백서 피부 fibroblast 배양에서 triamcinolone acetonide가 collagen 대사에 미치는 영향

Abstract

[한글]

피부조직의 창상 치유과정은 collagen 대사 조절과 밀접한 관계가 있는 매우 복잡한 생물학적 현상으로서 그 근본적인 기전을 밝히려는 연구가 활발하다. 또한 창상 치유후에 생성되는 keloid나 비후성 반흔의 생성기전에 대한 연구 또한 활발하다. 이들 병발 부위의 조직 병리학적 소견으로는 collagen과 같은 과량의 결체조직 성분이 침착되어 있으며 생화학적 소견으로는 prolylhydroxylase나 galactosylhydroxylysyl glucosyltransferase 등의 효소활성이 증가된다고 보고된 바 있다.

현재 임상에서는 이들 병발 부위에 합성 glucocorticoid를 국소적으로 투여하여 좋은 효과를 나타내고 있는데, 이들 합성 glucocorticoid가 피부 fibroblast의 성장 및 fibroblast내 collagen 대사에 미치는 영향에 대한 보고들이 상반된 결과를 나타내고 있다.

따라서 본 실험에서는 glucocorticoid 중에서도 collagen 합성 억제 정도가 가장 강한 triamcinolone acetonide를 여러 농도로 처리한 후 백서 피부 fibroblast에서 collagen의 양적변동 및 collagen에 대한 mRNA를 정량하여 다음과 같은 결과를 얻었다. 생후 2일된 백서 피부로부터 fibroblast를 분리 배양하였으며, 계대 배양한 이 fibroblast에 triamcinolone acetonide를 저농도 (0.5, 1, 2, 4 μM)로 10일간 처리하여 실험에 사용하였다. Collagen의 정량은 collagen에 특이하게 존재하는 hydroxyproline을 정량하여 collagen을

간접적으로 정량하였다.

저농도의 triamcinolone acetonide를 처리시 배양용기당 hydrosyproline의 양은 0.5, 1, 2, 4 μM의 triamcinolone acetonide 농도에서 대조군에 비하여 각각 179.1, 200.5, 229.7, 171.9%로 현저히 증가되었으며, 각 배양용기내 μg DNA 당 hydroxyproline의 양도 각각 147.6, 166.7, 224.0, 133.1%로서 대조군에 비하여 collagen양을 증가시킨 것으로 사료된다.

그러나 triamcinolone acetonide를 50, 100, 150 μM의 고농도로 처리시에는 각 triamcinolone acetonide 농도에서 배양용기내 hydroxyproline의 양이 대조군에 비하여 각각 45.1, 43.8, 39.3%로 감소되었으며, μg DNA 당 hydroxyproline양도 역시 42.4, 37.7, 34.0

%로 collagen양이 대조군에 비하여 현저히 감소되는 양상을 보이고 있다. 각 농도에서 DNA양은 대조군에 비하여 각각 107.8, 117.6, 115.2%로서 150 μM triamcinolone acetonide에서도 저농도에서와 같이 세포성장 억제효과는 나타내지 않았다.

Collagen α1(Ⅰ)의 cDNA가 삽입된 plasmid를 가지고 [α**-32P] dCTP를 사용하여 nick translation방법에 의해 제조한 (32)**P로 label된 probe의 specific는 1.7x10**3 cpm/μg DNA였으며, 이 probe를 가지고 type 1 collagen의 α1(Ⅰ)에 대한 mRNA가 대조군에

비하여 현저히 감소하였으며 50 μM의 triamcinolone acetonide 처리시에는 대조군의 약 1/4로 감소되었다.

이상의 결과를 종합해 보면 저농도의 triamcinolone acetonide 처리시는 백서 피부 fibroblast의 collagen의 양이 증가하였으며, 이러한 증가는 전사(transcription) 이후 단계에서의 조절에 의한 결과로 사료된다.

반면에 고농도의 triamcinolone acetonide 처리시에는 collagen의 양이 현저히 감소되었는데 이는 collagen α1(Ⅰ) mRNA의 양적 감소 현상과 상처되는 결과로서, 이와같은 고농도의 triamcinolone acetonide 처리시에는 collagen 합성의 전사 단계에서 선택적으로

억제된다고 사료된다.

The Effects of Triamcinolone Acetonide on Collagen Metabolism in Cultured

Fibroblasts of Rat Skin

Won Jin Chung

Department of Medical Science The Graduate School, Yonsei University

(Directed by Professor Jae Duk Lew, M.D., Ph.D.

and Professor Yoon Soo Kim, M.D., Ph.D.)

The process of wound healing of skin tissue is closely related to the regulation

of collagen metabolism and is a complicate biological phenomenon. Therefore, the

study of the basic mechanism of wound healing is an active field. In addition,

there are many active investigations of the mechanism of keloid or hypertrophic

scar formation after wound healing. It has been reported that histopathological

changes, such as excess accumulation of collagen, a connective tissue component and

biochemical changes, such as increased activities of prolyl hydrosylase and

galactosylhydrosylysyl glucosyltransferase, were observed in keloid tissue. Now,

clinical use of glucocorticoids on the local dermal tissues to decrease collagen

synthesis gives a good response, but reports of the effect of synthetic

glucocorticoids on the growth and collagen fibroblast of rat skin were treated with

triamcinolone acetonide in various concentrations and the amounts of collagen and

collagen specific mRNA in fibroblast were measured.

Fibroblast were isolated and cultured from 2 day old rat skin, then subcultured

cells were incubated in media containing low concentrations of triamcinolone

acetonide (0.05, 1, 2, and 4μM) or high concentrations of triamcinolone acetonide

(50, 100, and 150μM) for 10 days and were used for the experiment.

The results are as follows:

1. The amount of collagen was measured indirectly by the measurement of

hydroxyproline which in present specifically in collagen. The amounts of

hydrosyproline in the culture flasks treated with 0.5, 1, 2, and 4μM triamcinolone

acetonide were 179.1, 200.5, 229.7 and 171.9 of the control respectively, and the

amounts of hydroxyproline per ug DNA were 147.6, 166.7, 224.0 and 133.1% of the

control, indicating the increased collagen content by the treatment of low

concentration of triamcinolone acetonide. DNA contents of fibroblasts treated with

0.5, 1, 2, and 4μM triamcinolone acetonide were 121.4, 120.3, 102.6 and 129.2% of

the contrlo.

2. However, the amounts of hydrosyproline in culture flasks treated with the high

concentrations of triamcinolone acetonide, 50, 100, and 150μM were 45.1, 43.8 and

39.3% of the control, respectively. The amounts of hydrosyproline per μg DNA were

also 42.4, 37.7 and 34.0% of the control, respectively, indicating a decreased

collagen content caused by high concentrations of triamcinolone acetonide

treatments. The DNA contents in each treatment group were 107.8, 117.6 and 115.2%

of the control, indicating that even 150μM triamcinolone acetonide did not inhibit

the cell growth but increased it slightly.

3. Treatment with 1μM triamcinolone acetonide caused a slight decrease in the

content of mRNA for theα1 chain of type Ⅰ collagen, but no great change was

observed. However, treatments of 50μM and 100μM triamcinolone acetonide resulted

in a decrease of mRNA for the α1 chain of type Ⅰ collagen to a quarter of the

control. This suggests that the increase of collagen content in rat skin

fibroblasts treated with low concentrations of triamcinolone acetonide mignt result

from posttrancscriptional regulation, and that the decrease of collagen content

caused by high concentrations of triamcinolone acetonide might result from

selective control in the transcriptional step as evidenced by the decreased amonut

of mRNA for α1(Ⅰ) collagen in conjuction with the decreased collagen content.

[영문]

The process of wound healing of skin tissue is closely related to the regulation of collagen metabolism and is a complicate biological phenomenon. Therefore, the study of the basic mechanism of wound healing is an active field. In addition, there are many active investigations of the mechanism of keloid or hypertrophic scar formation after wound healing. It has been reported that histopathological changes, such as excess accumulation of collagen, a connective tissue component and

biochemical changes, such as increased activities of prolyl hydrosylase and galactosylhydrosylysyl glucosyltransferase, were observed in keloid tissue. Now, clinical use of glucocorticoids on the local dermal tissues to decrease collagen synthesis gives a good response, but reports of the effect of synthetic

glucocorticoids on the growth and collagen fibroblast of rat skin were treated with triamcinolone acetonide in various concentrations and the amounts of collagen and collagen specific mRNA in fibroblast were measured.

Fibroblast were isolated and cultured from 2 day old rat skin, then subcultured cells were incubated in media containing low concentrations of triamcinolone acetonide (0.05, 1, 2, and 4μM) or high concentrations of triamcinolone acetonide (50, 100, and 150μM) for 10 days and were used for the experiment.

The results are as follows:

1. The amount of collagen was measured indirectly by the measurement of hydroxyproline which in present specifically in collagen. The amounts of hydrosyproline in the culture flasks treated with 0.5, 1, 2, and 4μM triamcinolone acetonide were 179.1, 200.5, 229.7 and 171.9 of the control respectively, and the amounts of hydroxyproline per ug DNA were 147.6, 166.7, 224.0 and 133.1% of the control, indicating the increased collagen content by the treatment of low concentration of triamcinolone acetonide. DNA contents of fibroblasts treated with 0.5, 1, 2, and 4μM triamcinolone acetonide were 121.4, 120.3, 102.6 and 129.2% of the contrlo.

2. However, the amounts of hydrosyproline in culture flasks treated with the high concentrations of triamcinolone acetonide, 50, 100, and 150μM were 45.1, 43.8 and 39.3% of the control, respectively. The amounts of hydrosyproline per μg DNA were

also 42.4, 37.7 and 34.0% of the control, respectively, indicating a decreased collagen content caused by high concentrations of triamcinolone acetonide treatments. The DNA contents in each treatment group were 107.8, 117.6 and 115.2%

of the control, indicating that even 150μM triamcinolone acetonide did not inhibit the cell growth but increased it slightly.

3. Treatment with 1μM triamcinolone acetonide caused a slight decrease in the content of mRNA for theα1 chain of type Ⅰ collagen, but no great change was observed. However, treatments of 50μM and 100μM triamcinolone acetonide resulted in a decrease of mRNA for the α1 chain of type Ⅰ collagen to a quarter of the control. This suggests that the increase of collagen content in rat skin fibroblasts treated with low concentrations of triamcinolone acetonide mignt result

from posttrancscriptional regulation, and that the decrease of collagen content caused by high concentrations of triamcinolone acetonide might result from selective control in the transcriptional step as evidenced by the decreased amonut

of mRNA for α1(Ⅰ) collagen in conjuction with the decreased collagen content.