|Budget Amount *help
¥8,000,000 (Direct Cost : ¥8,000,000)
Fiscal Year 1998 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Fiscal Year 1997 : ¥6,500,000 (Direct Cost : ¥6,500,000)
To study the role of bcl-2 family in chondrocyte dfferentiation, we established a novel chondroprogenitor cell line, N 1511, derived from p53-deficient mouse rib. N 1511 cells reproduce the entire cartilage development process in chemically defined medium. In alpha-minimum essential medium (alphaMIEM) containing 10% fetal calf serum, cells exhibited characteristics of the mesenchymal phenotype, such as expression of collagen type I (alpha1) mRNA, but not collagen IX (alpha1) mRNA.When cells were treated with serum-free alphaMEM supplemented with 10^<-6>M insulin and 50 ng/ml human recombinant (hr) BMP-2, expression of collagen II (alpha1) mRNA and collagen IX (alpha1) mRNA, [^<35>S]sulfate uptake, alkaline phosphatase activity, expression of col X (alpha1) mRNA, and matrix calcification were sequentially upregulated over a 6-day period. Reverse transcription-polymerase chain reaction revealed that the amount of collagen IIB, which is cartilage specific, relative to collagen IIA, which is found in several other cell types, was increased during cytodifferentiation. HrBMP-2 promoted chondroprogenitor cell survival in a dose-dependent manner : removal of hrBMP-2 induced apoptotic cell death within 9 h. In this model, expression of bcl-2 family was examined by RT-PCR.Although bcl-2 expression was reduced in 24 h after confluent, bax and bcl-X^L expression were stable. Protein expression of Bcl-2 was determined immunohistochemically using day 14 p.c. rat embryo femur. Results showed that significant amount of Bcl-2 protein is not expressed in growth plate cartilage.