실험적 급성 혈청병이 Cholesterol 식이성 동맥경화증에 미치는 영향

Abstract

[한글]

동맥경화증에 관한 연구는 오래전 부터 여러분야에서 광범위하게 진행되어 그 병인론에 대해서 몇가지의 가설로 요약되나(Walton, 1975), 아직도 인체에 발생되는 동맥경화증의 원인 및 발생에 미치는 제 요인에 대해서는 완전히 규명되지 못한 상태에 있고 본 교실에서도 1960년도 부터 여러 관점에서 동맥경화증에 대한 일련의 연구를 실시해 왔다.

근래에 와서 혈역동학적 변화(Texon, 1974)와 동맥벽의 손상에 대한 연구가 많은데 최근 면역학적 손상에 대한 증거가 증가되기 시작했고 면역학적 기전이 관련되어 일어나는 동맥경화증은 인체에서 일어날 수 있는 항원항체반응, 특히 자가면역 반응이 많은 노년기 질환의 원인직 요인이 될 수 있다는 근래의 학설과 관련되어 많은 학자들의 관심이 되고는 있으나 극히 최근의 몇몇의 연구뿐으로 아직 연구업적은 소수인 실정이다 (Lamberson 및 Fritz, 1971, 1974; Poston 및 Davies, 1974).

따라서 본 연구에서 급성혈청병성 면역학적 손상이 cholesterol 식이성 동맥경화증의 초기변화에 어떤 기전으로 관여 하는지를 규명하고저 본 실험을 시도하였다.

실험재료 및 방법

체중 2kg내외의 웅성가토 33마리를 사용하여 모두 이를 5군으로 나누었다.

제Ⅰ군 정상대조군 3마리

제Ⅱ군 Bovine Serum Albumin 주사군 8마리

제Ⅲ군 Cholesterol 단독 투여군 6마리

제Ⅳ군 Cholesterol 투여 및 BSA 주사군 9마리

제Ⅴ군 Cholesterol 투여, BSA 및 Prednisolone 주사군 7마리

급성혈청병을 유발하기 위하여 BSA를 정맥주사 하였고, cholesterol은 매일 경구투여 하였으며 Prednisolone은 근육주사하였다. BSA제거가 가장 많이 되었다고 생각되는 시기(A군)와 이로부터 일주일 후에(B군)도살 하였다.

도살 즉시 대동맥의 육안, 광학, 면역 및 전자현미경으로 검사하였다.

실험성적 및 결론

실험 성적을 연구분석하여 다음과 같은 결론을 얻었다.

1. 육안적 초기 동맥경화증 변화는 계Ⅳ군의 대동맥 기시부에서 뚜렷하였으며, 제 Ⅴ군에서는 미약하였고, 제Ⅲ군에서는 거의 찾아 볼 수 없었다.

2. 초기변화로 생각되는 내막의 부종성 융기는 Ⅱ군에서 Ⅴ군까지 모두 관찰할 수 있었으나 제Ⅳ군에서 가장 현저하였고, 제Ⅴ군에서 억제되는 경향이 있었으며, Ⅱ군 및 Ⅲ군에서는 경하였다.

3. 지질 침착 및 foam 새포의 침윤은 Ⅲ군에서 다소 있었으나 Ⅳ군에서 현저하였고 Ⅴ군에서는 감소된 경향이 있었으며 Ⅱ군에서는 관찰할 수 없었다.

4. 혈소판 및 섬유소의 내막 부착은 Ⅳ군에서 뚜렷했으며 Ⅲ군 및 Ⅴ군에서는 경미한 경향을 보였다.

5. 면역 형광항체법으로 항원(BSA) 및 C3, Immunoglobulins의 침착을 확인했으며, Ⅱ, Ⅳ 및 Ⅴ군은 C3가 관여된 면역학적 손상이 있었음을 확인했다.

6. 전자현미경으로 내막세포, 평활근 세포의 세포질내에서 지질구를 관찰하였고 내막하의 현저한 단백질성 물질 및 섬유소의 축적과 내막세포의 접착부의 분리 및 내막세포의 편평화등을 특히 Ⅳ군에서 현저하게 관찰할 수 있었다. 또 혈소판 및 섬유소의 내막 부착 및 내막하 침착을 관찰할 수 있었으며 섬유소는 또 형광항체법으로 확인했다.

이상의 결과를 종합 분석하면 대동맥의 면역학적 손상은 식이성 동맥경화증을 촉진시킴을 알 수 있으며 짧은 기간이긴 하지만 일단 면역학적 손상을 받으면 그 손상이 없어져도 동맥경화증이 지속된다고 해석할 수 있다.

초기변화로 생각되는 fatty dots 및 streaks, 내막의 부종성 융기 및 혈소판, 섬유소의 내막 축적등의 변화를 모두 관찰할 수 있었으나, 급성 혈청병 유발시에는 보체의 침착, 내막의 부종성융기 및 혈소판, 섬유소 축적등이 내막손상에 크게 관여되고 또 내막의 투과력의 증가로 가토 식이성 동맥경화증에 촉진적으로 관계하는 것으로 사료된다.

[영문]

During the past three decades we have learned a great deal about atherosolerosis, and a multititude of etiologic factors acting singly or synergically may be involved in its pathogenesis. The blood components, hemodynamic factors, and the

vessel wall itself, all play a distinct and important role in the inception and progression of the lesions in the experimental animals as well as in man(Haust, 1971)

Numerous theories of atherogenesis have been proposed, but many of them are no longer favored. However, four have been widely entertained in recent yeara; (1) the degenerative theory, (2) the thrombogenic theory, (3) the platelet aggregation theory, (4) the insudative theory (Walton), 1975).

Injury to the arterial wall has been shown to enhance the atherogenic effect of diet-induced hyperlipemia, such as physical injury, chemical injury, irradiation, enzymatic injury, and allergic injury.

Recently there is increasing attention given to the idea that various forms of immune injury can exaggerate the atherogenic effect of diet-induced hyperlipemia (Bieber, et al,, 1970; Hardin, et al., 1973; Poston and Davies, 1974; Friedman, et

al., 1975). Immune complex arteritis is a potent adjunct to cholesterol feeding in experimental atherogenesis, and so may be produced by dietary antigens in man. Therefore, the large quantity of proteins eaten in well nourished western communities could contribute to the generally high incidence of ischemic vascular disease, and also some have suggested that hypersentivity to milk protein might be an atherogenic factor (Poston and Davies, 1974: Gresham, 1975). In the human being,

particularyly in the older age group, there is both high incidence and severe atherosclerosis but also there is the frequent occurence and severe atherosclerosis but also there is the frequent occurence of autoimmune diseases. Because of this

recently suggestion and theoretical proposal about the immunologic enhancement much attention has been given to its study. However, the importance of immune and inflammatory mechanisms in atherosclerosis still remains to be determined in man.

There are reports atout early changes of atherosclerosis by light or ultrastructural examinations, but no detailed study is found about the early changes of atherosclerosis and about pathogenetic mechanisms by ultrastructural study, particularly in the immunologic injury.

The present study, therefore, is designed to investigate the pathogenetic mechanism involved in the early atherosclerotic changes induced by acute serum sickness in high cholesterolfed rabbits using the gross, light, immunofluorescent and electron microscopic studies.

Materials and Methods

A. Materials :

A total of thirty three albino male rabbits weighing approximately 2 Kg. each were used. For the induction of "one shot" acute serum sickness, crystalized Bovine Serum Albumin (BSA) from the Miles Laboratory were used. A total of 15Gm. of

crystalized BSA contained in 150 ml. phosphate buffer saline after autoclaving was processed by membrane filtration (Lee, et al., 1974). A high-titered rabbit anti-BSA serum was used for the daily capillary precipitation reactions. Prednisolone from the Chong Kun Dang Laboraory was used for the anti-inflammatory and anti-immunologic effects, and cholesterol from the Merk Laboratory was used for the induction of cholesterol atherosclerosis.

B. Methods :

The animals for the experiment were divided into five groups; Group Ⅰ, 3 rabbits was a Normal control; Group Ⅱ was an experimental control given injections of BSA, 8 rabbits; Group Ⅲ was an experimental control given cholesterol feeding only, 6

rabbits; Group Ⅳ was an experimental group given injections of BSA combined with cholesterol feeding, 9 rabbits; Group Ⅴ was

the prednisolone-treated group. Each group was subdivided into group A and B. Subgroup A was the rabbits sacrificed the day after complete or maximum BSA clearance from the circulation, and subgroup B was the rabbits sacrificed one week after subgroup A. The 24 rabbits given BSA were injected intravenously with 0.5 Gm,

crystalized BSA contained in 5 ml. phosphate buffer saline. Beginning on the fourth day and continuing daily thereafter, the 7 rabbits of Group Ⅴ were given 1.5mg. per Kg. of prednisolone intramuscularly. The rabbits were given 2.5 Gms of cholesterol each day parenterally. Clearance of BSA from the circulation was

checked in all rabbits by daily capillary preipitation reactions using high-titered rabbit anti-BSA serum. Sections of the ascending aorta were examined grossly, and after fixation in 10% formalin, the tissue was stained with a saturated solution of

equal parts of Sudan Ⅳ and Oil red 0 in 70% isopropanol. Part of the aorta was frozen in dry-ice-acetone mixture for immunofluorescent examination. The aortic tissue for immunofluorescent examination was cut into three or four micron

thickness, and were examined by a direct immunofluorescent antibody technique with specific fluorescein-tagged antisera fractions, such as FITC-conjugated anti-rabbit immunoglobulins (IgG, IgM, IgA), and anti-rabbit beta 1 C globulin, anti-rabbit

fibrinogen and rabbit anti-BSA. All of the above reagents, such as high titered rabbit anti-BSA serum, and PITC-conjugated anti-rabbit fractions were supplied by the Dr. Germuth Experimental Laboratory.

The aortic tissue far histopathologic examinations were fixd in 10% formalinand stained with hematoxylin and eosin, and also with Oil red 0, Masson's Trichrome, and phosphotungustic acid hematoxylin stains.

Ultrastructural examination was done with the usual processing and double staining methods, and the Hitachi HU-11E-1 electron microscope was used.

Summary

Correlative studies were made by light, immunofluorescent and electron microscopic examinations in order to ascertain the influence of experimental acute serum sickness on cholesterolfed atherosclerosis in the rabbit, and the following results are obtained.

1. The early gross atherosclerotic changes were obvious, particularly in the ascending aorta of Group Ⅳ, but very mild in Group Ⅴ, and hardly visible in Group Ⅲ.

2. The intimal edematous elevation considered as early microscopic atherosclerotic change was characteristically found in all groups except the normal control group, but it was prominent in Group Ⅳ. lesser in Group Ⅴ, and mild in Groups Ⅱ and Ⅲ.

3. The appearance of lipid and foamy cell in the aortic wall was marked in Group Ⅳ, lesser in Group Ⅴ, and mild in Group Ⅲ, but was not found in Group Ⅱ.

4. Aggregation of platelet and fibrin on the endothelial surface was frequently found in Group Ⅳ, and occasionally found in Groups Ⅲ and Ⅴ.

5. Immunofluorescent study confirmed the deposits of BSA (antigen), immunoglobulins, complement (C3), and fibrinogren, in Groups Ⅱ, Ⅳ, and Ⅴ, and the complement-mediated immunopathologieal injury of the aorta.

6. Electron microscopic study showed lipid droplets in the endothelial and smooth muscle cell cytoplasm, marked accumulation of proteinous material and fibrin in the subendethelial area particularly in Group Ⅳ. Aggregation of platelets and fibrin

on the endothelial surface and in the subendothelial area was seen, and moreover, the fibrin twas also identified by the immunefluorescent method. Also impressive was the rather tight junction of the endothelial cell in the Group Ⅴ, but loose

junction in Group Ⅳ, and a still leaser degree in Group Ⅲ.

These results indicate that immunopathological injury of the aorta enhances the cholesterolfed atherosclerosis, and may be interpreted that atherosclerosis once developed by immunological injury might not be reversible aftcr withdrawal of the immunological injury even through it is of very short duration. The early microscopic changes such as fatty dots/streaks, intimal edematous elevation, and aggregation of platelets and fibrin on the endothelial surface are observed to various degrees. It may be assumed that in rabbits with acute serum sickness, initmal edematous elevation, and aggregation of platelet and fibrin may greatly enhance the development of cholesterol-fed atherosclerosis, and moreover, particularly increased endothelial permeability due to immune complexes and vasoactive amines released from platelets and participation of fibrin might promote

and enhance the cholesterol fed atherosclerosis in rabbits.