Inhibitory action mechanism of cartilage-specific functional martix/chondromodulin-I on growth of vascular endothelial cells.

软骨特异性功能性martix/chondromodulin-I对血管内皮细胞生长的抑制作用机制。

基本信息

批准号：
05837010
负责人：
HIRAKI Yuji
金额：
$ 1.15万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05837010/
关键词：
Angiogenesis Growth Inhibitor Extracellular Matrix Cellular Differentiation Chondromodulin-I Chondrocytes

项目摘要

Last year, we established CHO cells which express recombinant bovine chondromodulin-I (bChM-I) precursor cDNA by the gene transfer technique and selection. However, bChM-I expressed in the cells wa recovered in the conditioned medium as a complex form with serum albumin, a major component in fetal bovine serum (FBS) added as an indispensable supplement in the culture medium. Therefore, we tried to isolate uncomplexed form of recombinant bChM-I.First, we tried to lower the FBS concentration in the culture medium to avoid absorption of the recombinant molecules by serum albumin. However, the expression level of the recombinant molecules in the culture medium was dependent on the FBS concentration in the medium. Thus it was not successful to optimize the culture conditions. Taking advantage of selective binding of albumin on a blue-sepharose column, we tried to purify recombinant bChM-I from the albumin-bChM-I complex. The complex form of bChM-I was successfully absorbed on the blue-sepharose column. However, recovery of bChM-I from the column was found to be very poor ((]sy.apprxeq.[)5%) even in the strong dissociative conditions in the presence of 1 M guanidine hydrochloride. Under these circumstances, we change our strategy to use natural form of bChM-I purified from fetal bovine cartilage for studying its action on angiogenesis in vivo. For this purpose, we studied ectopic bone formation which requires angiogenesis for replacement of induced cartilage into bone. Within 3 weeks, the control implants without cartilage-extracts induced mature bony tissue at the site of implantation. Then, we tested the DBP pellets mixed with purified bChM-I bound to heparin-sepharose beads in nude mice. Histological examination of the implanted DMP pellets clearly indicated that purified ChM-I inhibited blood vessel invasion into cartilage as found in crude cartilage-extracts.

去年，我们通过基因转移技术和选择建立了表达重组牛软骨调节素-I（bChM-I）前体cDNA的CHO细胞。然而，细胞中表达的bChM-I以与血清白蛋白的复合物形式在条件培养基中回收，血清白蛋白是作为培养基中不可缺少的补充物添加的胎牛血清(FBS)的主要成分。因此，我们尝试分离非复合形式的重组bChM-I。首先，我们尝试降低培养基中的FBS浓度以避免血清白蛋白吸收重组分子。然而，培养基中重组分子的表达水平取决于培养基中的FBS浓度。因此优化培养条件并不成功。利用白蛋白在蓝色琼脂糖柱上的选择性结合，我们尝试从白蛋白-bChM-I复合物中纯化重组bChM-I。 bChM-I 的复合物形式成功吸附在蓝色琼脂糖柱上。然而，发现即使在存在 1 M 盐酸胍的强解离条件下，bChM-I 从柱中的回收率也非常差 ((]sy.apprxeq.[)5%)。在这种情况下，我们改变策略，使用从胎牛软骨中纯化的天然形式的bChM-I来研究其对体内血管生成的作用。为此，我们研究了异位骨形成，异位骨形成需要血管生成来将诱导软骨替换为骨。 3周内，不含软骨提取物的对照植入物在植入部位诱导出成熟的骨组织。然后，我们在裸鼠中测试了与肝素琼脂糖珠结合的纯化 bChM-I 混合的 DBP 颗粒。植入的 DMP 颗粒的组织学检查清楚地表明，纯化的 ChM-I 抑制血管侵入软骨，如在粗制软骨提取物中发现的那样。