IN VITRO INITIATION OF BIOLOGICAL CALCIFICATION

生物钙化的体外引发

• 批准号: 7103042
• 负责人: ADELE L BOSKEY
• 金额: $ 38.18万
• 依托单位: HOSPITAL FOR SPECIAL SURGERY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-08-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7103042
• 关键词: X ray crystallography; apoptosis; blocking antibody; bone morphogenetic proteins; calcification; cell age; cell growth regulation; chick embryo; chondrocytes; embryo /fetus tissue /cell culture; enzyme inhibitors; extracellular matrix proteins; gene expression; infrared spectrometry; interferometry; metalloendopeptidases; normal ossification; parathyroid hormone related protein; phosphoproteins; phosphorylation; protease inhibitor; protein degradation; protein structure; proteoglycan; transmission electron microscopy

DESCRIPTION (provided by applicant): Conditions in which cartilage calcification is aberrant affect both the new born and the elderly (e.g., birth defects, growth deformities, rickets, and unwanted cartilage calcification in end-stage osteoarthritis and "tissue engineered" cartilage repair. To understand and manage these conditions it is essential to under- stand physiologic cartilage calcification. Based on results of the previous funding period we plan to test the hypotheses (1) that cartilage calcification does not require prior apoptosis in the mouse, as we have already demonstrated in the chick, (2) that there is a family of extracellular phosphorylated matrix proteins that are important for regulation of mineralization in the growth plate, (3) that.aggrecan degradation is required for physiologic cartilage calcification to occur, and (4) that there are certain key regulators and matrix factors that differentiate the culture that will be mineralizable from non-mineralizable cultures. To test these hypotheses and gain additional insights into the mechanism of chondrocyte-mediated calcification we propose to use a mouse cell line along with the chick limb-bud mesenchymal cells to allow access to additional crucial reagents. With the recent availability of the chick gene array we are able to look for the "key factors" that differentiate mineralizing and non-mineralizing cultures in the chick and confirm them in the mouse. Our primary outcome for all these studies will be the amount and properties of the mineral that is deposited. The specific aims are: 1: To verify that chondrocytes apoptosis is not essential for cartilage calcification in the mouse cell line known to under go endochondral ossification. 2: To expand our ongoing studies of the importance of modification of matrix proteins by specific matrix metalloproteases (MMPs) and cathepsins. 3: To determine which phosphoproteins made by hypertrophic chondrocytes and found in calcified cartilage are responsible for the initiation and regulation of cartilage calcification using a combination of immuno-localization, immuno-blocking, and addition of extraneous protein to cultures, and to verify any critical role with knockdown experiments using RNAi. 4: To use the chick gene array to discover novel factors that distinguish mineralizing from non-mineralizing micromass cultures, and verify these in both the chick and the mouse cell lines by immuno-localization, immuno-blocking, over expressing or blocking (RNAi) the expression of specific genes, and demonstrating their effects on mineralization.

描述（由申请人提供）：软骨钙化异常的情况影响新生儿和老年人（例如，出生缺陷、生长畸形、佝偻病以及终末期骨关节炎和“组织工程”软骨修复中不需要的软骨钙化。为了了解和处理这些情况，了解生理性软骨钙化是必不可少的.基于上一个资助期的结果，我们计划测试以下假设：（1）小鼠软骨钙化不需要预先凋亡，正如我们已经在鸡中证明的那样，（2）存在一个细胞外磷酸化基质蛋白家族，其对生长板中矿化的调节很重要，（3）聚集蛋白聚糖降解是发生生理性软骨钙化所必需的，和（4）存在某些关键调节因子和基质因子，其将可矿化的培养物与不可矿化的培养物区分开。为了验证这些假设，并进一步了解软骨细胞介导的钙化机制，我们建议使用小鼠细胞系沿着鸡肢芽间充质细胞，以获得额外的关键试剂。随着最近鸡基因阵列的可用性，我们能够寻找区分鸡的矿化和非矿化培养物的“关键因素”，并在小鼠中确认它们。所有这些研究的主要结果将是沉积的矿物的数量和性质。具体目标是：一曰：在已知进行软骨内骨化的小鼠细胞系中，验证软骨细胞凋亡对于软骨钙化不是必需的。2：扩大我们正在进行的研究的重要性，修饰基质蛋白的特定基质金属蛋白酶（MMP）和组织蛋白酶。第三章：使用免疫定位、免疫阻断和向培养物中添加外源蛋白的组合，确定由肥大软骨细胞产生并在钙化软骨中发现的哪些磷蛋白负责软骨钙化的启动和调节，并使用RNAi通过敲除实验验证任何关键作用。第四章：利用鸡基因阵列发现区分矿化和非矿化微团培养物的新因子，并通过免疫定位、免疫阻断、过表达或阻断（RNAi）特定基因的表达在鸡和小鼠细胞系中验证这些因子，并证明它们对矿化的影响。