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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筛选技术，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在条件培养基中与血清白蛋白（胎牛血清（FBS）的主要成分）作为培养基中不可缺少的补充物添加到培养基中，以复合物形式恢复。因此，我们试图分离重组bChM-I的非络合形式。首先，我们试图降低培养基中FBS的浓度，以避免重组分子被血清白蛋白吸收。然而，重组分子在培养基中的表达水平取决于培养基中FBS的浓度。因此，对培养条件的优化并不成功。利用白蛋白在蓝脂糖柱上的选择性结合，我们试图从白蛋白- bchm - 1复合物中纯化重组bchm - 1。bchm - 1的配合物在蓝葡糖柱上被成功吸附。然而，即使在1 M盐酸胍存在的强解离条件下，从色谱柱中回收bchm - 1的回收率也很差（[]y.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来研究其在体内血管生成的作用。为此，我们研究了异位骨形成，这需要血管生成来将诱导软骨置换成骨。在3周内，不含软骨提取物的对照种植体在种植部位诱导了成熟的骨组织。然后，我们在裸鼠身上测试了与纯化的bChM-I结合的DBP微丸。植入DMP微球的组织学检查清楚地表明，纯化的ChM-I可以抑制软骨粗提物中血管侵入软骨。