Inhibitory Effect of an N-terminal Recombinant Fragment of Human Thrombospondin-2 on Migration of Human Dermal Microvascular Endothelial Cells = 사람 Thrombospondin-2 단백 아미노 말단 절편의 사람 피부 미세혈관 내피세포 이동 억제 효과

다국어 초록 (Multilingual Abstract)

Thrombospondin-2(TSP-2) has been known to modulate tumor growth, wound healing, and inflammation through inhibition of angiogenesis based on the findings from TSP-2 knock-out mice and overexpression studies. However, the molecular mechanisms of human TSP-2(hTSP-2) for inhibition of angiogenesis or the feasibility to develope as a new therapeutic angiogenesis inhibitor for curing cancer have not been intensively studied because so far the whole molecule of hTSP-2 is not available due to difficulties in extraction of useful amount of hTSP-2 from tissues and limitations on production of full length recombinant hTSP-2 in mammalian or insect cells. Therefore, an N-terminal 80 kDa fragment of hTSP-2 encompassing N-terminal globular region through typeⅠ repeats(hTSP-2/NTF) was expressed in human embryonal kidney 293 EBNA cells to investigate whether this fragment has antiangiogenic effect on primary cultured human endothelial cells(ECs). The recombinant vector was constructed as follows: an 1.67 kbp of cDNA of hTSP-2(nt 213 to 1883) was amplified by PCR from human placenta cDNA library and ligated into KpnⅠ and Bgl Ⅱ sites of the modified pCEP 4 expression vector. The recombinant protein was purified from the serum fee conditioned medium of transfected 293 EBNA cells using ammonium sulfate precipitation and gelatin- and heparin-sepharose columns. Yield was 2.1 ㎎/l of conditioned medium and the purified protein was confirmed by N-terminal peptide sequencing. To see the fragment has anti-angiogenic activity in vitro, migration assay was performed on human dermal microvascular ECs(HDMECs) with the recombinant protein, demonstrating inhibition of migration by 30 % at 1 ㎎/ml of hTSP-2/NTF as compared to control group. This effect was not neutralized by anti-CD 36 antibody, suggesting a different receptor other than CD 36, to which TSP-1 binds and inhibits EC migration, may be involved in the inhibitory mechanism of hTSP-2/NTF on the migration of HDMECs. Futher study to see in vivo antiangiogenic activity of hTSP-2/NTF is on-going to evaluate the validity of this fragment as a new antiangiogenic agent.