| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1994: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1993: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
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.
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