小鼠肠系膜动脉平滑肌细胞的制备及莲子心新碱对其收缩的抑制作用  

Preparation of Murine Mesenteric Artery Smooth Muscle Cells and The Inhibitory Effect of NeoL on Their Contraction

作　　者：王鑫 李源 万朋 杨光明[1] 潘扬[1] WANG Xin;LI Yuan;WAN Peng;YANG Guangming;PAN Yang(School of Pharmaceutical Science,Nanjing University of Chinese Medicine,Nanjing 210023 Jiangsu,China)

机构地区：[1]南京中医药大学药学院,江苏南京210023

出　　处：《中药新药与临床药理》2022年第8期1001-1008,共8页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金项目(81373295)。

摘　　要：目的建立小鼠肠系膜动脉平滑肌细胞(MASMCs)分离、培养和鉴定的方法,并探讨莲子心新碱(NeoL)对其收缩的抑制作用及机制。方法采用胶原酶消化法分离制备小鼠原代MASMCs;免疫荧光法检测细胞的纯度和分化状态;细胞计数法检测传代细胞的生长特性;CCK-8法检测MASMCs细胞活力;成像技术法观察NeoL对KCl诱导MASMCs收缩的影响;蛋白免疫印迹法及免疫荧光法检测MASMCs中ROCK1蛋白的表达情况。结果MASMCs生长曲线近似“S”形,第4~7天处于对数生长期,可用于传代,细胞的成活率达95%。经鉴定,MASMCs为典型的平滑肌细胞且纯度高。NeoL对MASMCs增殖能力有显著抑制作用,IC50值为1.327μmol·L^(-1)。与124 mmol·L^(-1)KCl诱导的模型组比较,0.3、0.6μmol·L^(-1)NeoL干预后MASMCs的细胞长度均显著延长(P<0.01),ROCK1蛋白表达明显下调(P<0.05,P<0.001)。结论成功构建了小鼠MASMCs模型,细胞纯度高,多次传代后细胞特征稳定;NeoL对KCl诱导的MASMCs收缩具有抑制作用,可能与其下调ROCK1蛋白表达有关。Objective To establish a method for the isolation,culture and identification of murine mesenteric artery smooth muscle cells(MASMCs),and to investigate the inhibitory effect and preliminary mechanism of contraction by neolepine of lotus seeds(NeoL).Methods The mouse primary MASMCs were isolated and prepared by collagenase digestion;the purity and differentiation status of the cells were detected by immunofluorescence;the growth characteristics of the passaged cells were determined by cell counting;the cell viability of MASMCs was detected by CCK-8;the effect of NeoL on KCl-induced contraction of MASMCs was observed by imaging technique;the expression of ROCK1 protein in MASMCs was detected by protein immunoblotting and immunofluorescence.Results The growth curve of MASMCs was approximately“S”shaped,and they were in the logarithmic phase of growth from 4-7 days,which could be used for passaging,with a viability rate of 95%.MASMCs were identified as typical smooth muscle cells with high purity.NeoL significantly inhibited the proliferation of MASMCs with an IC50 value of 1.327μmol·L^(-1).Compared with the model group induced by 124 mmol·L^(-1)KCl,the cell length of MASMCs was significantly longer after 0.3μmol·L^(-1)and 0.6μmol·L^(-1)NeoL intervention(P<0.01)and protein expression of ROCK1 was significantly down-regulated(P<0.05,P<0.001).Conclusion The mouse model of MASMCs was successfully constructed with high cell purity and stable cell characteristics after multiple passages;NeoL had an inhibitory effect on KCl-induced contraction of MASMCs,which may be related to the down-regulation of ROCK1 protein expression.

关 键 词：肠系膜动脉平滑肌细胞 胶原酶消化法 莲子心新碱 收缩抑制 ROCK1蛋白 小鼠 

分 类 号：R285.5[医药卫生—中药学]