실험적 이소성 골형성에서의 phosphatase 활성 변동

Abstract

[한글]

고관절 성형술을 실시하거나 신경손상을 입은 환자에서 근육 또는 건(터)등 결체 조직에서 뼈가 형성될 수 있으며 이를 이소골형성(heterotopic ossification)이라 한다.

실험적으로는 탈석회화된 골기질을 동결건조하여 흰쥐의 전복벽 근육내에 이식할 경우 이식부위의 간엽 세포들이 분화증식하여 이소골이 형성되므로 실험모델로 널리 이용되고 있다. Alkaline phosphatase는 골형성과정에 있어 중요한 역할을 하는 효소로서 그들의 생리적 기능에 대하여는 인(燐)농도를 증가시켜 인화칼슘이 벼에 축적되게 하는 기능과 protein kinase와 더불어 연골세포의 분화 및 증식을 조절하는 기능들이 제시되고 있다.

Indomethacin은 골절치유 및 이소골형성에서 골형성 초기의 특징인 염증반응을 억제하여 간엽 세포 증식을 감소시키며, 이와는 달라 cholecalciferol (vitamin D^^3)의 대사산물인 calcitriol(1,25-dihydroxycholecalciferol)은 골흡수능을 증가시킬 뿐 아니라 골무

기질의 항상성 및 뼈의 교체율 조절에도 관여한다고 한다.

이번 실험에서는 이소골형성시 세포분화에 대한 alkaline phosphatase 및 여러 phosphoamino acid phosphatase와의 상관관계를 규명하고 아울러 이에대한 cholecalciferol과 indomethacin의 영향을 관찰함으로써 이소골형성에 대한 phosphatase의 역할을 규명하고자

하였다.

실험동물로는 몸무게 100g 안팎의 Sprague-Dawley계 흰쥐를 암수구별없이 사용하였고 cholecalciferol (400ng/kg/day) 또는 indomethacin (2mg/kg/day)을 골기질 이식 1주전부터 피하주사하여 이식후 이소골형성과정을 4주간 비교관찰하였다. 이소골형성은 흰쥐의

전복벽 근육내에 6개의 골절편을 이식하고 1주, 2주, 3주 및 4주에 동물을 희생시킨 다음 4개의 골절편은 단백질 및 효소측정에, 2개의 골절편은 calcium 측정에 사용하였다.

실험성적은 다음과 같다.

1. Phosphoamino acid 분해 활성은 calf intestinal alkaline phosphatase 보다 흰쥐의 골조직내에 함유된 phosphatase가 현저히 높았다.

2. 골기질 이식후 정상흰쥐의 골조직내 calcium함량은 2주에 가장 높았으며, cholecalciferol투여로 증가하였고, indomethacin 투여로 감소하였다.

3. 대조군의 골조직내 단백함량은 이식후 2주에 가장 높았으며 cholecalciferol 투여로 증가하였나, indomethacin 투여군에서는 초기에 증가한 다음 감소하였다.

4. 대조군의 alkaline phosphatase 활성은 2주에 가장 높았으며 indomethacin 투여로 현저히 감소되었다.

5. 정상흰쥐의 phosphoserine phosphatase 활성은 이식후 2주에 가장 높았으며 cholecalciferol 투여군에서는 대조군과 차이가 없었으나 indomethacin 투여군에서는 감소경향을 나타내었다.

6. Phosphothreonine phosphatase 활성은 cholecalciferol 투여군에서 증가경향을 나타내었으나 indomethacin 투여군에서는 대조군과 별 차이가 없었다.

7. Phosphotyrosine phosphatase 활성은 cholecalciferol 투여군에서 증가하고 indomethacin 투여군에서는 감소하였다.

8. 이소골형성에 있어 calcium 함량 변동은 phosphotyrosine phosphatase 변동과 의의 있는 상관관계를 나타내었으며, alkaline phosphatase는 phosphoserine phosphatase와 가장 강한 상관관계를 나타내었다.

이상의 결과로 보아 이소골형성에는 alkaline phosphatase 및 phosphotyrosine phosphatase가 관여하며, 특히 초기 골세포 형성에는 phosphotyrosine phosphatase가 중요한 역할을 할 것으로 추측된다.

[영문]

Experimental heterotopic bone formation, induced by the implantation of demineralized bone matrix, is a useful model to study new bone formation and bone turnover. Alkaline phosphatase is known to be intimately related to the mineralization of bone and ossifying growth·plate cartilage. It has been suggested that alkaline phosphatase stimulates bone formation by raising the local phosphate concentration necessary for C^^a^^3(PO^^4)^^2 deposition. Recently it was postulated that alkaline phosphatase regulates chondrocyte differentiation via dephosphorylation of phosphotyrosyl protein.

Indomethacin delays or inhibits fracture healing and heterotopic ossification by inhibiting the mesenchymal cell proliferation as well as the inflammatory response.

In contrast, cholecalciferol stimulates the rate of bone mineralization in response to increased osteoclastic bone resorption.

The present study, therefore, was undertaken to investigate the roles of various phosphatases, especially phosphoamino acid phosphatases, and the effect of cholecalciferol and indomethacin in the process of experimental heterotopic ossification.

Sprague·Dawley rats, weighing about 100g of either sex, were used. Bone matrices were prepared using diaphyses of the femur and tibia after decalcification and defatting procedures. Six bone matrices per rat were implanted in muscle pouches created in the upper part of the abdominal wall. Cholecalciferol (400ng/kg) or indomethacin (2mg/kg) was administered daily subcutaneously from 1 week prior to implantation until sacrifice, up to 4 weeks after implantation. Four implants were pooled for determination of protein and phosphatase activities, and two implants were used for calcium determination.

The results are summerized as follows.

1. The activities of phosphoamino acid phosphatases were 10 to 40 times higher in rat bone phosphatase than calf intestinal alkaline phosphatase.

2. The amount of calcium in the bone matrix was highest 2 weeks after implantation. Cholecalciferol treatment increased and indomethacin decreased the calcium content in the bone matrix.

3. The protein content of the bone matrix increased up to 2 weeks after implantation, then decreased gradually. Cholecalciferol treatment increased the protein content, however, indomethacin decreased it after a transient increase 1 week after implantation.

4. The activity of alkaline phosphatase peaked 2 weeks after implantation and was reduced by indomethacin treatment.

5. Phosphoserine phosphatase activity in the implanted bone matrix was little changed either by cholecalciferol or indomethacin treatment.

6. The activity of phosphothreonine phosphatase tended to be increased by cholecalciferol treatment.

7. The acitivity of phosphotyrosine phosphatase was significantly increased by cholecalciferol treatment, but was markedly inhibited by indomethacin treatment.

8. The calcium content of the bone matrix implant was fairly correlated with the change of phosphotyrosine phosphatase activity, and the activity of alkaline phosphatase was best correlated with that of phosphoserine phosphatase.

From these results, it is suggested that alkaline phosphatase and phosphotyrosine phosphatase are the responsible for heterotopic ossification, and that phosphotyrosine phosphatase has an important role in the early phase of bone formation.