Streptozotocin 반복투여로 유발된 백서의 실험적 당뇨병에서 신사구체 변화에 대한 형태학적 연구

Abstract

[영문]

[한글]

인체 당뇨병은 인슐린 분비 부족이나 표적세포에서의 인슐린 작용부전으로 인해 대사장

애를 일으키며, 유전적, 환경적, 면역인자가 관여된다고 알려지고 있으나, 특히 IDDM에서

는 자가 면역반응이 중요한 병인요소로 대두되고 있다.

본 연구에서는 당뇨병의 자가면역성 실험유형을 이용하여 자가면역의 관련여부 및 취장

과 신사구체의 변화를 관찰하고자 본 연구에 착수하였다. 100gm 내외의 웅성백서 32마리

를 2군으로 대별하여 대조군은 0.5cc의 acidiffied saline을 1일 1회 연속 5일간 꼬리정

맥에 주사하였으며, 실험군은 pH 4.5의 streptozotocin을 40mg/kg로 같은 방법으로 주사

하였고, 당뇨병이 유발된 백서는 최종 약물 투여후 3,4,5,6일에 각각 5마리씩, 1,3,5,7개

월에 각각 3마리씩 도살하였으며, 대조군은 실험군과 동일시기에 각 3마리씩 도살하였다.

뇨당, 뇨단백, 뇨케톤체, 혈당 및 소도세포항체 검사와 함께 취장의 광학현미경적 및

신장의 광학현미경적, 면역형광현미경적, 전자현미경적 검색을 시행하여 다음과 같은 결

과를 얻었다.

1. 대조군에서 혈당은 정상법위였으며 뇨당, 뇨단백 및 뇨케톤체는 검출되지 않았다. S

treptozotocin투여군에서는 약물투여 3일째 도살군부터 고혈당이 나타나 7개월군까지 지

속되었으며, 전예에서 2+∼4+의 뇨당이 검출되었고, 20%에서 ±∼1의 뇨단백이 검출되었

으며 뇨케톤체는 검출되지 않았다.

2. 광학현미경 소견상 사구체 기저막은 실험군 전군을 통하여 대조군과 비교 관찰시 의

의있는 차이가 없었으며, streptozotocin투여 3,5,7개월째 도살군에서 맥관막내 국소적인

기질의 증가가 관찰되었다.Streptozotocin투여 3개월군부터 신세뇨곤 상피의 세포질내

당원침착이 관찰되었다.

3. Streptozotocin투여 3일째 도살군부터 취소도내에 소수의 호산구 및 호중성구의 침

윤, 취소도세포의 핵 농축 및 변성이 관찰되었으며, 7개월군에서는 간질내 호산구 침윤도

관찰되었다.

4. 면역형광현미경 소견상 소도세포항체는 대조군 및 실험군에서 음성이었으며, 사구체

맥관막내 면역글로불린의 침착이 streptozotocin투여 3일째 도살군부터 관찰되어 3개월

군에서 가장 현저하였다.

5. 전자현미경 검사상 streptozotocin 투여 3,4일군에서 상피세포 및 내피세포의 종창

이 나타났으며, 5,7개월군에서 상피하혹모양의 사구체 기저막의 비후, 사구체 기저막의

균열, lamina rara interna의 rarefaction 과 함께 맥관막 기질의 증가 및 전자고밀도 물

질의 침착이 관찰되었다.

이상의 소견을 종합하면 면역학적 견지에서 취장소도내의 호산구 침윤과 사구체 맥관막

내의 지속적인 면역글로불린의 침착 등을 분석 종합할 때 streptozotocin에 의한 취소도

세포독작용을 완전히 배제할 수는 없으나, 자가면역기전의 가능성을 생각할 수 있어 앞으

로 이에 대한 공범위한 연구는 흥미있을 것으로 사료된다.

A Morphologic Study of Glomerular Changes in Multiple Low Dose Streptozotocin

-induced Diabetic Rats

Jong Hyun Lee

Department of Medical Science The Graduate School, Yonsei University

(Directed by Professor In Joon Choi, M.D.)

Diabetes is a metabolic disorder characterized by fasting hyperglycemia and

glycosuria. Its pathogenesis is still unknown, but genetic, environmental and

immunologic factors are encountered and autoimmunity is considered to be an

important factor in the induction of IDDM.

The purpose of this study is to investigate the role of autoimmunity in the

evolution of diabetes mellitus and to observe morphologic changes of pancreatic

islets and renal gnomeruli using the experimental model proposed by Like and

Rossini(1976).

A total of 32 male rats, weighing about 100 gm, were divided into normal control

and streptozotocin-treated groups. The first group was injected with acidified

saline 0.5 cc through the tail vein for 5 consecutive days. The experimental group

was injected with streptozotocin 40 mg/kg, dissolved in 0.9% saline and acidified

with 0.025 M citric acid, as the same method as the control. The induction of the

diabetes was confirmed by examination of urine sugar using reagent strip every

other day after the drug administration.

Each 5 rats were killed on the 3rd, 4th, 5th & 6th days and each 3 rats on the

1st, 3rd, 5th & 7th months after the drug administration. The urine was collected

for the detection of g1ycosuria, proteinuria, ketonuria and the blood was collected

for glucose and for islet cell antibody.

For light microscopic examination paraffin b1ocks were sectioned at 5 micron

thickness and was stained with hematoxyline-eosin, PAS, PAMS, trichrome and alcian

blue for kidney and only hematoxylin-eosin for pancreas.

Islet cell antibody was demonstrated by indirect immunofluorescent technique on

unfixed 5 micron cryostat sections. Undiluted rat sera and 1 : 4 diluted

FITC-labelled rabbit-anti-rat-IgG were exposed on the normal rat pancreas. The

kidney was examined by direct immunofluorescent technique using 1 : 4 diluted

FITC-labelled rabbit-anti-rat-IgG using Leitz dialoux immunofluorescent microscope.

For electron microscopic examination conventional procedures were applied and the

observation was made with Hibachi H-500 transmission electron microscope.

The results obtained were as follows:

1. The b1ood sugar was within normal range in the control group, while in the

streptozotocin-treated groups, hyperglycemia was noted and maintained through the

course of experimental diabetes.

2. Glycosuria was noted in all rats of the streptozotocin-treated groups,

proteinuria became positive in 20% of the streptozotocin-treated groups, and

ketonuria was not detectable in all rats.

3. By light microscopic examination increase of mesangial matrix started to

appear in the streptozotocin-treated 3-month group with gradual increase and

without noticible changes in the glomerular basement membrane. Glycogen began to

acoumulate in the cytoplasm of the renal tubular epithelium in the

streptozotocin-treated 3-month group.

4. Infiltrations of a few eosinophils & neutrophils were noticed in the

pancreatic islets with nuclear pyknosis and cellular degeneration in the

streptozotocin-treated 3-day group, and eosinophils infiltrated the stroma of the

pancreas in the streptozotocin-treated 7-month group also.

5. Islet cell antibody was negative in both control and streptozotocin-treated

groups. Immnoglobulin deposition was found in the glomerular mesangium of the

streptozotocin-tre-ated 3-day group which gradually increased in amount and was

most prominent in the 3-month group.

6. By electron microscopical examination endothelial and epithelial swellings

were noted at the streptozotocin-treated 3-and 4-day groups. Subepithelial

hump-like thickening of the glomerular basement membrane and rarefaction of lamina

rara interna were associated with increase of mesangial matrix and presence of

electron dense deposit in the streptozotocin-treated 5-and 7-month groups.

In summary, from an immunologic point of view, although the cytotoxic effect of

the streptozotocin can't be completely excluded, the possible pathogenic role of

autoimmunity in the experimental induction and progression of diabetes mellitus is

to be stressed and is an interesting subject to further study.