豚鼠耳蜗螺旋动脉平滑肌细胞的原代培养及鉴定
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摘要
目的联合2种常用的细胞培养方法从豚鼠耳蜗螺旋动脉中提取平滑肌细胞,为相关实验研究提供材料。方法从豚鼠耳蜗中分离出螺旋动脉,应用1.25g/L的胰蛋白酶进行消化并将组织块贴壁,运用自然纯化法及根据不同细胞贴壁能力的差异性对细胞进行纯化,通过形态学观察及免疫荧光、免疫组化和Western blot对细胞进行鉴定。结果 85%的组织块存活,第4代平滑肌细胞纯度达95%。镜下可见细胞呈典型的"峰-谷"样生长,免疫荧光、免疫组化及Western blot结果显示平滑肌细胞特异性的肌动蛋白呈阳性表达。结论通过本研究方法,可以得到大量高纯度的豚鼠耳蜗螺旋动脉平滑肌细胞,有望为内耳循环系统紊乱导致的病理生理变化的体外研究及血管平滑肌药物评价提供一个理想的研究材料。
Objective To obtain vascular smooth muscle cells(VSMCs)from spiral modiolar artery of guinea pigs with a combination of two common cultural methods so as to provide materials for related experimental studies.Methods Spiral modiolar artery was separated from the cochlea of guinea pigs first and then digested with1.25 g/L of trypsin to be allowed to adhere.It was then purified by comprehensive application of natural purification and differential adherence ability.Finally,the cells were confirmed by morphological observation,immunofluorescence and immunocytochemistry analysis,as well as Western blot.Results 85% of the explants survived.The smooth muscle cells at passage 4 had a purity of over 95%.The cells were observed to present a typical"hill-and-valley"growth pattern under the microscope.Moreover,immunofluorescence,immunohistochemical staining and Western blot showed the specific and intense positive expression ofα-SM-actin,a cell type-specific marker.Conclusion By this protocol,we can obtain a variety of highly purified vascular smooth muscle cells from the spiral modiolar artery of guinea pigs,providing an ideal experiment material for studies on the pathogenesis of diseases related to the circulation disturbance of the inner ear in vitro,as well as evaluation of drug's effect on vascular smooth muscle.
Objective To obtain vascular smooth muscle cells(VSMCs)from spiral modiolar artery of guinea pigs with a combination of two common cultural methods so as to provide materials for related experimental studies.Methods Spiral modiolar artery was separated from the cochlea of guinea pigs first and then digested with1.25 g/L of trypsin to be allowed to adhere.It was then purified by comprehensive application of natural purification and differential adherence ability.Finally,the cells were confirmed by morphological observation,immunofluorescence and immunocytochemistry analysis,as well as Western blot.Results 85% of the explants survived.The smooth muscle cells at passage 4 had a purity of over 95%.The cells were observed to present a typical"hill-and-valley"growth pattern under the microscope.Moreover,immunofluorescence,immunohistochemical staining and Western blot showed the specific and intense positive expression ofα-SM-actin,a cell type-specific marker.Conclusion By this protocol,we can obtain a variety of highly purified vascular smooth muscle cells from the spiral modiolar artery of guinea pigs,providing an ideal experiment material for studies on the pathogenesis of diseases related to the circulation disturbance of the inner ear in vitro,as well as evaluation of drug's effect on vascular smooth muscle.
引文
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[13]梁志浩,吴康,金颖康,等.一种大鼠肺静脉平滑肌细胞快速培养方法的建立及评价[J].国际呼吸杂志,2017,37(1):45-50.
[2]ATTANASIO G,CAGNONI L,MASCI E,et al.Chronic cerebrospinal venous insufficiency as a cause of inner ear diseases[J].Acta Otolaryngol,2017,137(5):460-463.
[3]ZHANG X,ZHOU J,TAN G,et al.Clinical analysis of shortterm efficacy in senile sudden deafness[J].J Clin Otorhinol Head Neck Surg,2013,27(22):1272-1275.
[4]TAKEDA S,HATA R,CAO F,et al.Ischemic tolerance in the cochlea[J].Neurosci Lett,2009,462(3):263-266.
[5]WANGERMANN P,WONNEBERGER K.Neurogenic regulation of cochlear blood flow occurs along the basilar artery,the anterior inferior cerebellar artery and at branch points of the spiral modiolar artery[J].Hear Res,2005,209(1):91-96.
[6]LI L,MA KT,ZHAO L,et al.The characteristics of resting membrane potential on smooth muscle cells and endothelial cells in guinea pigs cochlea spiral artery[J].Chin J Applied Physiol,2012,28(2):128-132.
[7]JIANG ZG,Si JQ,LASAREV MR,et al.Two resting potential levels regulated by the inward-rectifier potassium channel in the guinea-pig spiral modiolar artery[J].J Physiol,2001,537(3):829-842.
[8]ZHOU Y,ZHOU JG,QIU QY,et al.Effects of ClC-3 chloride channel on H2O2-induced apoptosis in rat aortic vascular smooth muscle cells[J].Chin Pharmacolog Bulletin,2006,22(10):1175-1179.
[9]BIELA SA,SU Y,SPATZ JP,et al.Different sensitivity of human endothelial cells,smooth muscle cells and fibroblasts to topography in the nano-micro range[J].Acta Biomater,2009,5(7):2460-2466.
[10]SONG JW,GU W,FUTAI N,et al.Computer-controlled microcirculatory support system for endothelial cell culture and shearing[J].Anal Bioanalchem,2005,77(13):3993-3999.
[11]REIMANN K,KRISHNAMOORTHY G,WIER W G,et al.Gender differences in myogenic regulation along the vascular tree of the gerbil cochlea[J].PLoS One,2011,6(9):e25659.
[12]易斌,钱桂生,白莉,等.不同方法培养大鼠肺动脉平滑肌细胞的差异[J].第三军医大学学报,2005,27(24):2425-2427.
[13]梁志浩,吴康,金颖康,等.一种大鼠肺静脉平滑肌细胞快速培养方法的建立及评价[J].国际呼吸杂志,2017,37(1):45-50.