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甲状旁腺素相关肽核定位序列和C末端缺失导致小鼠皮肤衰老的机制研究

Deletion of PTHrP Nuclear Localization and Carboxyl Terminus Leads to Skin Aging

作者: 专业:人体解剖学 导师:韩群颖 年度:2009 学位:硕士  院校: 南京医科大学

Keywords

Parathyroid hormone related peptide, nuclear localization sequence, Bmi-1, angiogenesis, skin aging

        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。先前研究表明PTHrP是一多激素,即不同的片段具有不同功能。其中N-末端(1-36)发挥PTH样作用;中段(37-86)参与胎盘钙转运; PTHrP(87-107)为核定位序列(NLS) ;C-末端(108-139)具有抑制骨吸收作用。体外研究表明PTHrP的NLS可通过胞内分泌(intracrine)方式发挥促进细胞增殖和抑制细胞凋亡的作用,PTHrP的C-末端对于NLS入核后发挥作用是必不可少的。为研究PTHrP NLS和C-末端的在体功能,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,产生了PTHrP NLS和C-末端敲除小鼠(即PTHrP KI小鼠,简称KI小鼠)。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 NLS和C-末端缺失致皮肤衰老的作用机制尚不清楚。为研究PTHrP KI致小鼠皮肤衰老的作用机制,我们分别从整体、组织细胞和分子水平比较分析了生后1天(P1)、1周(1W)和2周(2W)龄同窝KI和野生型(WT)小鼠的皮肤表型差异。整体观察表明:KI小鼠出现皮肤变薄、角质层增厚、皮下脂肪和总胶原减少等衰老表型。利用甲苯胺蓝染色观察到KI小鼠的皮肤渗透性增加,皮肤屏障功能不全。上述结果证明PTHrP NLS和C-末端缺失能够导致小鼠皮肤衰老。通过增殖细胞核抗原(PCNA)免疫组化染色和Annexin V、PI双染流式细胞检测,结果显示KI小鼠皮肤细胞增殖降低、凋亡增加。Western blot结果显示细胞周期蛋白D1和E及细胞周期蛋白依赖性激酶(CDK)2和4在KI小鼠皮肤组织中表达水平明显降低;而CDK抑制因子(CDKI)包括p16、p19、p27和p53在KI小鼠皮肤组织中表达水平均明显升高。这些结果表明KI小鼠可以通过下调Cyclin D1、E和CDK2、4,上调p16、p19、p27和p53而使小鼠皮肤衰老。通过新鲜皮肤体视显微摄影和血小板/内皮细胞粘附分子(CD31)免疫组化染色观察发现:KI小鼠皮肤血管较WT明显疏而细小;实时荧光定量RT-PCR检测发现CD31、血管内皮细胞生长因子(VEGF)、血管生成素1和2(Ang-1、Ang-2)及血管生成素受体2(Tie-2)的mRNA水平均明显低于WT小鼠;与mRNA的改变一致,CD31、VEGF、Ang-1和Ang-2的蛋白表达水平在KI小鼠皮肤组织中的表达也明显降低。这些结果表明PTHrP NLS和C-末端的缺失导致皮肤血管发生障碍也是小鼠皮肤衰老的原因之一。鉴于原癌基因Bmi-1(B cell specific moloney murine leukemia virus insertion site 1)蛋白在PTHrP KI小鼠来源的胚胎成纤维细胞中表达水平明显下调,核转位和核转移减少。我们推测Bmi-1可能介导PTHrP KI引起的皮肤衰老。为验证这一假说,我们利用免疫共沉淀检测PTHrP和Bmi-1是否能够相互结合,并分析了同窝2周和4周龄Bmi-1基因敲除纯合子小鼠和野生型小鼠皮肤表型的差异。发现PTHrP和Bmi-1具有相互结合的能力。表型分析结果表明:Bmi-1 KO小鼠皮肤总胶原阳性百分率在2周龄时较WT小鼠明显降低。4周龄时,Bmi-1 KO小鼠皮肤明显变薄,角质层增厚,皮下脂肪减少,总胶原阳性百分率进一步降低。通过PCNA免疫组织化学染色发现Bmi-1 KO小鼠的皮肤细胞增殖明显降低。Western blot方法检测发现CDKI包括p16、p19和p27在2周和4周龄Bmi-1 KO小鼠皮肤组织中的表达均明显上调。这些结果表明PTHrP NLS和C-末端能够通过Bmi-1介导抑制CDKI包括p16、p19和p27在皮肤组织中的表达,刺激皮肤细胞的增殖,抑制皮肤细胞凋亡而使小鼠皮肤衰老。这一研究表明PTHrP NLS和C-末端能够通过Bmi-1刺激皮肤细胞的增殖,抑制皮肤细胞凋亡;并能够通过上调促血管发生因子在皮肤组织中的表达而促进皮肤血管发生,发挥抗皮肤衰老的作用。本研究率先阐明了PTHrP NLS和C-末端在抗皮肤衰老中的作用机制,为将PTHrP NLS和C-末端开发成抗皮肤衰老药物提供实验和理论依据。
    Many studies have demonstrated the important role played by the N-terminal region of PTHrP as is the N-terminus of PTH, by interacting with the common PTH/PTHrP receptor; however increasing evidence suggests that PTHrP is a polyhormone with different functions for different domains. The mid-molecular region, amino acids 37-86, is responsible for placental Ca2+ transport and C-terminus 108-139 inhibit osteoclast activity and bone resorption. Previous studies have shown nuclear localization of PTHrP in multiple cell types and have been identified a functional nuclear localization sequence (NLS) in the 87-107 region of the molecule. The intracrine action of PTHrP has been reported in vitro to include inhibition of cell apoptosis and stimulation of cell proliferation. To investigate the function of NLS of PTHrP in vivo, we generate an animal model with disruption of the NLS of PTHrP by introducing a premature termination codon TGA into the PTHrP gene and created a“knock-in”mouse expressing PTHrP (1-84), a form of the protein missing the NLS and its carboxyl terminus (PTHrP KI). Mice homozygous for the knock-in mutation displayed features of impaired growth and early senescence including muscle atrophy, atrophic skin with hyperkeratosis, reduced body fat, and osteoporosis. However, it is unknown what mechanism of PTHrP KI resulted in skin aging.To explore the mechanism of PTHrP KI caused skin aging, the phenotypes of skin were analyzed by the comparison of PTHrP KI mice at postnatal day 1, 7 and 14 to their age-matched wild-type (WT) littermates using histopathologic, cellular and molecular approaches. PTHrP KI mice displayed a premature aging phenotype in skin including thinner skin with hyperkeratosis of the epidermis, reduced collagen and subcutaneous fat deposition in skin matrix. The skin permeability was increased in PTHrP KI mice demonstrated by staining with toluidine blue and the result suggests that PTHrP KI resulted in a faulty permeability barrier. Consequently, deletion of the PTHrP NLS and carboxyl terminus leads to a skin premature senesence.The proliferation of dermatic cells was decreased and the apoptosis of dermatic cells were increased in PTHrP KI mice demonstrated by immunohistochemical staining for the proliferating cell nuclear antigen (PCNA), double staining with Annexin V and propidium iodide and flow Cytometry analysis, respectively. The expression levels of cell cycle protein D1 and E and cyclin dependent kinase (CDK)2 and 4 were down-regulated significantly, whereas the pressionlevels of cell cycle dependent kinase inhibitors (CDKI) including p16, p19, p27and p53 were up-regulated markedly in skin tissues from PTHrP KI mice. These results suggest that skin senesence occurred in PTHrP KI mice is associated with inhibiting dermatic cell proliferation and enhancing dermatic cell apoptosis through the down-regulation of cyclin D1 and E and CDK2 and 4 and the up-regulation of CDKI including p16, p19, p27and p53.The density of skin vessels was reduced in the PTHrP KI mice compared to WT littermates demonstrated by imaging with steromicroscopy in fresh skin samples and the immunohistochemical staining for platelet endothelial cell adhesion molecule-1 (CD31). The gene expression levels of CD31,vascular endothelial growth factor (VEGF), angiopoietin-1(Ang-1), angiopoietin-2(Ang-2) and endothelial cell receptor tyrosine kinases(Tie-2) were down-regulated significantly at the postnatal day 1, 7 and 14 in skin tissues from PTHrP KI mice demonstrated by the real-time RT-PCR. Consistent with gene expression, the protein expression levels of CD31, VEGF, Ang-1 and Ang-2 were also down-regulated in skin tissues from PTHrP KI mice demonstrated by Western blot. These results suggest that skin senesence occurred in PTHrP KI mice is also associated with the impairment of angiogenesis in the skin. According to our previous studys that the expression level of Bmi-1 and the nuclear translocation were reduced in mouse embryonic fibroblasts (MEFs) from PTHrP KI mice, we proposed whether Bmi-1 was as a down-stream target of PTHrP mediated skin senesence caused by PTHrP KI. To test our hypothesis, the interaction of PTHrP with Bmi-1 was assessed by co-immunoprecipitation and the dermatic phenotypes were compared between the wild-type and Bmi-1 knockout (KO) mice at 2 and 4 weeks of age using morphological, cellular and molecular approaches. The total collagen positive area was reduced significantly at 2-week-old Bmi-1 KO mice. The skin was thinner with hyperkeratosis of the epidermis, collagen and subcutaneous fat deposition in skin matrix were reduced in 4-week-old Bmi-1 KO mice compared to their WT littermates. The proliferation of dermatic cells was decreased demonstrated by immunohistochemical staining for PCNA in 2- and 4-week-old Bmi1 KO mice. The expression levels of cell cycle dependent kinase inhibitors including p16,p19 and p27 were up-regulated markedly in skin tissues from Bmi-1 KO mice demonstrated by Western blot. These findings indicates that Bmi-1 may be as a down-stream target of PTHrP mediated skin senesence caused by PTHrP KI by inhibiting the proliferation of dermatic cells and stimulating the apoptosis of dermatic cells through the up-regulation of CDKI including p16, p19, p27.This study demonstrated that PTHrP NLS and carboxyl terminus plays an important role in preventing skin aging by stimulating the proliferation of dermatic cells, inhibiting the apoptosis of dermatic cells and enhancing angiogenesis of the skin mediated by Bmi-1. This study provides an experimental and theoretical evidence that PTHrP NLS and carboxyl terminus could be used as a therapeutics to prevent skin aging.
        

甲状旁腺素相关肽核定位序列和C末端缺失导致小鼠皮肤衰老的机制研究

缩写词4-7
中文摘要7-9
英文摘要9-11
引言12-17
材料与方法17-39
结果39-50
    第一部分 甲状旁腺素相关肽核定位序列与C-末端缺失导致小鼠皮肤衰老39-46
        1.PTHrP KI对小鼠生长和皮肤屏障功能的影响39-40
        2.PTHrP KI对小鼠皮肤形态的影响40-41
        3.PTHrP KI对皮肤细胞增殖和凋亡的影响41-43
        4.PTHrP KI对皮肤血管发生的影响43-46
    第二部分 Bmi-1基因缺失导致小鼠皮肤衰老46-50
        1.PTHrP和Bmi-1具有相互结合的能力46-47
        2.Bmi-1基因敲除对小鼠生长和皮肤形态的影响47
        3.Bmi-1基因敲除对皮肤细胞增殖的影响47-50
讨论50-56
小结56-57
结论和意义57-58
参考文献58-64
综述64-72
    参考文献69-72
攻读学位期间发表文章情况72-73
致谢73
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