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An inhibitory effect of tumor necrosis factor-alpha antagonist in monocrotaline-induced pulmonary hypertensive rat model

Title
An inhibitory effect of tumor necrosis factor-alpha antagonist in monocrotaline-induced pulmonary hypertensive rat model
Other Titles
Monocrotaline 으로 유도된 폐동맥 고혈압 백서 모델에서Tumor Necrosis Factor-α 길항제의 억제 효과
Authors
권정현
Issue Date
2010
Department/Major
대학원 의학과
Publisher
이화여자대학교 대학원
Degree
Doctor
Advisors
홍영미
Abstract
Background: The pathogenesis of pulmonary hypertension includes hypertrophy of vascular and perivascular cells, inhibition of cellular apoptosis, constriction and proliferation of arterioles, and inflammation and remodeling of pulmonary arterioles. Tumor necrosis factor (TNF)-α is also suggested to contribute to the development of pulmonary hypertension. Infliximab, a chimeric monoclonal antibody to TNF-α has a clinical effect to promote cellular apoptosis and activation of the complement system. Therefore, infliximab would be helpful in pulmonary hypertension, however the exact mechanism is unknown. The changes in pathology and gene expressions of TNF-α, endothelin-1 (ET-1), endothelin receptor A (ERA), endothelial nitric oxide synthase (eNOS), matrix metalloproteinase (MMP) 2, and tissue inhibitor of matrix metalloproteinase (TIMP) were investigated to explore the effect of infliximab in a monocrotaline (MCT) induced pulmonary hypertension rat model. Methods : Six-week-old male Sprague-Dawley rats, weighing between 200-250 g, were allocated into three groups: control group (n=27), subcutaneous (sc) injection of normal saline (0.1 mL/kg); monocrotaline (M) group (n=36), single sc injection of MCT; monocrotaline + infliximab (M+I) group (n=27), sc injection of MCT (60 mg/kg) plus single sc injection of infliximab (5 mg/kg). The rats were weighed and sacrificed after 1, 5, 7, 14 and 28 days. The heart weight and histology of pulmonary arteries were examined. Microarray of lung tissues was analyzed using GeneSpring GX 7.3.1 (Agilent technologies, CA). The genes whose gene expressions were more than 2 times from control group were selected. Gene expressions of TNF-α, ET-1, ERA, eNOS, MMP2 and TIMP were investigated in a MCT induced pulmonary hypertension rat model by reverse transcription-polymerase chain reaction (RT-PCR). Results: The medial wall thickness in the M+I group was significantly lower than the M group on day 7. Increase in the number of intra-acinar muscular arteries after MCT injection was reduced by infliximab treatment on day 14 and 28. Gene expressions of TNF-α, ET-1, ERA and MMP2 were significantly lower in M+I group compared than M group on day 5. eNOS and TIMP showed late gene expression in M group on day 28. Conclusion : Administration of infliximab may induce early change in gene expressions of TNF-α, ET-1, ERA and MMP2 leading to an inhibitory effect on lung vascular hypertrophy in a MCT induced pulmonary hypertension rat model. Therefore it is speculated that infliximab may be helpful for the management of pulmonary hypertension.;목적: 폐동맥 고혈압의 기전으로 폐혈관의 내피세포로부터 Tumor necrosis factor(TNF)-α 분비가 증가되어 세포 증식 촉진, 세포 사멸 억제, 혈관 수축, 염증 반응을 통해 폐혈관의 재형성이 관여하는 것으로 알려져 있다. 이에 따라 TNF- α 의 길항제인 Infliximab의 치료 효과가 기대된다. 이에monocrotaline (MCT)으로 유발된 폐동맥고혈압 동물 모델에 infliximab을 투여하였을 때 해부학적 변화와 폐동맥 고혈압과 관련된 유전자 발현의 변화를 알아보고자 본 연구를 실시하였다. 대상 및 방법: 250g 내외의 6된 웅성 Sprague-Dawley 백서를 대조군, M군, monocrotaline + infliximab (M+I) 군으로 나누었다. M군과 M+I 군에는 MCT 수용액 60 mg/kg를 배부에 1회 피하 주사하였고, M+I 군에서는 infliximab 5 mg/kg을 MCT투여 후 1일째 피하주사로 1회 투여하였다. 대조군에는 동량의 생리식염수를 주사하였다. Microarray chip 실험을 선행하여 6개 유전자 (TNF α, ET-1, ERA, eNOS, MMP2, TIMP) 의 뚜렷한 발현의 변화를 확인한 후, 이들 유전자에서 reverse transcription-polymerase chain reaction (RT-PCR)을 시행하였다. 1, 5, 7, 14, 28 일에 각각 동물의 체중을 측량한 후 희생시켜 우심실과 좌심실의 무게를 측정하였고, RT-PCR을 시행하여 유전자 발현의 변화를 비교하였다. 결과: MCT로 유발된 폐세동맥의 중막 비후 소견이 infliximab투여 1주 후에 현저하게 저하되었다. 또한 호흡성 세기관지 이하의 폐포에 분포하는 근육성 폐세동맥의 숫자도 MCT+I 군에서 MCT군에 비해 14, 28일에 현저하게 감소하였다. TNF-α, ET-1, ERA, MMP2, TIMP 의 유전자 발현은 MCT군에서 대조군에 비해5일째 유의한 증가를 보였고, TNF-α, ET-1, ERA, eNOS, MMP-2 유전자 발현은 5일째 infliximab 투여 후에 유의하게 감소하였다. 결론: infliximab 투여는 MCT로 유발된 폐동맥 고혈압 백서 모델에서 폐혈관 중막 비후의 감소와 호흡성 세기관지 이하의 혈관 수를 감소시켰고, ET-1, ERA, MMP2 염증성 사이토카인 유전자 발현이 유의하게 감소하였다.
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