大鼠心肌细胞和心肌细胞系H9C2表达弹性蛋白
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摘要
目的检测弹性蛋白在大鼠心肌细胞和H9C2心肌细胞系内的表达。方法取新生和成年SD大鼠各5例的新鲜心脏,冷冻切片;取20只新生3 d SD大鼠心脏,通过0.05%胰蛋白酶和0.075%Ⅱ型胶原酶消化法,获取原代心肌细胞;免疫组织化学和免疫荧光技术检测大鼠心肌组织、原代心肌细胞和H9C2心肌细胞系的弹性蛋白表达;ELISA法检测培养的H9C2心肌细胞系上清中的弹性蛋白。结果在大鼠心肌细胞、H9C2心肌细胞系和新生、成年大鼠心肌组织上均发现心肌肌钙蛋白T和弹性蛋白的共表达。免疫组织化学结果发现,弹性蛋白在成纤维细胞、平滑肌细胞和细胞间质内均有表达。H9C2心肌细胞培养液的上清中检测到少量的弹性蛋白。结论大鼠心肌细胞表达弹性蛋白,可能参与心肌细胞的势能和细胞间质弹性纤维的构建。
Objective To detect the expression of elastin in cardiomyocytes and cardiomyocyte cell line H9 C2. Methods The five fresh hearts of newborn and adult SD rats are made into frozen slices.Primary cardiomyocytes were isolated from the hearts of the twenty neonatal 3 days SD rats by the digestion of 0.05% trypsin and 0.075% type Ⅱ collagenase.The technology of immunochemical and immunofluorescence were used to detect the expression of elastin in rat myocardial tissue, primary cardiomyocytes and cardiomyocyte cell line H9 C2.Elastin in supernatant of cultured cardiomyocyte cell line H9 C2 was detected by ELISA. Results The co-expression of cardiac troponin T and elastin was found in rat cardiomyocytes, cardiomyocyte cell line H9 C2,neonatal and adult SD rat myocardial tissues.The result of immunohistochemistry also revealed the expression of elastin in fibroblasts, smooth muscle cells, and interstitial substance.A small amount of elastin was detected in the supernatant of cardiomyocyte cell line H9 C2. Conclusion Rat cardiomyocytes express elastin,it may be involved in the potential energy of myocardial cells and the construction of intercellular elastic fibers.
Objective To detect the expression of elastin in cardiomyocytes and cardiomyocyte cell line H9 C2. Methods The five fresh hearts of newborn and adult SD rats are made into frozen slices.Primary cardiomyocytes were isolated from the hearts of the twenty neonatal 3 days SD rats by the digestion of 0.05% trypsin and 0.075% type Ⅱ collagenase.The technology of immunochemical and immunofluorescence were used to detect the expression of elastin in rat myocardial tissue, primary cardiomyocytes and cardiomyocyte cell line H9 C2.Elastin in supernatant of cultured cardiomyocyte cell line H9 C2 was detected by ELISA. Results The co-expression of cardiac troponin T and elastin was found in rat cardiomyocytes, cardiomyocyte cell line H9 C2,neonatal and adult SD rat myocardial tissues.The result of immunohistochemistry also revealed the expression of elastin in fibroblasts, smooth muscle cells, and interstitial substance.A small amount of elastin was detected in the supernatant of cardiomyocyte cell line H9 C2. Conclusion Rat cardiomyocytes express elastin,it may be involved in the potential energy of myocardial cells and the construction of intercellular elastic fibers.
引文
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[2] Kocica MJ,Corno AF,Carreras-Costa F,et al.The helical ventricular myocardial band:global,three-dimensional,functional architecture of the ventricular myocardium[J].Eur J Cardiothorac Surg,2006,29 (Suppl 1):S21-S40.
[3] Mecham RP,Madaras J,McDonald JA,et al.Elastin production by cultured calf pulmonary artery endothelial cells[J].J Cell Physiol,1983,116(3):282-288.
[4] Davidson JM,Hill HE,Alford JL,et al.Developmental changes in collagen and elastin biosynthesis in porcine aorta[J].Dev Biol,1986,118(1):103-111.
[5] Sauvage M,Hinglasis N,Mandet C,et al.Localization of elastin mRNA and TGF-β1 in rat aorta and caudal artery as a function of age[J].Cell Tissue Res,1998,291(2):305-314.
[6] Li SH,Sun Z,Guo L,et al.Elastin overexpression by cell-based gene therapy preserves matrix and prevents cardiac dilation[J].J Cell Mol Med,2012,16(10):2429-2439.
[7] Cai XH,Guo ZhK,Mao HL,et al.Histological architecture of reticular fibers and age-related changes of myocardial tissue in rats[J].Acta Anatomica Sinica,2009,40(1):127-129.(in Chinese)蔡新华,郭志坤,毛会丽,等,大鼠心肌组织中网状纤维组织学构筑及增龄变化[J].解剖学报,2009,40(1):127-129.
[8] Kocica MJ,Corno AF,Carreras-Costs F,et al.The helical ventricular myocardial band:global,three-dimensional,functional architecture of the ventricular myocardium[J].Eur J Cardiothorac Surg,2006,29(1):21-40.
[9] Yu Y,Bao S,Guo Z,et al.Kinetic alterations of collagen and elastic fibres and their association with cardiac function in acute myocardial infarction[J].Mol Med Rep,2018,17(3):3519-3526.
[10] Zhai XB,Wu JF,Guo ZhK,et al.Aging changes of ventricular elastic fibers and elastin in normal rats[J].Acta Anatomica Sinica,2013,44(1):106-110.(in Chinese)翟新博,武俊芳,郭志坤,等.正常大鼠心室肌弹性纤维和弹性蛋白的增龄变化[J].解剖学报,2013,44(1):106-110.
[11] Yu YX,Guo ZhK.Research progress on myocardial elastic fibers[J].International Journal of Cardiovascular Disease,2018,45(2):73-75.(in Chinese)余悦欣,郭志坤.心肌弹性纤维研究进展[J].国际心血管病杂志,2018,45(2):73-75.
[12] Maria A,Hisayuki H,Zhou HY,et al.Notch inhibition enhances cardiac reprogramming by increasing MEF2C transcriptional activity[J].Stem Cell Reports,2017,8(3):548-560.
[13] Faraco J,Bashir M,Rosenbloom J,et al.Characterization of the human gene for microfibril-associated glycoprotein (MFAP2),assignment to chromosome 1p36.1-p35,and linkage to D1S170 [J].Genormes,1995,25(3):630-637.
[14] Fujimoto N,Tajima S,Ishibashi A.Expression of microfibril-associated glycoprotein-1 (MAGP-1) in human epidermal keratinocytes[J].Arch Dermatol Res,2000,292(1):21-26.
[15] Henderson M,Polewski R,Fanning JC,et al.Microfibril-associated glycoprotein-1 (MAGP-1) is specifically located on the beads of the beaded-filament structure for fibrillin-containing microfibrils as visualized by the rotary shadowing technique[J].J Histochem Cytochem,1996,44(12):1389-1397.