자연적으로 유발된 고혈압 백서에서 안지오텐신 전환효소를 억제하는 Small Hairpin RNA 투여 효과
Effect of Small Hairpin RNA Molecules Targeting Angiotensin-converting Enzyme Gene in Spontaneously Hypertensive Rats
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연구배경: RNA interference는 이중 가닥의 RNA (double-stranded RNA)가 dicer에 의해 절단되어 생성되는 21-25 뉴클레오타이드 크기의 작은 RNA조각으로 상보적인 서열을 갖는 mRNA에 특이적으로 결합하여 단백질 발현을 억제하는 것으로 밝혀졌다. 본 연구에서는 자연적으로 유발된 고혈압 쥐(spontaneously hypertensive rat, SHR) 모델에서 안지오텐신 전환효소(angiotensinconverting enzyme, ACE)에 대한 small hairpin RNA (shRNA)가 주입된 렌티바이러스 벡터를 투여하여 혈역학적, 해부학적 변화를 알아보고, 이와 같은 조절 효과가조직학적 변화 또는 유전자 수준에서 어느 시기에 어느정도 일어나는지를 규명하고자 하였다. 방법: 12주의 자성 성숙 Wistar-Kyoto 백서를 대조군으로 SHR을 고혈압군(H군)으로 shRNA가 주입된 렌티바이러스 벡터를 투여한 치료군을 ACE-interfering RNA (iRNA) (A군)으로 하였다. shRNA 투여는 렌티바이러스에 형질주입된 상태로 1 mL를 실험 시작일에 SHR의 꼬리에 정맥투여하였다. 대조군 및 실험군 모두 21일, 35일에 도살하였다. 혈청 알도스테론, ACE, 안지오텐신치를측정하였고, reverse transcription-polymerase chain reaction과Western blot분석을 이용하여 endothelin (ET)-1,ACE, angiotensin II receptor (AT II R) 1A, neutrophil cytosolic factor (NCF), caspase-3, Bax, Bcl-2 유전자 발현을 측정하였다. 결과: 혈압은 H군에서 유의하게 증가하였고 A군에서유의하게 감소하였다. 혈청 안지오텐신치는 H군에서 유의하게 증가하였고, A군에서 유의하게 감소하였다. ACE 농도는 세 군 간에 유의한 차이가 없었다. ACE 농도는 5주에, ET-1와 Bcl-2 단백질 농도는 ACE-iRNA 치료 후 3주, 5주째 유의하게 감소하였디. Bax 단백질 농도는ACE-iRNA 치료 후 3주에 유의하게 감소하였다. AT II R, NCF, caspase-3 단백질 농도는 ACE-iRNA 치료 후 유의한 감소가 없었다. 결론: ACE에 대한 shRNA가 주입된 렌티바이러스 벡터는 항고혈압 효과가 뚜렷하였고, ACE, ET-1, Bax,Bcl-2 유전자 발현의 감소를 초래하였다. 향후 shRNA 주입 양을 증량시킨 연구가 추후에 이루어져야 할 것으로생각한다.
Abstract
Background: Interfering RNA (iRNA) represents a recent breakthrough in effective blocking of the target genes in mammalian cells. Angiotensin-converting enzyme (ACE) has been shown to play an important role in the pathogenesis of hypertension. The purposes of this study were to investigate the effects on blood pressure, myocardial hypertrophy and gene expressions of iRNA targeting ACE. Methods: Twelve week old male Wistar-Kyoto rats were grouped as follows: control group (C group), spontaneously hypertensive rat (SHR) group (H group), and ACE-iRNA group (A group) in which SHR was treated with recombinant lentiviral vectors carrying small hairpin RNA targeting ACE. Reverse transcriptionpolymerase chain reaction and western blot analysis of ACE, endothelin (ET)-1, angiotensin (AT) II receptor type 1A,neutrophil cytosolic factor, caspase 3, Bax, and Bcl-2 were performed in the heart tissues. Serum AT, ACE, and high sensitive-C reactive protein were estimated. Results: Systolic blood pressure was significantly decreased in the A group compared with the H group in weeks 3 and 5. Serum AT level was significantly lower on day 1, weeks 3 and 5 after ACE-iRNA treatment. ACE protein contents were significantly lower after ACE-iRNA treatment in week 5. ET-1 and Bcl-2protein contents were significantly lower after ACE-iRNA treatment in weeks 3 and 5. Bax protein contents were significantly lower after ACE-iRNA treatment in week 3. Conclusions: Recombinant lentiviral vectors carrying shRNA targeting ACE prevented hypertension. Serum AT and gene expressions such as ACE, ET-1, Bax, and Bcl-2 were significantly decreased after ACE-iRNA treatment.
참고문헌 (27)
- Matzke M, "RNA : guiding gene silencing", Science, 293, pp.1080~1083 (2001)
- Hannon GJ, "RNA interference", Nature, 418, pp.244~251 (2002)
- Zamore PD, "RNA interference : big applause for silencing in Stockholm", Cell, 127, pp.1083~1086 (2006)
- Escors D, Breckpot K, "Lentiviral vectors in gene therapy : their current status and future potential", Arch Immunol Ther Exp(Warsz), 58, pp.107~119 (2010)
- Trittibach P, "Lentiviral-vector-mediated expression of murine IL-1 receptor antagonist or IL-10 reduces the severity of endotoxin-induced uveitis", Gene Ther, 15, pp.1478~1488 (2008)
- Jazwa A, "New vectors and strategies for cardiovascular gene therapy", Curr Gene Ther, 7, pp.7~23 (2007)
- Navar LG, "Intratubular renin-angiotensin system in hypertension", Hypertension, 57, pp.355~362 (2011)
- Ferreira AJ, "New cardiovascular and pulmonary therapeutic strategies based on the angiotensin-converting enzyme 2", angiotensin-(1-7), Int J Hypertens, pp.2012, 2012[DOI]
- Gunaruwan P, "Effects of bradykinin on venous capacitance in health and treated chronic heart failure", Clin Sci(Lond), 116, pp.443~450 (2009)
- Sink KM, "Angiotensin-converting enzyme inhibitors and cognitive decline in older adults with hypertension : results from the Cardiovascular Health Study", Arch Intern Med, pp.1195~1202 (2009)
- Martinez MV, "The association of right ventricular function and pulmonary arterial compliance in patients with idiopathic pulmonary arterial hypertension", J Am Coll Cardiol, 59(13), pp.1605~ (2012)
- Arif H, "Drug compliance after stroke and myocardial infarction : a comparative study", Neurol India, 55, pp.130~135 (2007)
- Sijen T, "On the role of RNA amplification in dsRNA-triggered gene silencing", Cell, 107, pp.465~476 (2001)
- Freeman EJ, "Angiotensins differentially activate phospholipase D in vascular smooth muscle cells from spontaneously hypertensive and Wistar-Kyoto rats", Am J Hypertens, 8, pp.1105~1111 (1995)
- Lopez B, "Biochemical assessment of myocardial fibrosis in hypertensive heart disease", Hypertension, 38, pp.1222~1226 (2001)
- An SJ, "Endothelin-1 expression in vascular adventitial fibroblasts", Am J Physiol Heart Circ Physiol, 290, pp.700~708 (2006)
- Yokoyama H, "Role of blood pressure reduction in prevention of cardiac and vascular hypertrophy", Am J Hypertens, 18, pp.922~929 (2005)
- Fortuno MA, "Overexpression of Bax protein and enhanced apoptosis in the left ventricle of spontaneously hypertensive rats : effects of AT1 blockade with losartan", Hypertension, 32, pp.280~286 (1998)
- Fortuno MA, "p53-mediated upregulation of BAX gene transcription is not involved in Bax-alpha protein overexpression in the left ventricle of spontaneously hypertensive rats", Hypertension, 33, pp.1348~1352 (1999)
- Cha JH, "Changes of gene expressions in spontaneously hypertensive rat model after losartan treatment", Korean Circ J. (2012)
- He J, "RNA interference targeting the ACE gene reduced blood pressure and improved myocardial remodelling in SHRs", Clin Sci(Lond), 116, pp.249~255 (2009)
- Vazquez J, "Selective silencing of angiotensin receptor subtype 1a(AT1aR)by RNA interference", Hypertension, 45, pp.115~119 (2005)
- Phillips MI, "Prolonged reduction of high blood pressure with an in vivo, nonpathogenic, adeno-associated viral vector delivery of AT1-R mRNA antisense", Hypertension, 29(1), pp.374~380 (1997)
- Puddu GM, "Gene-based therapy for hypertension: do preclinical data suggest a promising future?", Cardiology, 108, pp.40~47 (2007)
- Gupta R, "Human studies of angiogenic gene therapy", Circ Res, 105, pp.724~736 (2009)
- Shim TJ, "Establishment of a eNOS gene therapy model using PLGA nanosphere in spontaneous hypertensive rat", J Korean Soc Hypertens, 16, pp.28~38 (2010)
- Kim SJ, "Association of angiotensin converting enzyme, angiotensinogen, and angiotensin II type 1 receptor gene polymorphisms with left ventricular hypertrophy in Korean hypertensive patients", J Korean Soc Hypertens, 11, pp.44~52 (2005)