甲狀旁腺素相關肽核定位序列與C-末端缺失導致小鼠腦發(fā)育異常
發(fā)布時間：2018-07-25 11:44
【摘要】： 甲狀旁腺素相關肽(PTHrP)與甲狀旁腺素(PTH)的N-末端在序列及空間構象上高度同源并可通過相同的受體——PTH／PTHrP受體(又稱PTH 1型受體)發(fā)揮作用。研究表明PTHrP是一多激素，即不同的結構域具有不同功能。其中間段(37-86)參與胎盤鈣轉運；C-末端(108-139)具有抑制骨吸收作用。PTHrP(87-107)為核定位序列(NLS)，體外研究表明PTHrP的NLS可通過細胞內分泌(intracrine)方式發(fā)揮促進細胞增殖和抑制細胞凋亡作用，PTHrP的C-末端對于NLS入核后的作用發(fā)揮必不可少。但NLS和C-末端的在體功能尚不清楚。為了研究NLS的在體功能，我們通過在小鼠PTHrP基因第84位氨基酸序列后敲入(Knock-In)一氨基酸翻譯的終止子——TGA，使其只表達PTHrP(1-84)，而不表達NLS和C-末端，這樣就產生了PTHrP NLS和C-末端敲除小鼠(即PTHrP KI小鼠，簡稱KI小鼠)。純合子KI小鼠出現(xiàn)生長阻滯及肌萎縮、皮膚過度角化、全身脂肪減少和骨質疏松等衰老表型。但是PTHrP KI對腦發(fā)育的影響尚未研究。 PTHrP及其受體在中樞神經系統(tǒng)廣泛表達且在大腦皮質、海馬、小腦等部位高表達。為了研究PTHrP KI對小鼠腦發(fā)育的影響，我們采用胚胎(E18．5)、生后1天(P1)、P7、P14的同窩KI和野生型(WT)小鼠，分別從整體水平、組織細胞水平和分子水平研究PTHrP的NLS和C-末端缺失導致的小鼠腦發(fā)育異常。整體觀察表明：KI小鼠的腦較輕、吻尾徑縮短、嗅球短小、大腦皮質前部較薄、小腦較小。說明PTHrP的NLS和C-末端在維持腦正常發(fā)育中起重要作用。 通過增殖細胞核抗原(PCNA)免疫組化染色，發(fā)現(xiàn)KI小鼠的室下帶、海馬和小腦中PCNA陽性細胞數(shù)都較同窩的WT小鼠明顯減少；Caspase-3免疫組化和TUNEL染色陽性細胞在KI小鼠齒狀回部位明顯增加；Western Blot結果表明，KI小鼠的細胞周期依賴性激酶抑制因子(CDKI)P16、P21、P27、P53都明顯升高。這些結果說明PTHrP的NLS和C-末端可通過下調CDKI表達而抑制腦細胞凋亡、刺激神經干細胞增殖，從而促進腦發(fā)育。 神經元特異性核蛋白(NeuN)免疫組化結果表明，KI小鼠齒狀回的NeuN陽性神經元百分率從出生至P14天均較WT小鼠低；在大腦皮質，其陽性細胞百分率在E18．5和P1較WT小鼠為低，，而從P7至P14，其陽性百分率則與WT小鼠相近，但其平均大小則明顯小于同窩WT小鼠。通過膽堿乙酰轉移酶(ChAT)免疫組化染色發(fā)現(xiàn)，P7和P14天的KI小鼠腦干面神經核ChAT陽性產物總灰度值較WT小鼠明顯降低。實時熒光定量RT-PCR結果表明在P1至P14天的KI小鼠腦鈣結合蛋白D-28k的mRNA水平均較WT小鼠明顯下調。鈣結合蛋白D-28k的陽性產物面積和總灰度值在P14天的KI小鼠齒狀回、紋狀體和小腦蒲肯野細胞均明顯降低。這些結果說明PTHrP的NLS和C-末端能夠促進神經元的分化和成熟，并可通過上調鈣結合蛋白D-28k而促進正常神經元功能的發(fā)揮并具防止神經元退變的作用。 膠質纖維酸性蛋白(GFAP)免疫組化結果表明，在海馬、小腦和P14天的室下帶部位KI小鼠的GFAP陽性細胞和纖維與WT小鼠無明顯差異。但在室下帶部位KI小鼠的GFAP陽性結構從E18．5至P7天均較WT小鼠少，并伴有室下帶細胞向大腦皮質遷移的延遲和小腦皮質外顆粒層細胞內向顆粒層遷移延遲。髓鞘堿性蛋白(MBP)免疫組化檢測發(fā)現(xiàn)KI小鼠MBP陽性結構在腦不同部位均較WT小鼠明顯減少。MBP的Western Blot檢測結果與免疫組化結果一致。這些結果表明PTHrP的NLS和C-末端能夠：①促進星形膠質細胞在室下帶和小腦的分化，從而加速細胞從室下帶向大腦皮質或從小腦外顆粒層向內顆粒層遷移。②促進少突膠質細胞分化和髓鞘形成。 對新鮮腦表面血管的觀察發(fā)現(xiàn)P14天的KI小鼠腦血管較WT小鼠明顯細小。熒光定量RT-PCR檢測發(fā)現(xiàn)血小板／內皮細胞粘附分子(PECAM-1，又稱CD31)和血管內皮細胞生長因子(VEGF)的mRNA水平在P7和P14天KI小鼠均明顯低于WT小鼠。VEGF的Western Blot結果與熒光定量RT-PCR結果一致。這些結果說明PTHrP的NLS和C-末端可通過促進腦血管的發(fā)生而促進腦發(fā)育。 本課題對KI小鼠腦發(fā)育不良進行了較系統(tǒng)的研究，包括腦形態(tài)觀測、腦細胞增殖、分化、遷移、凋亡及腦血管發(fā)生檢測。結果證明PTHrP KI引起了腦形態(tài)異常、神經干細胞增殖能力降低和細胞凋亡增加。這些改變可能與上調P16、P21、P27和P53等CDKI有關。PTHrP KI也造成了腦細胞遷移、分化和成熟的延遲及髓鞘形成和腦血管發(fā)生異常。這些發(fā)現(xiàn)充分說明PTHrP的NLS和C-末端可通過促進腦細胞增殖、遷移、分化、血管發(fā)生和抑制腦細胞凋亡而在小鼠腦發(fā)育中起重要作用。PTHrP NLS和C-末端的這些作用提示其在中樞神經系統(tǒng)疾病(如神經退行性病變和中風等)的治療方面具有潛在的臨床應用價值。
[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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R363
本文編號：2143719
[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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R363
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