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Development of a novel tissue culture system for the intervertebral disc

新型椎间盘组织培养系统的开发

基本信息

批准号：
13671540
负责人：
CHIBA Kazuhiro
金额：
$ 2.3万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

Purpose of the current study was to establish a reliable, cost effective and reproducible in vitro tissue culture system for the intervertebral disc to investigate the metabolism of the intervertebral disc as a whole organ, thereby obtaining information which may lead to the elucidation of possible mechanisms of intervertebral disc degenerationRabbit intervertebral discs were extirpated and embedded in alginate gel and were cultured for up to 4 weeks. At weekly intervals, the contents of DNA, sulfated proteoglycans, antigenic keratan sulfate,hyaluronic acid and collagen in the cultured disc tissues were measured to evaluate the tissue integrity, Incorporation rates of 3SS sulfate were also measured at each time point to investigate proteoglycan synthesis and newly synthesized radiolabelled proteoglycans were characterized by separation with sepharose CL2B column sieve chromatography. Intervetebral disc cells cultured in medium alone were used as controls throughout the experimentsThe annulus fibrosus and ucleus pulposus of the intervertebral disc cultured in alginate gel maintained a higher content of various extracellular matrix molecules and sustained a higher rate of proteoglycan synthesis than those cultured in medium alone at all time points. Newly synthesized proteoglycans in the discs embedded in alginate gel also maintained their molecular sizes close to intact discsIncorporation of whole disc tissues in alginate gel proved useful to maintain the integrity of intervertebral disc and may be useful for studying the metabolism of intervertebral disc as a whole organ

本研究的目的是建立一种可靠的、经济的、可重复的椎间盘组织体外培养系统，以研究椎间盘作为一个整体器官的代谢，从而获得可能导致椎间盘退变的可能机制的信息。每隔一周检测培养椎间盘组织中DNA、硫酸化蛋白多糖、硫酸角质素、透明质酸和胶原的含量，以评价组织完整性。在每个时间点检测3SS硫酸盐掺入率，以研究蛋白多糖的合成，并用SepharoseCL2B柱筛层析对新合成的放射性标记蛋白多糖进行表征。在所有实验时间点，在藻酸盐凝胶中培养的椎间盘纤维环和髓核的各种细胞外基质分子的含量和蛋白多糖的合成速率都比在单独培养基中培养的椎间盘细胞高。将整个椎间盘组织包埋在藻酸盐凝胶中，可以有效地保持椎间盘的完整性，并有助于研究椎间盘作为一个整体的代谢。