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간흡충 감염 백서의 혈청내 IgE의 변동 및 Allergen의 분리에 관한 연구

Issue Date
1980
Description
의학과/박사
Abstract
[한글] 본 실험연구에서는 간흡충(Clonorchis sinensis)을 백서에 감염시켜 혈청내 IgE를 측정하고 allergen을 분리 증명하고져 시행하였다. 실험동물로는 150g내외의 백서를 사용하였고 실험동물에 대한 간흡충의 감염은 간흡충의 제2중간 숙주인 참붕어(Pseudorasbora parva)로부터 분리한 메타셀카리아 50개를 위내에 직접 주입하였고, 5주후에 대변에서 간흡충의 난자를 검출하여 감염을 확인하였다. 실험군은 동일연령의 비감염 건강대조군과 염실험군으로 구분하여 대조군은 실험시작 후(감염군에 감염시킨 동일시기)1,2,3,4,주후에, 실험군은 1,2,3,4,5 및 6주후에 채혈하여 혈청을 분리하고 -70℃에 보관하여 IgE측정과 passive cutaneous anaphylaxis(PCA) 반응검사에 사용하였다. IgE의 측정은 Rousseaux-Prevost등(1977)의 방법을 약간 수정하여 시행하였다. 즉 항IgE혈청에 대한 실험혈청내 IgE와 표준IgE와의 경쟁(competition)이 있음을 이용하여 radio immunoassay를 시행하고 IgE치를 구하였다. 성충항원을 분획하기 위하여 20% polyacrylamide gel 면역전기영동법을 시행하였으며 여기서 얻어진 gel을 수평으로0.5cm씩 절단하여 0.01% NaCl용액으로 분획항원 성분을 추출하고 농축, 냉동건조시켜 allergen이 되는지 여부를 Evan's blue염색상으로 식별하는 PCA시험으로 관찰하였다. PCA시험상 양성반응을 보인 혈청중 일부는 IgE의 온도에 대한 변화를 보기 위해 56℃에서 1시간 가온한 다음 동일한 방법으로 PCA시험을 시행하였다. PCA시험상 양성반응을 보인 분획항원의 분자량을 측정하기 위하여 6%∼20%의 gradient polyacrylamide gel 면역전기영동법을 이용하였다. 간흡충 감염백서의 혈청내 항체생성은 이중한천확산법상 감염 2주후부터 관찰되었으며 이후 6주까지 계속되었고 대조군과 감염군의 혈청내 IgE치는 대조군에서 1,2,3, 그리고 4주후에 각각 0.08±0.04㎍/ml 내지 0.04±0.02㎍/ml였으며 감염군에서는 1주후 건강대조군과 비슷하였으나 2주후에는 급격히 상승하였으며 4주후에는 최고치에 도달하였고 5주 및 6주 후에는 약간씩 감소하였으나 대조군에 비해 20∼30배 가량 높았다. PAC시험상 양성반응을 보인 분획항원은 주행이 가장 빨랐던 부분으로서 이 부분이 allergen성분을 함유하고 있음을 알 수 있었으며 분자량은 14,400 또는 그 이하임을 알 수 있었고 염색 된 침강대는 하나 뿐이었다. 또한 IgE치가 높았던 백서의 혈청을 56℃에서 1시간 가온했을 때 PCA시험상 음성 반응을 보였다. 이상의 결과는 간흡충-숙주간의 IgE의 역할을 규명하는데 기초적 자료가되고 간흡충의 수용성 allergen분획은 간흡충증의 면역학적 진단에 이용될 수 있을 것으로 생각된다.
[영문] The present study aimed to determine the serum IgE level among rats in experimental clonor chiasis and to isolate the allergen from the whole worm. Fifty metacercariae isolated from flesh of second intermediate host, Pseudorasbora parva, were directly introduced to the stomach of rats and the infection was confirmed by detection of the eggs of Clonorchis sinensis in feces five weeks after infection. Sera were collected in 1,2,3,4,5 and 6 weeks after the infection respectively. As control, sera were also sampled from the non-infected at the same time. Sera were preserved at -70℃ for IgE determination and for passive cutaneous anaphylaxis(PCA) reaction. In quantitative analysis of serum IgE level the technique reported by Rousseaux-Prevost et al.(1977) was applied slight modification. In radioimmunoassay the solubility difference between free and antibody-bound IgE in a 33% saturated ammonium sulfate solution was used. Monoclonal IgE and goat antiserum specific to the epsilon chain was supplied by Professor Capron, Institut Pasteur de Lille, France. Standard IgE was used as reference in the range from 0.01 to 1.42㎍/㎖ and the standard power curve fit(y=0.00114x**-3.08, r**2=94%, DF=6) was calculated. Whole worm extract of adult Clonorchis sinensis was perpared by the following process: crushing the freezed worm, ultracentrifugation(18,000rpm for 1 hour), dialysis against chilled destilled water(4℃ for 48 hours) and freeze-drying. Electrophoresed 20% polyacrylamide gel with whole worm antigen was horizontally sectioned in width of 0.5cm and then eluted in a 0.01% NaCl solution for collecting the fractionde antigenic component. The fractioned antigens were injected concurrently with 1% Evan's blue solution into tail vein of rat which was sensitized 48 hours previously with Clonorchis infected rat serum for PCA reaction. Thirty minutes after the injection of each fractioned antigen the rats were sacrificed and skined to observe the colouration on the skin. Positive antigen on PCA reaction was re-electrophoresed in 6%∼20% gradient polyacrylamide gel to determine the molecular weight with reference proteins of electrophoresis calibration kit, Pharmacia Co., Sweden. To evaluate the effect of heating on reaginic antibodies, IgE, the PCA positive serum was also heated at 56℃ for 1 hour was used. As results, serum IgE levels in non-infected control rats showed ranges of 0.04±0.02㎍/㎖∼0.08±0.04㎍/㎖ during 4 weeks. But in the infected rats, IgE levels rapidly increased in 2 weeks after infection(1.21±0.66㎍/㎖) reaching to the peak on 4gh week(1.90±1.42㎍/㎖). Though the IgE level slightly decreased from the 4th week, the value still kept approximately 20 times of the values in control series. In PCA reaction the fractioned positive antigen(allergen) was eluted from the lowest park of polyacrylamide gel. The molecular weight of this fraction was measured at about 14,400. The fraction was temporarily determined as an allergen of Clonorchis sinensis because it induced the postitive reaction in passive cutaneous anaphylaxis. To finalize the conclusion verification of IgE antibodies in animal model shall be required. Nevertheless, the result in the present study is highly suggestive that allergen can be utiltzed as means of immunodiagnosis in the Clonorchis sinensis infection.
URI
http://ir.ymlib.yonsei.ac.kr/handle/22282913/115774
Appears in Collections:
2. 학위논문 > 1. College of Medicine (의과대학) > 박사
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