배양된 사람 추간반 세포에서 Matrix Metalloproteinase-3의 생성과 추간반 퇴행성 변화의 관계

Abstract

[한글]

추간반의 퇴행성 변화의 정확한 기전은 아직 밝혀져 있지 않은 상태이다. 추간반의 퇴행성 변화에는 생역학적 요소와 생화학요소가 관여한다고 알려져있으며 최근 생화학적 요소에 중요 부분인 단백 분해 효소의 역할에 대해 활발한 연구가 진행되고 있다. 단백 분해 효소는 추간반의 주요 구성 성분인 기질의 core protein과 collagen을 분해시켜 퇴행성 변화를 일으킨다고 알려져 있다. Matrix metalloproteinase(MMP)는 이러한 단백 분해 효소 중의 하나로 추간반 조직에서 분비가 확인된 후 관심을 받고 있으며 이 중 Matrix met

alloproteinase-3(MMP-3)이 여러 MMP의 전구체를 활성화시키며 넓은 기질 특이도를 가지고 골 관절염 연골의 proteoglycan의 분해에 관여한다고 증명된 바 있다. 그러나 아직 퇴행성 변화와의 직접적인 관계는 논란이 있으며 그 조절기전은 불명확하다. 본 연구에서는 추간반의 퇴행성 질환으로 수술 받은 환자의 추간반 조직(수핵부)을 이용하여 퇴행의 정도와 MMP-3의 분비와 상관 관계가 있는지 알아보고 MMP-3의 억제 인자인/Tissue inhibitor of metalloproteinase-1(TIMP-1)과 추간반의 퇴행도와의 관계를 알아보았다. 조직 배양과 세포 배양한 결과를 비교하여 추간반 세포의 역할을 규명해 보고자하였고 추간반 탈출의 유형과 MMP-3과 관계가 있는지 알아보았다. / 수술을 시행한 환자의 추간반 퇴행의 정도를 자기 공명 영상에서 Thomson의 분류에 따라 분류하였다. 수술 시 가능하면 추간반 수핵부 만을 떼어냈고 실험실에서 두 부분으로 나누어 조직배양과 세포배양을 시행하였다. 수핵부 추간반 조직을 일정한 크기로 나누어 배양 배지에서 48시간 배양 후 조직표본과 배양배지를 얻었다. 세포배양은 조직을 잘게 부순 후 효소 처치하여 추간반 세포를 분리한 다음 세포 배양하여 세포와 배양배지를 얻었다. 조직 표본과 세포 표본은 MMP-3과 TIMP-1 단클론 항체를 이용하여 면역화학적 방법으로 염색 후 양성세포비율을 구했고 배양 배지에서 효소결합면역흡착검사 방법으로 MMP-3과 TIMP-1의 농도를 측정하였다. / 조직 배양 결과 MMP-3의 농도와 양성세포 비율은 퇴행의 정도가 증가할수록 통계학적으로 유의

하게 증가하다가 퇴행의 정도가 가장 심한 군에서는 감소하는 양상을 보였다. 이와는 반대로 세포 배양 결과 MMP-3의 농도와 양성 세포의 비율은 퇴행의 정도가 증가할수록 감소하는 양상을 보였다. 조직 배양과 세포 배양 모두에서 퇴행의 정도와 TIMP-1의 농도와 양성세포 비율은 유의한 차이를 보이지 않았다. 추간반 탈출 유형과 MMP-3 농도와 양성 세포비율은 유의한 차이가 없었다. / 결론적으로 조직 배양 결과, 추간반의 퇴행의 정도와 MMP-3의 분비량과는 밀접한 관계가 있으며 MMP-3은 추간반의 퇴행성 변화의 초기 또는 중기에 관여할 것으로 판단된다. 세포 배양의 결과, 퇴행의 정도가 증가할수록 MMP-3의 분비량이 감소한 것은 추간반 세포의 MMP-3 분비는 세포의 환경과 밀접한 관계가 있으며 향후 MMP-3 분비의 조절 인자에 대해서는 연구가 필요할 것이다. TIMP-1과 퇴행의 정도와는 무관하며 추간반의 탈출 유형과 MMP-3의 분비량도 무관하다고 판단된다.

[영문]

The pathophysiology of intervertebral disc degeneration is complex and not yet fully understood. It was believed that degeneration of intervertebral disc is resulted not only from biomechanical factors but also from biochemical factors.

Recently, the role of proteinase which causes in disc degeneration is been investigated. It is established that various proteinases involve in matrix degradation of intervertebral disc. Matrix metalloproteinase(MMP) is one of proteinases which cause in the degradation of matrix and collagen. It was confirmed from various studies that MMP was formed in intervertebral disc. Among of them, matrix metalloproteinase-3(MMP-3) is known to be capable of degrading many components of extracellular matrix. However the relationship between intervertebral disc degeneration and the MMP-3 production from disc cell is not clearly defined, and the controlling mechanism of MMP-3 production is not yet clearly revealed. To determine the relationship between intervertebral disc degeneration and MMP-3 production from disc cell, human disc tissues and cells obtained during the operation of degenerative disc disease were cultured. The levels of MMP-3 and TIMP-1(antagonist of MMP-3) released in the medium were measured with the use of an enzyme immunoassay. Immunohistologic staining was also performed in tissue and cell culture specimen. The degree of disc degeneration was classified 5 grades according to Thompson''s classification. The levels of MMP-3, TIMP-1 and their positive cell ratio on immunohistochemical study were compared between each grade. The relationship between three types of disc herniation and MMP-3 production was also investigated. As a results, the level of immunoreactive MMP-3 and the immunoreactive cell ratio were increased as degeneration advanced, but were

decreased in severely degenerated disc (grade 5 by Thompson''s classification) on the tissue culture system. But on the cell culture system, the level of immunoreactive MMP-3 and immunoreactive cell ratio were decreased as degeneration advanced. The level of immunoreactive TIMP-1 and immunoreactive cell ratio were not different significantly on both culture systems. There is no significant difference between the types of disc herniation and MMP-3 production.

In conclusion, the degree of disc degeneration is significantly correlated with the MMP-3 production in disc tissue and MMP-3 may involve in initial or intermediate phase of disc

degeneration. The result of this study suggest that the production of MMP-3 in disc cell may be affected by cellular environment. Further study is recommended for clarifying the regulation of MMP-3 production in disc cell. The relationship

between the production of TIMP-1 and the degree of disc degeneration is not clear.

Key Words: intervertebral disc, degeneration, matrix metalloproteinase-3, tissue inhibitor of metalloproteinase-1, tissue culture, cell culture The pathophysiology of intervertebral disc degeneration is complex and not yet fully understood. It was believed that degeneration of intervertebral disc is resulted not only from biomechanical factors but also from biochemical factors. Recently, the role of proteinase which causes in disc degeneration is been investigated. It is

established that various proteinases involve in matrix degradation of intervertebral disc. Matrix metalloproteinase(MMP) is one of proteinases which cause in the degradation of matrix and collagen. It was confirmed from various studies that MMP was formed in intervertebral disc. Among of them, matrix

metalloproteinase-3(MMP-3) is known to be capable of degrading many components of extracellular matrix. However the relationship between intervertebral disc degeneration and the MMP-3 production from disc cell is not clearly defined, and the controlling mechanism of MMP-3 production is not yet clearly revealed. To

determine the relationship between intervertebral disc degeneration and MMP-3 production from disc cell, human disc tissues and cells obtained during the operation of degenerative disc disease were cultured. The levels of MMP-3 and TIMP-1(antagonist of MMP-3) released in the medium were measured with the use of an enzyme immunoassay. Immunohistologic staining was also performed in tissue and cell culture specimen. The degree of disc degeneration was classified 5 grades according to Thompson''s classification. The levels of MMP-3, TIMP-1 and their positive cell ratio on immunohistochemical study were compared between each grade. The relationship between three types of disc herniation and MMP-3 production was also investigated. As a results, the level of immunoreactive MMP-3 and the immunoreactive cell ratio were increased as degeneration advanced, but were

decreased in severely degenerated disc (grade 5 by Thompson''s classification) on the tissue culture system. But on the cell culture system, the level of immunoreactive MMP-3 and immunoreactive cell ratio were decreased as degeneration advanced. The level of immunoreactive TIMP-1 and immunoreactive cell ratio were not different significantly on both culture systems. There is no significant difference between the types of disc herniation and MMP-3 production. In conclusion, the degree of disc degeneration is significantly correlated with the MMP-3 production in disc tissue and MMP-3 may involve in initial or intermediate phase of disc degeneration. The result of this study suggest that the production of MMP-3 in disc cell may be affected by cellular environment. Further study is recommended for clarifying the regulation of MMP-3 production in disc cell. The relationship between the production of TIMP-1 and the degree of disc degeneration is not clear.