Resolution of Epithelial Cell Hyperplasia

上皮细胞增生的解决

• 批准号: 8502494
• 负责人: Yohannes Tesfaigzi
• 金额: $ 50.44万
• 依托单位: LOVELACE BIOMEDICAL & ENVIRONMENTAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-15 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8502494
• 关键词: Abbreviations; Adenovirus Vector; Affect; Aftercare; Air; Alanine; Alcian Blue; Apoptosis Inhibitor; Appearance; BCL2 gene; BH3 Domain; Cell Culture Techniques; Cell Death; Cells; Chronic; Chronic Bronchitis; Codon Nucleotides; Cystic Fibrosis; Development; Disease; Embryo; Endotoxins; Epithelial; Epithelial Cells; Epithelium; Equus caballus; Ethnic Origin; Exposure to; Fibroblasts; Gender; Genetic Polymorphism; Genetic Variation; Genotype; Half-Life; Health; Hematoxylin and Eosin Staining Method; Human; Hyperplasia; Individual; Inflammatory Response; Liquid substance; MUC5AC gene; Malignant neoplasm of lung; Mediating; Messenger RNA; Metaplasia; Mucins; Mucous body substance; Mus; Mutate; Noxae; Pathway interactions; Peptides; Periodic acid Schiff stain method; Phenotype; Play; Process; Proline; Proline-Rich Domain; Proteins; Puma; Rattus; Regulation; Resolution; Risk; Role; Single Nucleotide Polymorphism; Smoker; Smoking; Source; Staging; TP53 gene; Testing; Variant; Wild Type Mouse; airway epithelium; arginylarginine; arginylproline; base; carcinogenesis; cigarette smoke-induced; cigarette smoking; clinically significant; cohort; cystic fibrosis patients; effective therapy; mRNA Expression; novel strategies; particle; pro-apoptotic protein; prolyl-proline; repaired; response; restoration

DESCRIPTION (provided by applicant): Epithelial cell hyperplasia in response to an inflammatory response is accompanied by mucous cell metaplasia (MCM), the appearance of mucous cells in airways that are normally devoid of these cells. MCM is one of the major factors for increased mucous hypersecretion in chronic bronchitis. We have shown that Bcl-2, an inhibitor of apoptosis, sustains MCM in rats and mice and may have clinical significance because it is expressed in mucous cells of patients with cystic fibrosis or chronic bronchitis. A polymorphism in the p53 gene at codon 72 (Arg/Pro) differentially affects Bcl-2 expression; p53Pro increases more efficiently than p53Arg, the pro-apoptotic protein Noxa that reduces the bcl-2 mRNA half-life. Deleting or mutating the proline residue within the corresponding region of murine p53 had the same effect in mouse embryo fibroblasts. Consistent with these findings, smokers with the p53Pro/Pro genotype were at lower risk of developing chronic bronchitis than smokers with the p53Arg/Arg genotype. Preliminary results with normal human bronchial epithelial cells (NHBECs) from five Arg/Arg and five Pro/Pro individuals showed that in the untreated state NHBECArg/Arg cultures showed reduced Noxa mRNA levels compared to NHBECPro/Pro cultures and reduced Puma mRNA levels when cultures were treated with environmental cigarette smoke (ETS). Therefore, we want to test the central hypothesis that bronchial cells with the p53Pro/Pro variant resolve epithelial cell hyperplasia and MCM more efficiently than bronchial cells with p53Arg/Arg by inducing Noxa expression that mediates the reduction of Bcl- 2 levels. Specific Aim 1 will determine whether differentiated NHBECArg/Arg, NHBECArg/Pro, and NHBECPro/Pro cultures will show differences in secreted mucus, MUC5AC, Noxa, and Puma mRNA expression, and the development of MCM before and after exposure to ETS extract. Similarly, mouse tracheal epithelial cells from mice with wild-type and mutated p53 will be tested for secreted mucus, and expression of Muc5ac, Noxa and Puma mRNAs, and MCM in the untreated state and after treatment with ETS extract. In addition, five representative NHBECArg/Arg and NHBECPro/Pro cultures will be infected with adenoviral vectors expressing Ad-p53Pro or Ad-p53Arg, respectively, to determine whether the phenotypes for these NHBECs will be modulated. Specific Aim 2 will increase and reduce Noxa and/or Puma expression in NHBECPro/Pro and NHBECArg/Arg cultures to determine their roles in the development of MCM. In addition, the role of these proteins will be examined by exposing noxa-/-, puma-/- and wild-type mice to ETS and by treating NHBECArg/Arg cultures with Noxa- and/or Puma-derived peptides. These studies will provide strategies for novel therapies that may facilitate the restoration of the aberrant repair process and reduce epithelial cell hyperplasia and MCM in chronic bronchitis.

描述（由申请人提供）：响应炎症反应的上皮细胞增生伴随着粘液细胞化生（MCM），粘液细胞在通常没有这些细胞的气道中的粘液细胞出现。 MCM是慢性支气管炎中粘液过度分泌增加的主要因素之一。我们已经表明，凋亡的抑制剂Bcl-2在大鼠和小鼠中维持MCM，并且可能具有临床意义，因为它在患有囊性纤维化或慢性支气管炎患者的粘液细胞中表达。密码子72（arg/pro）在p53基因中的多态性会差异地影响Bcl-2的表达。 p53pro比p53arg（降低Bcl-2 mRNA半衰期的促凋亡蛋白NOXA）更有效地增加。在鼠p53相应区域内删除或突变脯氨酸残基在小鼠胚胎成纤维细胞中具有相同的作用。与这些发现一致，与患有p53arg/arg基因型的吸烟者相比，患有p53Pro/Pro基因型的吸烟者患慢性支气管炎的风险低。来自五个ARG/ARG和五个Pro/Pro个体的正常人支气管上皮细胞（NHBEC）的初步结果表明，在未经处理的状态NHBECARG/ARG培养物中，与NHBECPRO/Pro培养物相比，NOXA mRNA水平降低了，与培养物与环境烟雾相比降低了puma mRNA，并降低了puma mRNA水平。因此，我们希望测试中心假设，即具有p53Pro/Pro变体的支气管细胞可以比p53arg/arg的支气管细胞更有效地分辨上皮细胞增生和MCM，从而诱导NOXA表达介导BCL-2水平的降低。具体目标1将确定分化的NHBECARG/ARG，NHBECARG/PRO和NHBECPRO/PRO培养是否会显示出分泌的粘液，MUC5AC，NOXA和PUMA mRNA的差异，以及在暴露于ETS提取物之前和之后的MCM的发展。类似地，将测试来自野生型和突变p53小鼠的小鼠气管上皮细胞的分泌粘液，并在未处理的状态和用ETS提取物处理后的MUC5AC，NOXA和PUMA mRNA和MCM的表达。此外，将分别用表达AD-P53PRO或AD-P53ARG的腺病毒载体感染五个代表性的NHBECARG/ARG和NHBECPRO/PRO培养物，以确定是否会调节这些NHBEC的表型。特定的目标2将在NHBECPRO/PRO和NHBECARG/ARG培养物中增加和减少NOXA和/或PUMA表达，以确定它们在MCM发展中的作用。此外，将通过将NOXA - / - ，PUMA - / - 和野生型小鼠和用NOXA-和/或PUMA衍生的肽处理NHBECARG/ARG培养物来检查这些蛋白质的作用。这些研究将为新型疗法提供策略，以促进异常修复过程的恢复，并减少慢性支气管炎中上皮细胞增生和MCM。