생쥐 대동맥에서 혈관 내피세포 및 민무늬근육세포의 효과적 분리 = An Effective Isolation of the Vascular Endothelial and Smooth Muscle Cells from the Mouse Aorta

국문 초록 (Abstract)

특정 유전자들이 없는 생쥐 또는 정상적인 생쥐의 대동맥으로부터 각각 분리된 세포들을 이용하여 서로 비교분석하면 죽상경화증 등의 질병 기전을 밝히는 데 유용한 모델로 쓰일 수 있다.
본 실험에서는 4~6주된 C57BL/6J 정상 생쥐 대동맥에서 혈관 내피세포와 민무늬근육세포를 효과적으로 분리하는 방법을 확립하기 위해서, 바깥막만을 제거하여 내막과 중간막으로 구성된 대동맥조직을 얻었다. 이 조직을 제1형 또는 제2형 아교질분해효소용액으로 처리하여 각각에서 분리된 내피세포와 민무늬근육세포의 특성을 조사하였다. 이들 세포의 성장 양상은 도립현미경으로 관찰하였다. 내피세포의 표지자인 CD31과 민무늬근육세포의 표지자인 α-smooth muscle actin은 공초점현미경으로 검경하였고, 효소용액의 종류에 따른 이들 세포의 분리 효과는 전체세포 수 중각 표지자에 양성반응을 보인 세포 수를 백분율로 표시하였다.
제1형 아교질분해효소용액으로 분리된 내피세포들은 배양 10일에 배양용기의 70~80%를 세포들이 덮었고, 제2형 아교질분해효소용액의 경우 40~50%가 세포들로 덮였다.
민무늬근육세포들은 배양 13일에 제1형 및 제2형 아교질분해효소용액 모두에서 배양용기의 70~80%가 세포들로 덮였다. 제1형 아교질분해효소용액으로 분리된 세포 중 CD31 양성반응인 내피세포의 백분율은 91.1±2.865%**였고, 제2형 아교질분해효소용액을 처리한 경우는 86.4±3.641%였다(**p⁄0.05, n=5). α-smooth muscle actin 양성반응인 민무늬근육세포의 백분율은 제1형 아교질분해효소용액을 사용하였을 경우 87.9±5.713%, 제2형 아교질분해효소용액의 경우 86.6±4.778%로 두 군 사이에 유의한 차이가 없었다.
이상을 요약하면 대동맥조직을 제2형 아교질분해효소용액으로 처리하는 것에 비해 제1형 아교질분해 효소용액을 사용하는 것이 혈관내피세포를 분리하는 데 있어 더욱 효과적이었다.

다국어 초록 (Multilingual Abstract)

The mechanism of the disease such as artherosclerosis is easily elucidated by the comparison among cells isolated from each aorta of knockout mouse and wild type mouse, respectively. This study was aimed at effectively harvesting the endothelial and smooth muscle cells from 4~6 weeks old wild type C57BL/6J mouse aorta. The tunica adventitia was completely removed to get the aortic tissues only consisting of the tunica intima and the tunica media under the stereoscope. These aortic tissues were treated with type I collagenase or type II collagenase solution, respectively, and then the endothelial or smooth muscle cell was isolated. CD31 marker of the endothelial cell and α-smooth muscle actin marker of the smooth muscle cell were identified with confocal microscope. The percentages of the labelled cells by each marker represented the extent of purification of endothelial or smooth muscle cells, respectively, for harvested cells according to the collagenase solutions. 70~80% of culture vessel was covered with the endothelial cells 10 days after the treatment of the type I collagenase solution, while 40~50% of culture vessel covering with the cells after the treatment of the type II collagenase solution. 70~80% of culture vessel was covered with the smooth muscle
cell regardless of the type of the collagenase solution on the 13th day. Percentages of the CD31 positive cells after the treatment with the type I or the type II collagenase solution was 91.1±2.865%** and 86.4±3.641%, respectively (**p⁄0.05, n=5). Percentages of the α-smooth muscle actin labelled cells after the treatment with the type I or the type II collagenase solution were 87.9±5.713% and 86.6±4.778%, respectively, and these values were not significantly different.
Taken together, the aortic tissues using the type I collagenase solution comparing with using the type II collagenase solution were much more effective in the isolation of the endothelial cells.

참고문헌 (Reference)

• 1 Matthews C, "Vascular smooth muscle cells undergo telomere-based senescence in human atherosclerosis: Effects of telomerase and oxidative stress" 99 : 156-164, 2006
• 2 Gorenne I, "Vascular smooth muscle cell senescnce in atherosclerosis" 72 : 9-17, 2006
• 3 Zetter BR, "The endothelial cells of large and small blood vessels" 30 : 24-28, 1981
• 4 Fillinger MF, "The effect of endothelial cell coculture on smooth muscle cell proliferation" 17 (17): 1058-1068, 1993
• 5 Fajardo LF, "The complexity of endothelial cell" 92 : 241-250, 1989
• 6 Wagner DD, "The Weibel-Palade body: The storage granule foe von Willebrand factor and P-selectin" 70 : 105-110, 1993
• 7 Bentzon JF, "Smooth muscle cells in atherosclerosis originate from the local vessel wall and not circulating progenitor cells in apoE Knockout mice" 26 : 2696-2702, 2006
• 8 Battle T, "Selective isolation of rat aortic wall layers and their cell types in culture-application to converting enzyme activity measurement" 26 : 943-955, 1994
• 9 Minamino T, "Ras induces vascular smooth muscle cell senescence and inflammation in human atherosclerosis" 108 : 2264-2269, 2003
• 10 Ramos K, "Primary cultures of rat aortic endothelial and smooth muscle cells : an in vitro model to study xenobioticinduced vascular cytotoxicity" 23 : 288-296, 1987
• 11 Suga SI, "Phenotype-related alteration in expression of natriuretic peptide receptors in aortic smooth muscle cells" 71 : 34-39, 1992
• 12 Methia N, "Localized reduction of atherosclerosis in von Willebrand factor-deficient mice" 98 : 1424-1428, 2001
• 13 Virmani R, "Lessons from sudden coronary death: a comprehensive morphologi cal classification scheme for atherosclerotic lesions" 20 : 1262-1275, 2000
• 14 Ryan US, "Isolation, culture and subculture of bovine pulmonary artery endothelial cells: mechanical methods" 10 : 3-5, 1986
• 15 McGuire PG, "Isolation of rat aortic endothelial cells by primary explant techniques and their phenotypic modulation by defined substrata" 57 : 94-105, 1987
• 16 Marelli-Berg FM, "Isolation of endothelial cells from murine tissue" 244 : 205-215, 2000
• 17 Yun YE, "Isolation of Endothelial cells and smooth muscle cell from rat aorta" 2006
• 18 Ge S, "Isolation and culture of microvascular endothelial cells from murine spinal cord" 177 : 209-214, 2006
• 19 Su X, "Isolation and characterization of murine retinal endothelial cells" 9 : 171-178, 2003
• 20 Kevil CG, "In vitro culture and characterization of gene targeted mouse endothelium" 151-157, 2001
• 21 Gimbrone MA Jr, "Human vascular endothelial cells in culture. Growth and DNA synthesis" 673-684, 1974
• 22 Hewett PW, "Human lung microvessel endothelial cells: isolation, culture and characterization" 46 (46): 89-102, 1993
• 23 Nicosia RF, "Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay of angiogenesis in vitro" 63 : 115-122, 1994
• 24 Bowden J, "Expression of vascular endothelial growth factor in basal cell carcinoma and cutaneous squamous cell carcinoma of the head and neck" 585-589, 2002
• 25 Magid R, "Expression of matrix metalloproteinase-9 in endothelial cells is differentially regulated by shear stress. Role of c-Myc" 32994-32999, 2003
• 26 Edgell CJ, "Endothelium specific Weibel-Palade bodies in a continuous human cell line" 26 : 1167-1172, 1990
• 27 Kim JY, "Endothelial cells from the rat aorta in culture" 31 (31): 1998
• 28 Grant ME, "Current concepts of basement membrane structure and function" 1 : 819-842, 1981
• 29 Jaffe EA, "Culture of human endothelial cells derived from umblical veins: identification by morphological and immunological criteria" 52 : 2745-2756, 1973
• 30 Tull SP, "Cellular pathology of atherosclerosis: Smooth muscle cells promote adhesion of platelets to cocultureed endothelial cells" 98 : 98-104, 2006
• 31 Wagner DD, "Cell biology of von Willebrand factor" 6 : 217-246, 1990
• 32 Hay ED, "Cell biology of the extracellular matrix" Plenum Press 409-, 1981
• 33 Ross R, "Cell biology of atherosclerosis" 57 : 791-804, 1995
• 34 Kleinman HK, "Biological activity of laminin" 27 : 317-325, 1985
• 35 Van Mourik JA, "Biogenesis and exocytosis of Weibel-Palade bodies" 117 (117): 113-122, 2002
• 36 Kobayashi M, "A simple method of isolating mouse aortic endothelial cells" 12 (12): 138-142, 2005
• 37 Cha ST, "A method of isolation and culture of microvascular endothelial cells from mouse skin" 70 : 198-204, 2005
• 38 Wolinsky H, "A lamellar unit of aortic medial stucture and function in mammals" 20 (20): 99-111, 1967