FSH PROCESS CORE

FSH 工艺核心

• 批准号: 8449610
• 负责人: JEFFREY V. MAY
• 金额: $ 34.23万
• 依托单位: WICHITA STATE UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8449610
• 关键词: Adherent Culture; Aging; Amino Acid Sequence; Amino Acids; Area; Back; Binding; Bioinformatics; Biological; Biological Assay; Carbohydrates; Cell Culture Techniques; Cell Line; Cell model; Cells; Chemicals; Chemistry; Chromatography; Collaborations; Conditioned Culture Media; Contraceptive Agents; Cryopreserved Cell; Data; Databases; Deposition; Development; Doctor of Philosophy; Estradiol; Family; Family suidae; Fractionation; Funding; Gene Expression; Genetic Vectors; Glycopeptides; Glycoproteins; Goals; Gonadal Hormones; Gonadotropins; Hormones; Human; Human Chorionic Gonadotropin; Human Follicle Stimulating Hormone; Hybrids; Hypothalamic structure; Individual; Infertility; Ions; LH Receptors; Label; Laboratories; Lead; Liquid substance; Mammalian Cell; Molecular Biology; Monoclonal Antibodies; Neurons; Nitrogen; Ovarian; Peptide Mapping; Peptide Sequence Determination; Peptides; Pituitary Gland; Play; Postdoctoral Fellow; Preparation; Primary Cell Cultures; Principal Investigator; Procedures; Process; Production; Property; Protein Chemistry; Proteins; Quality Control; Radioimmunoassay; Recombinants; Reporting; Reproductive system; Research; Research Personnel; Role; Scientist; Services; Source; Staging; Steroid biosynthesis; Steroids; Structure; System; Techniques; Testing; Time; Training Support; Transformed Cell Line; Update; base; comparative efficacy; data sharing; experience; feeding; granulosa cell; meetings; member; milligram; pituitary gonadal axis; programs; protein expression; receptor binding; research study; response; scale up; steroid hormone; tissue/cell culture; urinary; wasting

The goal of the FSH process core laboratory (Core B) is to provide instumentation, support and training in protein chemistry techniques adapted to glycoprotein characterization. This core laboratory has extensive experience with gonadotropin purification and characterization. The gonadotropic hormones play a central role in regulating the reproductive system. They are released in response to episodic stimulation by hypothalamic neuronal activity to coordinate the later stages of follicle development initiated by endogenous gonadal cycles. In turn, gonadal hormones, particularly the steroid hormone estradiol feed back to the pituitary and alter gonadotropin synthesis and posttranslational processing. While steroid hormones are used therapeutically in contraceptive procedures, it is the gonadotropins that are used to treat infertility. The core will provide three support functions for the proposed project, as follows: Function #1. Produce hFSH glycofoms. Di-glycosylated, tetra-glycosylated and other hFSH glycoforms will be expressed by Dr. Bin Shuai in mammalian cell lines. Dr. Shuai will provide FSH-containng conditioned culture medium to Dr. Butnev. Function #2. Purify and chemically characterize hFSH glycoform preparations. Dr. Vladimir Butnev will purify and chemically characterize hFSH glycoforms. This will include isolating hFSH glycoforms from pituitary extracts and conditioned culture meduim, determining chemical purity, determining protein quantity, assessing protein integrity, as well as determining carbohydrate composition. FSH characterization is critical for interpreting the biological studies performed in Projects 1-3. FSH glycoform quality control data will be posted monthly on the Glycomics data-sharing page maintained by Core C. Function #3. Provide FSH assay and cell culture support for Project researchers. Dr. Jeffrey V. May will perform receptor-binding and steroidogenic assays in the well-characterized porcine granulosa cell primary culture system to confirm biological activity of purified hFSH'glycoforms. The preparations will then be released to project investigators. These assays will also be made available to project investigators as a service. Projects 1 and 2 will perform some experiments in a common cell model. Initially, this will be porcine granulosa cells. Dr. May will evaluate human granulosa cell lines that become available. For example project investigators wasted valuable time determining that KGN cells, a supposed human granulosa cell line, were unsuitable for our studies. If this project is funded, then Core B will evaluate cell lines and suitable lines will be made available to the projects. This core will also provide support for monoclonal antibody expression to support project 1. Dr. May will serve as overall Core Leader based upon his experience in primary cell culture as well as protein chemistry and molecular biology.

FSH流程核心实验室（核心B）的目标是提供Intsumentation，Support和培训 在适应糖蛋白表征的蛋白质化学技术中。这个核心实验室有广泛的 具有促性腺激素纯化和表征的经验。促性腺激素激素发挥了中心 在调节生殖系统中的作用。它们是根据情节刺激而释放的 下丘脑神经元活性，以协调内源性发育的后期阶段 性腺周期。反过来，性腺激素，特别是类固醇激素雌二醇回到 垂体和改变促性腺激素的合成和翻译后加工。使用类固醇激素 在避孕程序上，用于治疗不育症的是促性腺激素。核心 将为拟议项目提供三个支持功能，如下：功能＃1。产生HFSH 糖蛋糕。二糖基化，四糖基化和其他HFSH糖型将由Bin博士表达 Shuai在哺乳动物细胞系中。 Shuai博士将向博士提供FSH-Containng条件培养基。 但是。功能＃2。纯化和化学表征HFSH糖型制剂。弗拉基米尔博士 丁纳夫将纯化和化学表征HFSH糖型。这将包括隔离HFSH糖型 从垂体提取物和条件培养物中，确定化学纯度，确定蛋白质 数量，评估蛋白质完整性以及确定碳水化合物组成。 FSH表征 对于解释项目1-3中进行的生物学研究至关重要。 FSH糖基型质量控制数据 将每月发布在由CoreC。功能＃3维护的糖数据共享页面上。提供 FSH分析和细胞培养支持项目研究人员。 Jeffrey V. May博士将表演 特征良好的猪颗粒细胞原代培养物中的受体结合和类固醇生成测定 确认纯化的HFSH'Glycoforms的生物活性的系统。然后，准备工作将被释放到 项目调查人员。这些测定也将作为服务项目提供。项目 1和2将在公共细胞模型中执行一些实验。最初，这将是猪颗粒细胞。 梅博士将评估可用的人颗粒细胞系。例如项目调查人员 浪费了宝贵的时间，确定KGN细胞是一种假定的人颗粒细胞系，不适合 我们的研究。如果该项目是资助的，则核心B将评估细胞系，并将制作合适的线 可用于项目。该核心还将为单克隆抗体表达提供支持 项目1。May博士将根据他在初级细胞文化方面的经验作为总体核心领导者 作为蛋白质化学和分子生物学。