甲状旁腺素相关肽核定位序列与C-末端缺失导致小鼠脑发育异常

发布时间：2018-07-25 11:44

【摘要】： 甲状旁腺素相关肽(PTHrP)与甲状旁腺素(PTH)的N-末端在序列及空间构象上高度同源并可通过相同的受体——PTH／PTHrP受体(又称PTH 1型受体)发挥作用。研究表明PTHrP是一多激素，即不同的结构域具有不同功能。其中间段(37-86)参与胎盘钙转运；C-末端(108-139)具有抑制骨吸收作用。PTHrP(87-107)为核定位序列(NLS)，体外研究表明PTHrP的NLS可通过细胞内分泌(intracrine)方式发挥促进细胞增殖和抑制细胞凋亡作用，PTHrP的C-末端对于NLS入核后的作用发挥必不可少。但NLS和C-末端的在体功能尚不清楚。为了研究NLS的在体功能，我们通过在小鼠PTHrP基因第84位氨基酸序列后敲入(Knock-In)一氨基酸翻译的终止子——TGA，使其只表达PTHrP(1-84)，而不表达NLS和C-末端，这样就产生了PTHrP NLS和C-末端敲除小鼠(即PTHrP KI小鼠，简称KI小鼠)。纯合子KI小鼠出现生长阻滞及肌萎缩、皮肤过度角化、全身脂肪减少和骨质疏松等衰老表型。但是PTHrP KI对脑发育的影响尚未研究。 PTHrP及其受体在中枢神经系统广泛表达且在大脑皮质、海马、小脑等部位高表达。为了研究PTHrP KI对小鼠脑发育的影响，我们采用胚胎(E18．5)、生后1天(P1)、P7、P14的同窝KI和野生型(WT)小鼠，分别从整体水平、组织细胞水平和分子水平研究PTHrP的NLS和C-末端缺失导致的小鼠脑发育异常。整体观察表明：KI小鼠的脑较轻、吻尾径缩短、嗅球短小、大脑皮质前部较薄、小脑较小。说明PTHrP的NLS和C-末端在维持脑正常发育中起重要作用。通过增殖细胞核抗原(PCNA)免疫组化染色，发现KI小鼠的室下带、海马和小脑中PCNA阳性细胞数都较同窝的WT小鼠明显减少；Caspase-3免疫组化和TUNEL染色阳性细胞在KI小鼠齿状回部位明显增加；Western Blot结果表明，KI小鼠的细胞周期依赖性激酶抑制因子(CDKI)P16、P21、P27、P53都明显升高。这些结果说明PTHrP的NLS和C-末端可通过下调CDKI表达而抑制脑细胞凋亡、刺激神经干细胞增殖，从而促进脑发育。神经元特异性核蛋白(NeuN)免疫组化结果表明，KI小鼠齿状回的NeuN阳性神经元百分率从出生至P14天均较WT小鼠低；在大脑皮质，其阳性细胞百分率在E18．5和P1较WT小鼠为低，，而从P7至P14，其阳性百分率则与WT小鼠相近，但其平均大小则明显小于同窝WT小鼠。通过胆碱乙酰转移酶(ChAT)免疫组化染色发现，P7和P14天的KI小鼠脑干面神经核ChAT阳性产物总灰度值较WT小鼠明显降低。实时荧光定量RT-PCR结果表明在P1至P14天的KI小鼠脑钙结合蛋白D-28k的mRNA水平均较WT小鼠明显下调。钙结合蛋白D-28k的阳性产物面积和总灰度值在P14天的KI小鼠齿状回、纹状体和小脑蒲肯野细胞均明显降低。这些结果说明PTHrP的NLS和C-末端能够促进神经元的分化和成熟，并可通过上调钙结合蛋白D-28k而促进正常神经元功能的发挥并具防止神经元退变的作用。胶质纤维酸性蛋白(GFAP)免疫组化结果表明，在海马、小脑和P14天的室下带部位KI小鼠的GFAP阳性细胞和纤维与WT小鼠无明显差异。但在室下带部位KI小鼠的GFAP阳性结构从E18．5至P7天均较WT小鼠少，并伴有室下带细胞向大脑皮质迁移的延迟和小脑皮质外颗粒层细胞内向颗粒层迁移延迟。髓鞘碱性蛋白(MBP)免疫组化检测发现KI小鼠MBP阳性结构在脑不同部位均较WT小鼠明显减少。MBP的Western Blot检测结果与免疫组化结果一致。这些结果表明PTHrP的NLS和C-末端能够：①促进星形胶质细胞在室下带和小脑的分化，从而加速细胞从室下带向大脑皮质或从小脑外颗粒层向内颗粒层迁移。②促进少突胶质细胞分化和髓鞘形成。对新鲜脑表面血管的观察发现P14天的KI小鼠脑血管较WT小鼠明显细小。荧光定量RT-PCR检测发现血小板／内皮细胞粘附分子(PECAM-1，又称CD31)和血管内皮细胞生长因子(VEGF)的mRNA水平在P7和P14天KI小鼠均明显低于WT小鼠。VEGF的Western Blot结果与荧光定量RT-PCR结果一致。这些结果说明PTHrP的NLS和C-末端可通过促进脑血管的发生而促进脑发育。本课题对KI小鼠脑发育不良进行了较系统的研究，包括脑形态观测、脑细胞增殖、分化、迁移、凋亡及脑血管发生检测。结果证明PTHrP KI引起了脑形态异常、神经干细胞增殖能力降低和细胞凋亡增加。这些改变可能与上调P16、P21、P27和P53等CDKI有关。PTHrP KI也造成了脑细胞迁移、分化和成熟的延迟及髓鞘形成和脑血管发生异常。这些发现充分说明PTHrP的NLS和C-末端可通过促进脑细胞增殖、迁移、分化、血管发生和抑制脑细胞凋亡而在小鼠脑发育中起重要作用。PTHrP NLS和C-末端的这些作用提示其在中枢神经系统疾病(如神经退行性病变和中风等)的治疗方面具有潜在的临床应用价值。
[Abstract]:The N- terminal of parathyroid hormone related peptide (PTHrP) and parathyroid hormone (PTH) is highly homologous in sequence and space conformation and can play a role through the same receptor, PTH / PTHrP receptor (also known as PTH 1 receptor). The study shows that PTHrP is a multi hormone, that is, different domains have different functions. The segment (37-86) participates in the placental calcium transfer. C- terminal (108-139) has the inhibition of bone absorption by.PTHrP (87-107) as the nuclear location sequence (NLS). In vitro studies show that PTHrP NLS can play a role in promoting cell proliferation and inhibiting apoptosis through cell endocrine (intracrine), and the C- terminal function of PTHrP is essential to the function of NLS after nucleation. But the NLS and C- ends are in body. The function of NLS is not clear. In order to study the body function of NLS, we knock into the terminator of the Knock-In one amino acid translation after the PTHrP gene sequence of the mouse PTHrP gene, which makes it express only PTHrP (1-84), but does not express the end of NLS and C-, thus producing PTHrP NLS and C- end knockout mice (i.e. PTHrP KI mice, short for short) Mice in the homozygote KI mice showed growth block and muscular atrophy, hyperkeratosis, loss of body fat, and osteoporosis, but the effects of PTHrP KI on brain development have not been studied.
PTHrP and its receptors are widely expressed in the central nervous system and are highly expressed in the cerebral cortex, hippocampus, cerebellum and other parts. In order to study the effects of PTHrP KI on brain development in mice, we use embryos (E18.5), 1 days after birth (P1), P7, P14 of the same nest KI and wild type (WT) mice, respectively from the overall level, the tissue cell level and the molecular level of PTHrP respectively to study PTHrP. The overall observation showed that the brain of KI mice was lighter, the tail diameter of the KI was shorter, the olfactory bulb was short, the anterior part of the cerebral cortex was thinner and the cerebellum was smaller, indicating that the NLS and C- terminal of PTHrP played an important role in maintaining normal brain development in KI mice.
By immunohistochemical staining of proliferating cell nuclear antigen (PCNA), it was found that the number of PCNA positive cells in the subventricular zone of the KI mice, the hippocampus and the cerebellum were significantly lower than those of the WT mice with the same nest, and the Caspase-3 immunization and TUNEL staining positive cells in the dentate gyrus of KI mice increased significantly; Western Blot results showed that the cell cycle dependence of KI mice was dependent on the Western Blot results. The kinase inhibitory factor (CDKI) P16, P21, P27, and P53 all significantly increased. These results suggest that the NLS and C- terminals of PTHrP can inhibit the apoptosis of brain cells by down regulation of CDKI expression and stimulate the proliferation of neural stem cells, thus promoting brain development.
The immunohistochemical results of neuron specific nucleoprotein (NeuN) showed that the percentage of NeuN positive neurons in the dentate gyrus of KI mice was lower than that of WT mice from birth to P14 days, and in the cerebral cortex, the percentage of positive cells in E18.5 and P1 was lower than that of WT mice, while the positive percentage was similar to WT mice from P7 to P14, but the average size was obvious. Less than the same nest WT mice, the total gray value of ChAT positive products of KI mice in P7 and P14 days was significantly lower than that of WT mice through the immunohistochemical staining of choline acetyltransferase (ChAT). The real-time fluorescence quantitative RT-PCR results showed that the level of calcium binding egg white D-28k in KI mice from P1 to P14 days was significantly lower than that of the WT mice. The area of the positive product and the total gray value of the protein D-28k were significantly reduced in the dentate gyrus of KI mice in P14 days. The results showed that the NLS and C- terminal of the PTHrP could promote the differentiation and maturation of neurons, and could promote the function of normal neurons by up regulation of the calcium binding protein D-28k and prevent the function of the neurons. The role of neuron degeneration.
The immunohistochemical results of glial fibrillary acidic protein (GFAP) showed that there was no significant difference in the GFAP positive cells and fibers from the KI mice in the subventricular zone of the hippocampus, cerebellum and P14 days, but the GFAP positive structure of the KI mice in the subventricular zone was less than that of the WT mice from E18.5 to P7 days, with the delay in the migration of the subventricular bands to the cerebral cortex. The migration of the granular layer cells in the cerebellar cortex was delayed. The immunohistochemical detection of myelin basic protein (MBP) found that the MBP positive structure of the KI mice in different parts of the brain was significantly lower than that of the WT mice. The results of the Western Blot detection were in accordance with the immunohistochemical results. These results showed that the NLS and C- ends of PTHrP were capable of: 1. The differentiation of glial cells in the subventricular zone and the cerebellum accelerates the migration of the cells from the subventricular zone to the cerebral cortex or the granular layer of the cerebellum, and promotes the differentiation of oligodendrocytes and the formation of myelin.
The observation of the fresh brain blood vessels found that the KI mice in P14 days were significantly smaller than those of the WT mice. The fluorescence quantitative RT-PCR detection showed that the mRNA level of platelet / endothelial cell adhesion molecules (PECAM-1, also called CD31) and vascular endothelial growth factor (VEGF) in P7 and P14 days KI mice were significantly lower than those of WT mice. These results suggest that NLS and C-terminal of PTHrP can promote brain development by promoting cerebrovascular development.
A systematic study of brain dysplasia in KI mice, including brain morphological observation, brain cell proliferation, differentiation, migration, apoptosis and cerebrovascular detection, has been studied. The results show that PTHrP KI causes abnormal brain morphology, reduced proliferation of neural stem cells and increased apoptosis. These changes may be associated with the up regulation of CDKI, such as P16, P21, P27 and P53. .PTHrP KI also causes brain cell migration, differentiation and maturation delay and myelin formation and cerebrovascular abnormalities. These findings fully demonstrate that the NLS and C- terminals of PTHrP play an important role in the development of.PTHrP NLS and C- in mouse brain development by promoting brain cell proliferation, migration, differentiation, angiogenesis and inhibition of brain cell apoptosis. These results suggest that it has potential clinical value in the treatment of central nervous system diseases such as neurodegenerative diseases and stroke.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R363

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