The Growth Promoting Effect of Insulin-like Growth Factor-I(IGF-I) Purified from Human Serum F208 on the Rat Rib Chondrocytes

Lee, Sun Ho; Shin, Jeh Hoon; Oh, Phil soo; Lee, Hahng

Abstract

PURPOSE
The pathogenesis of short stature in growth hormone(GH) deficiency is believed to be based on the growth failure of growth plate chondrocytes by reduced growth hormone dependent insulin-like growth factor- I (IGF- I ) level in serum. Therefore, author studied the growth promoting effect of IGF- I purified from human serum on the chondrocytes, cultured from rat rib cartilage.
METHODS
Rat rib cartilage were treated with type II collagenase and hyaluronidase and were cultured in Ham's F-12 culture media containing 10% fetal calf serum. Growth promoting effect of IGF- I was measured by MTT dye by adding 20ng/ml IGF- I purified by protein-diol 120 column(YMC Co, Japan) from human serum, to 1*104 cultured chondrocytes separated into each of 96 well culture vessel.
RESULTS
1) When elution time of biotin labeled IGF- I by protein pak 300sw column was compared to elution time of standard molecular weight, IGF- I exists as large complex of 150Kd and small complex of 50Kd with free 7Kd form in serum before acid treatment. After acid treatment, IGF- I exists as small complex of 50Kd with free 7Kd form. 2) IGF- I purified from blood samples, as compared to genetic engineering product standard IGF- I , showed good parallelism in competition inhibition curve by purity analysis utilizing IGF- I antibody, and thus it is assumed that complex protein as inhibiting factor for purified IGF- I does not exist. Furthermore, complex protein was not present on the Western ligand method using biotin-labeled IGF- I ligand after purified IGF- I transferred to nitrocellulose paper following SDS-PAGE electrophoresis. 3)IGF- I of 20ng/ml showed 30% growth promoting effect, when rat rib chondrocyte culture with Dulbeco's modified Eagles medium(DMEM) is considered to show maximum growth promoting effect, while with pure culture medium, DMEM, showing minimum effect.
CONCLUSIONS
The results of this study suggest that IGF- I purified by this method assumes the role of growth promoting effect on the chondrocytes, and that the described method of radioimmuno assay of IGF- I also could effectively remove inhibiting protein complex, therefore allowing more accurate assay.