In vitro에서 demyelination 및 remyelination에 IGF-1이 미치는 효과와 PI3K와의 연관성

Abstract

[한글]

IGF-1은 근육 세포나 신경 세포들에서 anabolic effect를 보이고 있는 성장 인자이며 주로 간에서 합성되지만 glial 세포에서도 발현된다. Glial 세포 중 중추 신경계의 myelination을 담당하는 주요 세포는 oligodendrocyte(OL)인데 분화 전 형태인 oligodendrocyte precursor cell(OPC)이나 분화된 형태인 oligodendrocyte에 이상이 발생할 경우 demyelination/ remyelination의 균형이 깨져 myelin sheath가 정상적으로 유지되지 못하고 multiple sclerosis 같은 질병을 유발할 수 있다. 본 실험에서는 SD rat에서 primary culture로 OPC를 배양하여 OL로 분화시키고 in vitro myelination을 유도한 후, demyelination stress를 주거나 remyelination을 유도시킨 상황에서 주요 myelin 단백질에 생기는 변화와 이러한 변화에 미치는 IGF-1의 효과를 관찰하였다. 우선 In vitro myelination을 유도한 후 oligodendrocyte에서 myelin basic protein(MBP)와 myelin proteolipid protein(PLP)의 mRNA가 발현됨을 확인하였다. 이러한 MBP 및 PLP mRNA의 발현은 demyelination stress와 함께 감소했다가 remyelination 조건에서 다시 회복되었는데 demyelination stress를 주면서 IGF-1을 같이 첨가하면 MBP 및 PLP mRNA의 발현의 감소가 지연되거나 저지되는 결과를 보였다. 그리고 remyelination 진행 과정 중에 IGF-1을 첨가하면 MBP 및 PLP mRNA의 발현 회복이 가속화됨을 알 수 있었다. IGF-1에 의해 OPC의 유입이 증가하여 OL로 분화되는 과정이 촉진되는 효과도 관찰되었다. 그리고 특이적 저해제를 사용해 실험한 결과 IGF-1의 효과 중 MBP mRNA 발현을 조절하는 것과 OL의 수를 증가시키는 것은 PI3K/Akt를 통하는 것으로 나타났다.

[영문]Iusulinlike growth factor-1(IGF-1) is an growth factor that expressed mainly in liver and has anabolic effects on muscle cell, neuron, and astrocyte. It is also expressed in glial cell. Oligodendrocyte(OL) is a glial cell which is differentiated from oligodendrocyte precursor cell(OPC) and responsible for myelin sheath formation in central nerve system. These oligodendrocyte lineage cells lose their myelination capacity when exposed to several metabolic problems or toxic reagents. Abnormal demyelination, break of normal balance in demyelination/remyelination, and disease such as multiple sclerosis are originated in these defected OPC and OL. This report establishes the in vitro model of demyelination and remyelination condition with primary cultured rat oligodendrocyte. mRNA level of Myelin basic protein(MBP) and myelin proteolipid protein(PLP), two major myelin proteins, were tested by RT-PCR at each point of demyelination and remyelination, representative of myelination rate. IGF-1 was added in demyelination or remyelination condition to examine its function. To explore the downstream signal molecule of IGF-1, PI3K blocker LY294002 or calcineurine blocker cyclosporine A was used. OPC was differentiated into OL, which became mature with in vitro myelination condition, then expressed MBP and PLP mRNA. This MBP and PLP mRNAs were disappeared with demyelination stress and recovered with remyelination condition. OPC was increased and OL was decreased in demyelinated state, and OPC was decreased and OL was increased in remyelinated state. This result means by that OL died after demyelination, and OPC was recruited and differentiated into OL. However, during remyelination, recruited OPC was continuously differentiated into OL. With IGF-1 supplementation, the less defects in both MBP and PLP mRNA was detected. Furthermore OPC recruitment was increased in demyelination, and recovery of MBP/PLP mRNA and OPC differentiation were enforced with remyelination. These effects of IGF-1 were blocked with LY294002 but not with cyclosporine A, that is, IGF-1 affects on oligodendrocyte via PI3K signal. In conclusion, IGF-1 delayed demyelination and accelerated remyelination in primary cultured oligodendrocyte via PI3K/Akt signal pathway.