이소골 형성 과정에서 Phosphatase 동종 효소의 변동

Abstract

[한글]

근육 및 건(腱)등 결합조직에서 뼈가 형성되는 이소골 형성(heterotopic ossification)은 임상적으로 관절 성형술(arthroplasty)을 실시하거나 중추 신경손상을 입은 환자에서

나타난다.

골형성에 있어서의 석회화 및 재흡수 과정에는 뼈의 alkaline phosphatase및 acid phosphatase가 관여함은 잘 알려져 있다. 여러 종류의 골조직에서 alkaline또는 acid phosphatase를 검색하면 뼈의 형성 정도에 따라 각기 다른 동종효소가 나타남이 보고되어 있고,

또한 phosphoamino acid phosphatase도 시기에 따라 각기 다른 활성을 나타낸다고 한다.

현재까지 alkaline 및 acid phosphatase 동종효소에 관한 연구는 많으나 이소골 형성시기에 따른 동종효소의 변동에 대한보고는 거의 없는 상태이다.

그러므로 이번실험에서는 흰쥐의 대퇴골 및 경골로 골기질을 제조한 후 흰쥐 복근내에 이식하여 이소골 형성을 유도하였으며, 이소골 형성시기별로 혈청 및 골기질내 alkaline및 acid phosphatase동종효소를 등전집초법으로 분리하여 골형성 및 재성형에 관여하는 동종효소를 밝히고자 하였다.

실험결과를 요약하면 다음과 같다.

1. 이식 골기질내 calcium 함량은 2주에 급격히 증가하여 5주에 최고치를 나타내었다.

2. 이식 골기질내 alkaline phosphatase 활성은 이식후 2주에 가장 높았고, acid phosphatase활성은 3주에 최고치를 나타내었다.

3. 이식 골기질내 단백분획은 이식 전기간 동안 일정하였다.

4. 혈청 alkaline phosphatase는 등전점이 4.5인 하나의 분획으로서 이식 전기간 동안 일정하였다.

5. 정상 골조직의 alkaline phosphatase 동종효소는 4개로서 등전점은 4.5, 5.3, 5.6및 5.9이었다. 골기질 이식후 1주에는 동종효소의 주분획은 등전점 4.5인 산성분획이었으며 2주에는 등전점 5.0의 새 동종효소가 출현하여 6주까지 높은 활성을 나타내었다. 시일이 경과함에 따라 정상 골조직의 동종효소가 나타나서, 6주 후에는 정상 골조직의 alkaline phosphatase 동종효소와 비슷한 양상을 보였다.

6. 혈청 acid phosphatase동종효속는 6개로서 등전점이 4.5, 4.6, 4.7, 4.8, 4.9및 5.0이었으며 이식 기간중 각 분획의 활성도는 이식 골기질의 동종효소 변동과 비슷하였다.

7. 정상 골조직의 acid phosphatase 동종효소는 2개로서 등전점이 4.5 및 4.6이었다. 골기질이식 초기에는 등전점 5.0의 동종효소가 주분획을 나타내나 이식 3주 이후에는 등전점 5.6-6.0의 동종효소가 높은 활성을 유지하였다.

이상의 성적으로 보아 이소골 형성과정의 시기에 따라 등전점이 다른 alkaline phosphatase와 acid phoaphatase가 출현함을 관찰하였으며, 특히 등전점 5.0의 alkaline phosphatase동종효소와 등전점 5.6-6.0의 acid phosphatase 동종효소가 이소골 형성의 석회화와 재성형에 중요한 역할을 하리라 추측된다.

Changes of Phosphatase Isozymes in the Process of

Experimentally-induced Heterotopic Ossification

Jae Yung Hyun

Department of Medical Science, The Graduate School, Yonsei University

(Directed by Professor Byeong Mun Park and Associate Professor Young Soo Ahn)

The bone formation in periarticular connective tissues after total hip

arthroplasty or head injury is included in the category of heterotopic

ossification.

Various endogenous substances are responsible for the induction of new bone

formation. Among them, alkaline and acid phosphatases are the important enzymes

known to be related to the mineralization and resorption of bone.

It has been known that several isozymes of phosphatases might originate from

different organs and that, even within an organ system, several isozymes are

reported present.

In the present investigation, therefore, changes of isozyme patterns of

phosphatases were examined in the precess of experimentally-induced heterotopic

ossification in rats.

Bone matrices were prepared using diaphyses of the femur and tibia after

decalcification and defatting procedures. Six bone matrices per rat were implanted

in the abdominal muscle pouches, Implant from the rats were then taken out for

isozyme study every week for six weeks. The isozymes of alkaline and acid

phosphatages in serum and matrices were identified by isoelectric focusing and

phosphatase stainings.

The results obtained are as follows;

1. The calcium content in the bone matrix was increased markedly from the second

week after implantation and reached its highest levee at the fifth week.

2. The activities of alkaline and acid phosphatases were the highest at the

second and third weeks, respectively, after implantation.

3. The electrophoretic profiles of proteins in the bone matrices showed little

difference throughout the experimental periods.

4. Only one alkaline phosphatase isozyme with isoelectric paint (pl) of 4.5 was

identified in the serum, which is consistent throughtout the process of heterotopic

ossification.

5. Four isozymes of alkaline phosphatase were identified in normal bone and their

pl were 4.5, 5.3, 5.6 and 5.9. An isozyme with a pl of 4.5 was the prominent at the

first week of implantation and decreased thereafter. A new isozyme with pl pf 5.0

appeared at the second week and maintained high activity up to the sixth week. At

the sixth week of implantation, the pattern of isozymes of matrix alkaline

phosphatase was similar to that of normal bone except for the presence of a pl 5.0

isozyme.

6. Six isozymes of acid phosphatase were identified in the serum and their pl

were 4.5, 4.6, 4.7, 4.8, 4.9 and 5.0. New isozyme with pl of 6.0 was appeared at

the first week of implantation and its activity then remained higher up to the

sixth week.

7. Two isozymes of acid phosphatase were identified in normal bone and their pl

were 4.5 and 4.6. In the early stage of heterotopic ossification, a new isozyme

with pl 5.0 was the prominent one; whereas, in the later stage, pl 6.0 isozyme was

prominent.

From the above results, it is suggested that the different isozymes of alkaline

and acid Phosphatases are responsible for the different stages of heterotopic

ossification, and pl 5.0 isozyme of alkaline phosphatase and pl 5.6-6.0 isozymes of

acid phosphatase are closely related to the calcification and remodeling of bone

formation.

[영문]

The bone formation in periarticular connective tissues after total hip arthroplasty or head injury is included in the category of heterotopic ossification.

Various endogenous substances are responsible for the induction of new bone formation. Among them, alkaline and acid phosphatases are the important enzymes known to be related to the mineralization and resorption of bone.

It has been known that several isozymes of phosphatases might originate from different organs and that, even within an organ system, several isozymes are reported present.

In the present investigation, therefore, changes of isozyme patterns of phosphatases were examined in the precess of experimentally-induced heterotopic ossification in rats.

Bone matrices were prepared using diaphyses of the femur and tibia after decalcification and defatting procedures. Six bone matrices per rat were implanted in the abdominal muscle pouches, Implant from the rats were then taken out for isozyme study every week for six weeks. The isozymes of alkaline and acid phosphatages in serum and matrices were identified by isoelectric focusing and phosphatase stainings.

The results obtained are as follows;

1. The calcium content in the bone matrix was increased markedly from the second week after implantation and reached its highest levee at the fifth week.

2. The activities of alkaline and acid phosphatases were the highest at the second and third weeks, respectively, after implantation.

3. The electrophoretic profiles of proteins in the bone matrices showed little difference throughout the experimental periods.

4. Only one alkaline phosphatase isozyme with isoelectric paint (pl) of 4.5 was identified in the serum, which is consistent throughtout the process of heterotopic ossification.

5. Four isozymes of alkaline phosphatase were identified in normal bone and their pl were 4.5, 5.3, 5.6 and 5.9. An isozyme with a pl of 4.5 was the prominent at the first week of implantation and decreased thereafter. A new isozyme with pl pf 5.0 appeared at the second week and maintained high activity up to the sixth week. At the sixth week of implantation, the pattern of isozymes of matrix alkaline phosphatase was similar to that of normal bone except for the presence of a pl 5.0 isozyme.

6. Six isozymes of acid phosphatase were identified in the serum and their pl were 4.5, 4.6, 4.7, 4.8, 4.9 and 5.0. New isozyme with pl of 6.0 was appeared at the first week of implantation and its activity then remained higher up to the

sixth week.

7. Two isozymes of acid phosphatase were identified in normal bone and their pl were 4.5 and 4.6. In the early stage of heterotopic ossification, a new isozyme with pl 5.0 was the prominent one; whereas, in the later stage, pl 6.0 isozyme was

prominent.

From the above results, it is suggested that the different isozymes of alkaline and acid Phosphatases are responsible for the different stages of heterotopic ossification, and pl 5.0 isozyme of alkaline phosphatase and pl 5.6-6.0 isozymes of

acid phosphatase are closely related to the calcification and remodeling of bone formation.