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工艺核心实验室（Core B）的目标是提供仪器、支持和培训 在蛋白质化学技术适用于糖蛋白表征。该核心实验室拥有广泛的 有促性腺激素纯化和鉴定的经验。促性腺激素发挥着中枢作用， 调节生殖系统的作用。它们是在对偶发刺激的反应中被释放的， 下丘脑神经元的活动，以协调卵泡发育的后期阶段启动的内源性 性腺周期反过来，性腺激素，特别是类固醇激素雌二醇反馈到 垂体和改变促性腺激素合成和翻译后加工。虽然类固醇激素被用于 在避孕过程中，促性腺激素用于治疗不孕症。核心 将为拟议项目提供以下三个支持功能：功能#1。生产hFSH 糖形物。二糖基化、四糖基化和其他hFSH糖型将由Bin博士表达 在哺乳动物细胞系中的帅。帅博士将向博士提供含FSH的条件培养基。 布特涅夫功能#2。对hFSH糖型制剂进行纯化和化学表征。弗拉基米尔博士 Butnev将对hFSH糖型进行纯化和化学表征。这将包括分离hFSH糖型 从垂体提取物和条件培养液中，测定化学纯度，测定蛋白质 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糖型质控数据 将每月在Core C维护的糖组学数据共享页面上发布。功能#3。提供 为项目研究人员提供FSH测定和细胞培养支持。杰弗里·V·梅医生将在 猪颗粒细胞原代培养物中的受体结合和类固醇生成测定 系统以确认纯化的hFSH糖型的生物活性。准备工作将随后公布， 项目调查员。这些分析也将作为一项服务提供给项目调查人员。项目 图1和图2将在共同的细胞模型中进行一些实验。最初，这将是猪颗粒细胞。 博士May将评估可用的人类颗粒细胞系。例如，项目调查员 浪费了宝贵的时间来确定KGN细胞，一种假定的人类颗粒细胞系，不适合用于 我们的研究如果这个项目得到资助，那么核心B将评估细胞系，并将制作合适的细胞系 提供给项目。该核心还将为单克隆抗体表达提供支持，以支持 项目1.根据他在原代细胞培养方面的经验，May博士将担任总体核心领导人 as protein蛋白质chemistry化学and molecular分子biology生物.