNA损伤应激反应中,MRN复合物(由Mre11蛋白、Rad50蛋白、Nbs1蛋白共同组成)能够识别DNA损伤位点,通过调节损伤修复通路蛋白,参与DNA损伤修复。人类NbS1基因(编码Nbs1蛋白)突变将引起“染色体断裂综合症(ni jmegen breakage syndrome, NBS)",其临床表现主要包括：小颅畸形、免疫缺陷、生长发育缺陷、染色体不稳定性以及恶性肿瘤易患性等。为进一步研究“NBS综合症”患者小颅畸形的发生机制,本课题组前期研究中,通过“条件性基因敲除方法(conditional knockout)"将小鼠Nbn基因在中枢神经系统(central nervous system, CNS)神经元中特异性敲除。该小鼠模型(命名为NbnCNS-edl小鼠)的表型和人类“NBS综合症”患者的临床表现极为相似。因此该动物模型的制备为研究“NBS综合症”患者小颅畸形的发生机制提供了良好的实验基础。我们前期研究发现,生后早期NbnCNS-del小鼠大脑组织结构能够形成,但其内神经元出现明显的退行性变；而小脑的组织结构大部分不能形成,其内神经元退行性变更为严重。另外,我们发现,生后早期NbnCNS-edl小鼠胼胝体以及脑干区域的有髓神经纤维极向消失,密度显著降低,提示Nbsl可能对髓鞘形成起一定作用。本研究从神经胶质细胞对神经元的营养角度,探讨了生后早期NbnCNS-edl小鼠大脑皮质神经元及有髓神经纤维变性的分子机制。目前研究发现,Abn基因在小鼠CNS星形胶质细胞中的表达下调,导致神经生长因子(nerve growth factor,NGF)以及脑源性神经营养因子(brain derived neurotrophic factor,BDNF)的蛋白表达水平降低,而对神经营养素-3(neurotrophin-3,NT-3)的蛋白表达水平并未产生影响。另外,NGF的蛋白表达水平降低并未影响受体型酪氨酸蛋白激酶A(tyrosine kinase A,TrkA)以及P75神经营养素受体(P75neurotrophic receptor,P75NTR)的蛋白表达水平,但是NGF与两种受体的亲和力减弱。Nbn基因在小鼠CNS小胶质细胞中的表达下调,使小胶质细胞的功能降低,导致白细胞介素-1(interlukin-1, IL-1)及白细胞介素-6(interlukin-6, IL-6)的分泌减少。NGF以及BDNF的分泌减少下调了大脑皮质哺乳动物雷帕霉素靶向蛋白(mammalian rapamycin targeting protein, mTOR)以及S6核糖体蛋白(S6ribosomal protein, S6RP)的磷酸化水平,使星型胶质细胞对皮质神经元及有髓神经纤维的营养保护作用减弱；而IL-1及IL-6的分泌减少导致核因子κB (neuclei factor-κ B, NF-κB)的磷酸化水平降低,使小胶质细胞对皮质神经元及有髓神经纤维的炎性保护作用减弱。Nbn基因在小鼠CNS少突胶质细胞中的表达下调,抑制了髓鞘碱性蛋白(myelin sheath basic protein, MBP)以及髓鞘调控转录因子o1igo1的蛋白表达水平,导致髓鞘发育缺陷。神经营养作用以及炎性反应的减弱参与了髓鞘发育缺陷的分子机制。综合上述,Nbn基因在小鼠CNS中特异性敲除导致星形胶质细胞及小胶质细胞对大脑皮质神经元及有髓神经纤维的营养及炎性作用减弱,分别通过下调mTOR/S6RP、NF-κB信号转导通路的活性,引起大脑皮质神经元退行性变及髓鞘形成障碍。
[Abstract]:In the DNA damage stress response, the MRN complex (composed of Mre11 protein, Rad50 protein, Nbs1 protein) can identify the DNA damage site and participate in the repair of DNA damage by regulating the damage repair pathway protein. The mutation of the human NbS1 gene (the coded Nbs1 protein) will cause "dyor body fracture syndrome (Ni jmegen breakage)". The main manifestations include small cranial deformities, immunodeficiency, growth and development defects, chromosomal instability and susceptibility to malignant tumors.
In order to further study the mechanism of small craniofacial malformation in "NBS syndrome", in our previous study, the specific knockout of the mouse Nbn gene in the central nervous system (central nervous system, CNS) neurons was carried out by the conditional gene knockout method (conditional knockout). The mouse model (named as NbnCNS-edl mouse) The phenotype is very similar to that of the human "NBS syndrome". Therefore, the preparation of this animal model provides a good experimental basis for the study of the mechanism of small craniofacial malformation in "NBS syndrome" patients. Our previous study found that the brain tissue of NbnCNS-del mice could be formed in the early postnatal period, but the neurons in it appeared in the early stage. In addition, we found that the myelinated nerve fibers in the corpus callosum and the brain stem regions disappeared and the density decreased significantly in the early NbnCNS-edl mice, suggesting that Nbsl may play a role in the formation of myelin sheath.
In this study, from the nutritional angle of neuroglia cells to neurons, the molecular mechanism of neuronal degeneration in the cerebral cortex and myelinated nerve of NbnCNS-edl mice in the early postnatal period was investigated. The present study found that the expression of Abn gene in CNS astrocytes was down regulated, leading to the nerve growth factor (NGF) and the brain source. The protein expression level of brain derived neurotrophic factor (BDNF) was reduced, but it did not affect the protein expression level of neurotrophin -3 (neurotrophin-3, NT-3). In addition, the decrease of the protein expression level of NGF did not affect the type of tyrosine protein kinase A (tyrosine kinase) and neuronutrition. The protein expression level of P75neurotrophic receptor (P75NTR), but the affinity of NGF and two receptors weakened the expression of.Nbn gene in the mouse CNS microglia, reduced the function of the microglia and resulted in the decrease of the secretion of interleukin -1 (Interlukin-1, IL-1) and interleukin -6 (Interlukin-6, IL-6). And the decrease of BDNF secretion reduced the phosphorylation level of rapamycin target protein (mammalian rapamycin targeting protein, mTOR) and S6 ribosome (S6ribosomal protein, S6RP) in the cerebral cortex mammal, which weakened the protective effect of astrocytes on cortical neurons and myelinated nerve fibers; IL-1 and IL- were reduced. The decrease in secretion of 6 resulted in a decrease in the phosphorylation level of nuclear factor kappa B (neuclei factor- kappa B, NF- kappa B). The inflammatory protective effect of microglia on cortical neurons and myelinated nerve fibers weakened the expression of.Nbn gene in the mouse CNS oligodendrocytes, and inhibited the myelin basic protein (myelin sheath basic protein). The myelin sheath regulates the protein expression level of the transcription factor o1igo1, which leads to myelin development defects. Neurotrophic effects and diminished inflammatory responses are involved in the molecular mechanism of myelin development defects.
In addition, the specific knockout of the Nbn gene in the mouse CNS leads to the weakening of the nutritional and inflammatory effects of astrocytes and microglia on the cerebral cortex and myelinated nerve fibers. By downregulating the activity of mTOR/S6RP and NF- kappa B signal transduction pathway, the degeneration of cerebral cortex neurons and the obstruction of myelin formation are caused.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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