MIURA Nobuyuki Chiba University Hospital Instructor, 医学部・附属病院, 医員
KAKUTA Harumi Chiba University Hospital Instructor, 医学部・附属病院, 医員
OOTA Setsuo Chiba University Hospital Instructor, 医学部・附属病院, 医員
FUSE Akira NIH,DEPT.OF SAFETY RESEARCH, 安全性研究部, 室長 (60110300)
|Budget Amount *help
¥2,400,000 (Direct Cost : ¥2,400,000)
Fiscal Year 1996 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1995 : ¥1,500,000 (Direct Cost : ¥1,500,000)
We studied the sensitivity, binding affinity and signal transduction of thrombopoien (TPO) in megakaryocytic cell lines, CMK,CMS and CTS,which were established in our laboratory. When CMK,CMS and were incubated with TPO (provided by Genentech Inc.) at the concentrations of 1 ng/ml, 10 ng/ml and 100 ng/ml, CMK was enhanced in growth at dose dependent manner, but CMS and CTS were not. Same results were obtained using 3H-thymidine uptake method. Then the differentiation by TPO of these cells were studied using anti-platelet antibody, anti-CD61, by FACScan. The expression of this antigen was enhanced for CMK,but there were no changes for CMS and CTS.Study for the expression of c-Mpl, receptor of TPO,using FACScan revealed that all of these cells expressed c-Mpl on their cell surface.
The TPO binding to CMK,CMS and CTS were studied using ^<125>I-TPO (provided by Genentech Inc.) at 15ﾟC.The specific binding of ^<125>I-TPO to cells was determined by subtracted the non specific bound (cultured
with 100-folds excess of unlabeled TPO) from total bound (without unlabeled TPO). Assay time was 2 hours. When the inhibition test was done by culturing CMK cells with 0.2nM of ^<125>I-TPO and 0.002nM-200nM of unlabeled TPO,the binding of ^<125>I-TPO was inhibited about 50% by 0.2nM of unlabeled TPO,indicating that labeling procedure did not influence the binding activity. Scatchard analysis revealed that CMK exhibited the single class of receptor of 1,223 receptors per cell with a dissociation constant (Kd) of 223 pM.CMS also bind with ^<125>I-TPO,showing the possibility of some abnormality in signal transduction. Activation of Tyk2 was observed in both CMK and CMS.CTS did not bind with ^<125>I-TPO at all and show any activation of Tyk2, showing the possibility of some abnormality in recepter. On the other hand, the primary bone marrow megakaryocytes from normal person exhibited the single class of receptor of 12,140 receptors per cell with a dissociation constant (Kd) of 749pM.
These results indicated that CMK was useful for the study of TPO binding and response, CMS for the signal transduction of TPO and CTS for the abnormality of receptor. Less