CALBINDIN-D28K AND CALBINDIN-D9K AND 1,25(OH)(2) VITAMIN-D-3 RECEPTOR IMMUNOLOCALIZATION AND MINERALIZATION INDUCTION IN LONG-TERM PRIMARY CULTURES OF RAT EPIPHYSEAL CHONDROCYTES

被引:13
作者
BALMAIN, N
VONEICHEL, B
TOURY, R
BELQUASMI, F
HAUCHECORNE, M
KLAUS, G
MEHLS, O
RITZ, E
机构
[1] UNIV HEIDELBERG, DEPT PEDIAT, HEIDELBERG, GERMANY
[2] UNIV HEIDELBERG, DEPT INTERNAL MED, HEIDELBERG, GERMANY
关键词
CELL CULTURE; CHONDROCYTES; CALBINDINS-D; VITAMIN-D RECEPTOR; DIFFERENTIATION; IMMUNOCYTOCHEMISTRY;
D O I
10.1016/8756-3282(95)00132-W
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
Rat epiphyseal plate chondrocytes were grown on glass slides, as nonadhering monolayer cultures for up to 6 weeks. Chondrocyte growth, differentiation and maturation, matrix formation and mineralization, and the temporospatial distribution of the vitamin D-dependent calcium-binding proteins, calbindin-D9K and -D28K, and the 1,25(OH)(2)D-3 receptor (VDR), were all monitored. Chondrocytes became confluent in 2.5 weeks, differentiated to acquire a chondrocyte (polygonal) morphology, produced extracellular matrix, and finally formed a true monolayer mineralizing cartilaginous tissue, with all the stages of chondrocyte development within a single culture. beta-Glycerophosphate promoted initial matrix mineralization in 4 weeks and accelerated cell differentiation. High nominal calcium and ascorbic acid were needed for abundant matrix formation. VDR occurred at all differentiation stages, in the nuclei and nucleoli and in the cytoplasm. Calbindin-D28K and -D9K were not coexpressed. Calbindin-D28K was found in prechondroblasts, chondroblasts, and in newly differentiated chondrocytes. It was cytoplasmic in prechondroblasts and subsequently also in the perinuclear region and in nuclei, suggesting migration to the nuclear chromatin. Calbindin-D28K was nuclear only in newly differentiated chondrocytes in vitro and was not found in mature chondrocytes. In contrast, calbindin-D9K was present in the cytoplasm of mature and hypertrophic chondrocytes only. It was first in the cell body and eventually migrated within and to the far end of long cell processes with a decreasing cytoplasmic concentration showed by decreased immunostaining intensity, and ultimately hypertrophy of chondrocytes in culture. These in vitro patterns of calbindins-D and VDR accurately reflect their in vivo distributions. The genomic action of vitamin D, in vitro, resulted in the synthesis of nuclear VDR and calbindins-D. This in vitro model of chondrocyte differentiation provides a true cartilaginous mineralizing tissue in monolayer.
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收藏
页码:37 / 45
页数:9
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