망막색소상피세포에서 산소결핍에 의한 혈관신생인자의 발현 변화

Abstract

제목 : 망막색소상피세포에서 산소결핍에 의한 혈관신생인자의 발현 변화 목적 : 본 연구는 산소결핍 상태에서 망막색소상피세포가 혈관신생을 유도할 수 있는지를 조사하여 보고자 하였다. 방법 : 망막색소상피세포를 2시간과 24시간 저산소 환경에 노출시켜 배양하여, 혈관형성에 관여하는 VEGF와 PEDF의 발현과 생산을 역전사중합효소반응과 Western blotting으로 각각각 조사하였다. 혈관신생의 유도를 관찰하기 위하여 배양액을 이용하여 ECV-304에 대한 맥관형성의 유도를 확인하고 HFDMEM (human fetal dermal microvascular endothelial cell)의 이동능력을 검사하였다. 결과 : 산소결핍상태의 망막색소상피세포에서 PEDF 유전자의 발현과 생산은 감소하였고 (p〈 0.05), VEGF 유전자 발현과 생산은 증가하였다(p〈 0.05). 또한 배양액을 이용한 ECV-304의 맥관형성과 HFDMEM의 세포이동은 증가하였다. 결론 : 산소결핍상태의 망막색소상피세포는 VEGF를 증가시키고 PEDF를 감소시키며, 이러한 변화가 혈관신생을 유도할 수 있다고 생각된다.|The aim of this study is to elucidate the mechanism of neoangiogenesis of human retinal pigment epithelium (HRPE) under hypoxia. HRPE cells were cultured for 2 and 24 hours in the hypoxic chamber. Expression and production of angiogenesis-related factors including angiogenic factor, vascular endothelial growth factor (VEGF) and anti-angiogenic factor, pigment endothelial derived factor (PEDF) were checked by RT-PCR and Western blot, respectively. The induction of the neoangiogenesis by the culture supernatant harvested after exposure to hypoxic status was monitored by both tube formation in ECV 304 cell and migration assay of human fetal dermal microvascular endothelial cells. The competitive RT-PCR showed that the expression of PEDF gene in HRPE cells under hypoxic state was decreased compared to normoxic state (p<0.01) but the expression of VEGF gene was increased (p<0.01). This result corresponded to that of Western blot, showing the significant increase of VEGF production (p<0.01) and the decrease of PEDF production (p<0.01). Moreover, the tube formation and the migration assay displayed that the angiogenesis were increased according to the time of exposure to hypoxic stress. Taken together, HRPE cells under the hypoxic stress produce more VEGF and less PEDF, which lead to neoangiogenesis. This results suggest that the subretinal neovasculization under the hypoxic stress should be caused by breakdown of the balance of angiogenesis-related factors in HRPE cells.