组织块贴壁法和混合酶消化法分离原代血管平滑肌细胞的表型特征比较
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摘要
目的:探究组织块贴壁法、混合酶消化法分离培养的原代大鼠血管平滑肌细胞(VSMCs)在形态、生长、表型特征蛋白表达方面的不同,为合理使用VSMCs提供参考。方法:分别使用组织块贴壁法和混合酶消化法分离获得原代VSMCs,进行体外传代培养,观察细胞形态及细胞表型特征蛋白免疫荧光亮度。结果:混合酶消化法获得的原代VSMCs特征蛋白SMα-actin表达明显,表明在收缩表型特征方面优于组织块贴壁法,随着传代次数的增加,混合酶消化法在第7代仍部分保留收缩表型特性,而组织块贴壁法在第7代已丧失收缩表型特性,转变为合成型。结论:混合酶消化法在获得较稳定的收缩表型VSMCs方面比组织块贴壁法更具有优势,各实验室应根据自身的实验条件和实验设计准确选择。
Objective: To investigate the differences in morphology, growth and phenotypic characteristic protein expression of primary rat vascular smooth muscle cells(VSMCs) isolated and cultured by tissue block adhesion method and mixed enzyme digestion method, and provide reference for rational use of VSMCs. Methods: Primary VSMCs were isolated by tissue block adhesion method and mixed enzyme digestion method. The cell morphology and immunophenotypic expression of cell phenotypic protein were observed. Results: The expression of the characteristic protein SM α-actin of the primary VSMCs obtained by the mixed enzyme digestion method was obvious, indicating that the contractile phenotypic characteristics were significantly better than the tissue block adhesion method. With the increase of the number of passages, the mixed enzyme digestion method was still in the 7 th generation. Partially retained contractile phenotypic properties, and the tissue block adhesion method lost its contractile phenotypic properties in the 7 th generation and turned into a synthetic type. Conclusion: The enzymatic digestion method has more advantages than the tissue block adhesion method in obtaining stable phenotype VSMCs. Each laboratory should be accurately selected according to experimental conditions and experimental design.
Objective: To investigate the differences in morphology, growth and phenotypic characteristic protein expression of primary rat vascular smooth muscle cells(VSMCs) isolated and cultured by tissue block adhesion method and mixed enzyme digestion method, and provide reference for rational use of VSMCs. Methods: Primary VSMCs were isolated by tissue block adhesion method and mixed enzyme digestion method. The cell morphology and immunophenotypic expression of cell phenotypic protein were observed. Results: The expression of the characteristic protein SM α-actin of the primary VSMCs obtained by the mixed enzyme digestion method was obvious, indicating that the contractile phenotypic characteristics were significantly better than the tissue block adhesion method. With the increase of the number of passages, the mixed enzyme digestion method was still in the 7 th generation. Partially retained contractile phenotypic properties, and the tissue block adhesion method lost its contractile phenotypic properties in the 7 th generation and turned into a synthetic type. Conclusion: The enzymatic digestion method has more advantages than the tissue block adhesion method in obtaining stable phenotype VSMCs. Each laboratory should be accurately selected according to experimental conditions and experimental design.
引文
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[16] 赵志敏,王国坤,王杨,等.胶原酶消化法、组织块贴壁法培养的大鼠RASMCs生长、增殖及分化对比观察[J].山东医药,2017,57(28):33-36.
[2] Chi J,Meng L,Pan S,et al.Primary culture of rat aortic vascular smooth muscle cells:a new method[J].Med Sci Monit,2017,8(23):4014-4020.
[3] Petri MH,Laguna-Fernandez A,Tseng CN,et al.Aspirin-triggered 15-epi-lipoxin A(4) signals through FPR2/ALX in vascular smooth muscle cells and protects against intimal hyperplasia after carotid ligation[J].Int J Cardiol,2015,179:370-372.
[4] Ross R.The smooth muscle cell.Ⅱ.Growth of smooth muscle in culture and formation of elastic fibers[J].J Cell Biol,1971,50(1):172-186.
[5] Geisterfer AA,Peach MJ,Owens GK.Angiotensin Ⅱ induces hypertrophy,not hyperplasia,of cultured rat aortic smooth muscle cells[J].Circ Res,1988,62(4):749-756.
[6] Zhang Y,Qian X,Sun X,et al.Liuwei Dihuang,a traditional Chinese medicinal formula,inhibits proliferation and migration of vascular smooth muscle cells via modulation of estrogen receptors[J].Int J Mol Med,2018,42(1):31-40.
[7] Van der VP,de Bruin RG,Kraaijeveld AO,et al.Quaking,an RNA-binding protein,is a critical regulator of vascular smooth muscle cell phenotype[J].Circ Res,2013,113(9):1065-1075.
[8] Xie C,Guo Y,Zhu T,et al.Yap1 protein regulates vascular smooth muscle cell phenotypic switch by interaction with myocardin[J].J Biol Chem,2012,287(18):14598-14605.
[9] Kiyan Y,Limbourg A,Kiyan R,et al.Urokinase receptor associates with myocardin to control vascular smooth muscle cells phenotype in vascular disease[J].Arterioscler Thromb Vasc Biol,2012,32(1):110-122.
[10] Li H,Xiang Y,Fan L J,et al.Myocardin inhibited the gap protein connexin 43 via promoted miR-206 to regulate vascular smooth muscle cell phenotypic switch[J].Gene,2017,616:22-30.
[11] Zhuang J,Luan P,Li H,et al.The yin-yang dynamics of DNA methylation is the key regulator for smooth muscle cell phenotype switch and vascular remodeling[J].Arterioscler Thromb Vasc Biol,2017,37(1):84-97.
[12] Zhao J,Zhang W,Lin M,et al.MYOSLID is a novel serum response factor-dependent long noncoding RNA that amplifies the vascular smooth muscle differentiation program[J].Arterioscler Thromb Vasc Biol,2016,36(10):2088-2099.
[13] 李宪伟,姜维良.酶消化法分离培养大鼠血管平滑肌细胞方法的改良[J].哈尔滨医科大学学报,2011,45(1):58-60,63.
[14] 毛昱嘉,王文杰.胰酶消化法培养大鼠胸主动脉平滑肌细胞[J].中国动脉硬化杂志,2004,12(6):729-731.
[15] 刘幸平,沈岱菲,郑燕珊,等.二步酶消化法分离大鼠主动脉血管平滑肌细胞及活性鉴定[J].汕头大学医学院学报,2012,25(2):67-69,62.
[16] 赵志敏,王国坤,王杨,等.胶原酶消化法、组织块贴壁法培养的大鼠RASMCs生长、增殖及分化对比观察[J].山东医药,2017,57(28):33-36.